5-HT2C RECEPTORS: CELLULAR LOCALIZATION

AND CONTROL OF

DOPAMINERGIC PATHWAYS IN THE RAT

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

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candidate for the Ph.D. degree *.

(signed)______(chair of the committee)

______

______

______

______

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(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 10-13 Introduction to Serotonin 13-14 Evidence for Control of DA Activity 15-31 Roles of 5-HT2 Receptors in Modulating DA Activity 15-16 5-HT2A 16-21 Localization 16-18 18 18-19 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 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 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 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

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 , 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 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 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 (NE) and epinephrine – the other 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 in which cell bodies in one brain region send projections to another brain region where DA is released from terminals. The first to be characterized was the nigrostriatal pathway in which cell bodies in the (SNpc) project their to the caudate and , 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 (VTA) project axons to the (NA), and the tuberoinfundibular pathway in which cell bodies in the of the project axons to the (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 , specifically the prefrontal cortex (PFC) (Glowinski, 1975).

DA has binding affinity for  and  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). DA is cleared from the synapse by a transporter (DAT) that has been shown, in some regions, to be expressed far from synaptic release sites suggesting that DA diffuses from the synapse and acts as a neuromodulator at more distal sites (Sesack et al., 1998).

Early work examining the function of DA in the brain suggested an inhibitory role for DA. It was known that application of DA to the striatum resulted in inhibition of neurons in the striatum (Bloom et al., 1965). Once it was discovered that the highest levels of DA were found in the striatum, it was suggested that DA is involved in the control of motor function (Carlsson et al., 1959). Psychomotor stimulant drugs, such as , are known to increase extracellular levels of DA and concurrently increase motor behavior (van Rossum and Hurkmans, 1964; Sharp et al., 1987). Reserpine, a drug that interferes with the storage of monoamines in vesicles, is known to cause depletion of DA and other monoamines and Parkinsonian symptoms that can be alleviated by administration of the DA precursor L-Dopa (see Barbeau, 1961 for review).

While originally it was thought to be a disease of DA metabolism, it was eventually discovered that Parkinson’s disease is caused by the selective degeneration of the 12 dopaminergic nigrostriatal pathway (Carlsson, 1959; and see Hornykiewicz, 1965 for review).

Likewise, early work suggested that schizophrenia was a disorder of catecholamine metabolism and the buildup of a “toxin” in the brain (see Faurbye, 1968 and Stein, 1971 for review). As work in the field progressed it became known that early

(typical) antipsychotic medications used to treat schizophrenia blocked DA receptors

(Seeman et al., 1976), and it was suggested that schizophrenia may be a disorder caused by hyperactivity of DA systems (Glowinski, 1975). It was not known, however, whether the actions of medications on DA systems were involved in the beneficial aspects of the drugs or simply responsible for the extrapyramidal side effects

(reviewed in Matthysse, 1973). There is now evidence that DA transmission is increased in the striatum of schizophrenic patients (Laruelle et al., 1999) and specifically, that hyperactivity of the mesolimbic DA pathway is associated with the positive symptoms of the disease, such as hallucinations. On the other hand, hypoactivity of the mesocortical pathway has been linked to the negative symptoms (e.g. anhedonia) and impaired cognition associated with schizophrenia (Weinberger et al., 1987). Much of this knowledge came from the study of the second generation of antipsychotic drugs (e.g. ) that were labeled “atypical” based on the variance between their properties and side effect profiles and that of the older antipsychotic medications (e.g. ).

Most typical and drugs act as DA D2 receptor antagonists, blocking these receptors and subsequently causing an increase in extracellular DA. Atypical antipsychotic drugs cause a greater increase in DA release in the PFC and a smaller increase in the striatum, when compared with typical medications, which may be responsible for their enhanced ability to improve cognition and anhedonia and their 13 absence of extrapyramidal side effects (Moghaddam and Bunney, 1990; Pehek and

Yamamoto, 1994; Kuroki et al., 1999)

Lastly, DA has been strongly associated with reward. Specifically, stimuli that are rewarding, such as food and addictive drugs (e.g. cocaine), cause an increase in DA release from the mesolimbic pathway in the NA (Hernandez and Hoebel, 1988). It has been shown that DA is sufficient for the generation of feelings of reward. However stimulation of the frontal cortex or the NA, in the absence of DA, has been shown to be rewarding (see Wise and Rompre, 1989 for review). Thus, release of DA in the NA is not the final step in the reward pathway, but an intermediate one.

Introduction to Serotonin:

The compound 5-hydroxytryptamine (5-HT), termed serotonin, is made in cell bodies in the in the and send their axons in projections to regions throughout the brain including cerebral cortex, , , hippocampus, and hypothalamus as well as to the (Azmitia and Segal, 1978).

There are seven main types of 5-HT receptors (1-7) with subtypes of most of these for a total of at least 14 different receptors (Roth, 1994). In addition, there are a number of isoforms as a result of pre-mRNA editing of most of these receptors. With the exception of the ionotropic 5-HT3 receptor, all other 5-HT receptors are G-protein coupled receptors (metabotropic) and act through intracellular signaling pathways to hyperpolarize (in the case of 5-HT1 receptors) or depolarize (5-HT2/4/5/6/7) their host cells

(see Barnes and Sharp, 1999 for review). In contrast, the 5-HT3 receptor is an ion channel

(Yakel and Jackson, 1988; Derkach et al., 1989) and agonist binding at these receptors results in an inward flux of cations and thus an excitation of the host cell (Yakel and

Jackson, 1988). All of the 5-HT receptor subtypes have been shown to be expressed 14 postsynaptically. In addition, there is evidence for a presynaptic localization for some 5-

HT receptor subtypes as well. In particular, studies suggest that 5-HT1B receptors may act as heteroreceptors on GABAergic axon terminals in the VTA (O’Dell and Parsons,

2004; Yan and Yan 2001a), while 5-HT1A receptors act as autoreceptors in the raphe nuclei (Pazos and Palacios, 1985; Pompeiano et al., 1992). Thus, the effects of endogenous 5-HT, or serotonergic agonists, at particular 5-HT receptor subtypes depend on the cellular and subcellular localization of those receptors as well as on the signaling pathway to which they are coupled.

5-HT has been shown to be involved in the control of normal functions, such as sleep (Gannon and Millan, 2006), aging (McEntee and Crook, 1991; Nishimura et al.,

1998; Keuker et al., 2005; ), taste (Huang et al., 2006), and sexual behavior (Rodriguez et al., 1984; Giuliano and Clement, 2005). In addition, manipulation of the 5-HT system by the application of exogenous substances that bind to 5-HT receptors and/or the 5-HT transporter (SERT) has shown potential for the treatment of an array of diseases and conditions such as anxiety (Hendricks et al., 2003; DeVane et al., 2005), aggression

(Hendricks et al., 2003), drug abuse and withdrawal (Pandey and Pandey 1996; Parsons et al., 1998; De Deurwaerdere et al., 2005), Alzheimer’s disease (see McEntee and Crook,

1991 for review), migraine (for review see Goadsby, 2005), anorexia nervosa (Kaye et al.,

2005), and schizophrenia (see Meltzer, 1999 for review). Perhaps the most famous therapeutic action of serotonergic agents is the use of 5-HT reuptake blockers for the treatment of depression (for review see Heninger et al., 1984; Perry and Fuller, 1992;

Briley and Moret, 1993). While early blocked reuptake of DA, NE and 5-

HT, some modern treatments are selective for SERT, and thus for reuptake of 5-HT

(Torres et al., 2003). 15

Evidence for Serotonergic Control of DA Activity:

The cell bodies and the terminal regions of the mesocortical, mesolimbic, and nigrostriatal DA pathways are innervated by 5-HT neurons originating in the medial and dorsal raphe nuclei (Geyer et al., 1976; Azmitia and Segal 1978; Parent et al., 1981; Beart and McDonald, 1982; Herve et al., 1987; Nedergaard et al., 1988). Additionally, studies show that there are direct synaptic contacts between 5-HT terminals and DA cells in the (Herve et al., 1987; Nedergaard et al., 1988). Thus, 5-HT could potentially regulate the function of DA neurons via actions on midbrain DA cell bodies and/or DA terminals.

In fact, several studies have shown that manipulation of the serotonergic system can cause changes in dopaminergic behaviors and/or DA release in the brain. Early research showed that lesions of the raphe nuclei increased DA utilization in the NA and striatum and decreased DA utilization in the PFC (Herve et al., 1979, 1981). Likewise, application of 5-HT decreased stimulated-DA release from striatal slices (Westfall and

Tittermary, 1982) while acute treatment with a selective 5-HT reuptake inhibitor (SSRI) increased DA release in the PFC (Bymaster et al., 2002). Thus, it is likely that 5-HT receptors mediate the modulation of DA release by endogenous 5-HT and could provide a target for artificial manipulation of dopaminergic activity in particular brain regions.

Roles of the 5-HT2 Receptors in Modulating DA Activity:

The clinical efficacy of psychotherapeutic drugs that act on 5-HT systems may be due in part to their effects on DA systems. In particular, it has been shown that the 5-

HT2 receptor subtypes are capable of modulating dopaminergic activity and there is evidence that this regulation may be involved in the mechanism of action of atypical antipsychotic drugs (see Alex and Pehek, 2006). Work in this field was initially hampered 16

by lack of selective ligands that distinguished between the 5-HT2 receptor subtypes. The recent development of more selective drugs has aided these studies. It has been shown that the 5-HT2 receptor subtypes have unique localization patterns in the brain. In addition, they show subtle differences in coupling and may have different cellular and subcellular expression patterns. Also, studies have shown that 5-HT2A and 5-HT2C receptors differentially modulate DA release from the major dopaminergic pathways. This is important clinically since individual patients have a range of symptoms that may reflect dopaminergic dysfunction in some brain areas but not others and most therapeutics used to treat schizophrenia have affinity at both 5-HT2 receptor subtypes. In addition, undesirable medication side effects may be eliminated through the targeting of selective brain regions. This section will discuss the neuroanatomical basis for 5-HT2 receptor/DA interactions. Subsequently, the neuropharmacological evidence supporting a role for each of the 5-HT2 receptor subtypes in the modulation of the three major DA pathways will be summarized.

5-HT2A:

Localization. 5-HT2A receptors are positively coupled primarily to phospholipase

C through the Gq protein (Roth et al., 1984). Stimulation of these receptors with 5-HT or

5-HT2A agonists results in the production of inositol phosphates (Roth et al., 1984).

These receptors are capable of constitutive receptor activity, in vitro and in vivo, characterized by activation of second messenger pathways in the absence of agonist stimulation (Shapiro et al., 2002). Thus it has recently been discovered that many drugs labeled as antagonists at these receptors are actually inverse agonists due to their ability to not only block, but reverse this constitutive activity (Weiner et al., 2001). 17

Localization studies have shown that 5-HT2A receptors are expressed in several brain regions including the terminal regions of the DA pathways: the striatum, NA, and

PFC (Pazos et al., 1985; Pompeiano et al., 1994; Wright et al., 1995; Willins et al., 1997;

Cornea-Hebert et al., 1999). 5-HT2A receptors have also been shown to be expressed at low levels in the cell body regions of the DA pathways: the VTA and SN (Cornea-Hebert et al., 1999). 5-HT2A receptor expression has been most extensively studied in the cortex where they are localized primarily to the apical of pyramidal neurons but show some expression in parvalbumin-containing GABAergic interneurons as well (Willins et al., 1997; Hamada et al., 1998; Jakab and Goldman-Rakic, 1998; Cornea-Hebert et al.,

1999; Miner et al., 2003; Santana et al., 2004). Early lesion studies suggested that 5-HT2A receptors in the cortex are not expressed as presynaptic heteroreceptors on DA terminals or autoreceptors on the terminals of 5-HT neurons (Leysen et al., 1982, Leysen et al.,

1983). However, more recent work provides evidence that there may be some presynaptic 5-HT2A receptors on monoamine axons in the PFC (Miner et al., 2003). An additional study has shown that intracortical administration of a 5-HT2A antagonist is capable of blocking intracortical potassium (K+)-stimulated DA release in the PFC, which is known to result from the depolarization of nerve terminals (Pehek et al., 2001). Thus, there is support for a presynaptic localization of 5-HT2A receptors in the PFC. However, to date, most physiological evidence suggests a role for 5HT2A receptors on corticotegmental projection neurons (Bortolozzi et al., 2005; Pehek et al., 2006).

Likewise, in the striatum, the majority of the 5-HT2A receptors have been shown to be presynaptic receptors on terminals of afferents from the cortex and

(Bubser et al., 2001). A small percentage of the 5-HT2A receptors in the striatum are expressed on local interneurons (Bubser et al., 2001). Thus, mRNA levels for 5-HT2A 18 receptors in the striatum are low (Bubser et al., 2001). In the VTA, it has been shown that 5-HT2A receptors are expressed in both dopaminergic and non-dopaminergic

(presumably GABAergic) cell bodies (Doherty and Pickel, 2000; Nocjar et al., 2002).

Further work examining the cellular and subcellular localization of 5-HT2A receptors in the cell body and terminal regions would aid in their elucidation.

Nigrostriatal Pathway. The majority of the work examining a role for 5-HT2A receptors in regulating DA release from the nigrostriatal pathway has focused on modulation of stimulated release. For example, it has been shown that systemic administration of the 5-HT2A SR46349B blocks haloperidol-induced

DA release (Lucas and Spampinato, 2000) and attenuates amphetamine-induced DA release (Porras et al., 2002), in the striatum. Likewise, the mixed 5-HT2A/C receptor agonist DOI has been shown to potentiate amphetamine-induced DA release in the striatum (Ichikawa and Meltzer, 1995). The location of the receptors involved in mediating these effects is largely unknown.

Mesolimbic Pathway. In support of the immunohistochemical data showing 5-

HT2C receptors on DA neurons in the VTA (Nocjar et al., 2002), electrophysiological data provides evidence that stimulation of 5-HT2A receptors by 5-HT results in an excitation of dopaminergic neurons in this region (Pessia et al., 1994; Prisco et al., 1994). It has been shown that stimulation of the dorsal raphe nuclei results in an increase in DA in the NA that is blocked by systemic administration of a 5-HT2A receptor antagonist (De

Deurwaerdere and Spampinato, 1999). Systemic administration of the 5-HT2A receptor antagonist alone did not affect NA DA levels, suggesting that 5-HT2A receptors modulate phasic but not tonic activity of the mesolimbic pathway (De Deurwaerdere and

Spampinato, 1999). There is evidence that 5-HT2A receptors within the NA may play a 19

role in mediating these effects. In one study local administration of a 5-HT2A receptor antagonist blocked the 5-HT-induced increase in NA DA (Parsons and Justice, 1993).

Likewise, local administration of the 5-HT2A/ receptor agonist DOI has been shown to increase DA release in some regions of the NA (Bowers et al., 2000). 5-HT2A receptors within the NA are therefore likely to play some role in regulating local DA release.

However, local administration of a 5-HT2A receptor antagonist to the VTA, but not to the

NA shell, attenuated cocaine-induced hyperactivity in rats (McMahon et al., 2001).

Likewise, intra-VTA administration of a 5-HT2A receptor antagonist has also been shown to block amphetamine-induced locomotion and DA release in the NA (Auclair et al.,

2004). Importantly, 5-HT2A receptor antagonists have been shown to attenuate the discriminative stimulus properties of cocaine (McMahon and Cunningham, 2001). Thus,

5-HT2A receptor antagonism may be a desirable property of therapeutics for drug dependence.

Mesocortical Pathway. Evidence has accumulated that suggests that 5-HT2A receptor activation results in a facilitation of DA release in the PFC. Systemic administration of 5-HT2A receptor agonists increases PFC DA and this increase can be blocked with systemic or intracortical administration of the selective 5-HT2A receptor antagonist M100907 (Gobert and Millan, 1999; Pehek et al., 2001; Bortolozzi et al.,

2005). Likewise, intracortical administration of M100907 attenuates the -induced increase in PFC DA (Pehek et al., 2006). These results suggest that 5-HT2A receptors within the PFC are capable of modulating local DA release. Importantly, the administration of 5-HT2A receptor antagonists primarily affects stimulated, and not basal,

DA release in the PFC (Gobert et al., 2000; Pehek et al., 2001; Pehek et al., 2006). In support of the neurochemical data, studies have shown that 5-HT2A receptor blockade 20

attenuates DA-mediated behaviors. For example, systemic administration of 5-HT2A antagonists attenuates cocaine-induced hyperactivity (Filip et al., 2004).

Studies have examined the neural circuitry involved in the regulation of mesocortical DA release by PFC 5-HT2A receptors. It is possible that the relevant 5-HT2A receptors are localized on neurons in the PFC that project to the VTA and synapse on mesocortical DA neurons. Electron microscopy studies have shown that some cortical pyramidal neurons synapse on mesocortical DA cell bodies in the VTA (Sesack and Pickel, 1992). Also, it is known that stimulation of cortical 5-HT2A receptors increases pyramidal cell activity (Marek and Aghajanian, 1996). Lastly, it has been shown that systemic administration of the 5-HT2 receptor agonist DOI increases glutamate efflux in the VTA while concurrently increasing PFC DA (Pehek et al., 2006).

Intracortical infusions of M100907 block both increases (Pehek et al., 2006). Thus, stimulation of 5-HT2A receptors in the PFC may increase local DA release by increasing glutamate release in the VTA and subsequently exciting mesocortical projection neurons.

Taken together, these studies provide strong evidence that 5-HT2A receptors in the PFC regulate local DA release and suggest that feedback to the VTA may be involved.

However, the possibility that 5-HT2A receptors in the VTA also modulate mesocortical activity cannot be excluded

It is known that atypical antipsychotic drugs increase DA release in the PFC, a property that sets them apart from their typical predecessors. In addition, these atypical drugs have a high affinity for 5-HT2A receptors that has been shown to correlate with their efficacy (see Schmidt et al., 1995). Most typical and atypical antipsychotic drugs are D2 receptor antagonists. When D2 receptor antagonists and 5-HT2A receptor antagonists are administered together in animal studies, DA release in the PFC is enhanced (Westerink et 21 al., 2001; Liegeois et al., 2002). Given the work described here showing that mesocortical activity is augmented by 5-HT2A agonism, the effects of atypical antipsychotic drugs are difficult to understand. Thus, studies that are aimed at understanding how 5-HT2A antagonism increases the efficacy of these therapeutics are critical.

Summary. Stimulation of 5-HT2A receptors facilitates dopaminergic activity in the mesolimbic and mesocortical pathways. A trend for the same direction of effect has been observed for the nigrostriatal pathway, but future studies will help to clarify this role. There is no evidence supporting a role for 5-HT2A receptors in tonically regulating

DA release in the striatum, the NA or the PFC. Studies suggest that 5-HT2A receptors in the PFC play a role in regulating mesocortical release. Likewise there is evidence that 5-

HT2A receptors in both the NA and the VTA are capable of mediating effects on mesolimbic activity. The localization of 5-HT2A receptors that modulate nigrostriatal release is largely unknown.

5-HT2B:

Evidence suggests that 5-HT2B receptors are located primarily in the stomach fundus (Duxon et al., 1997). The limited number of these receptors in the brain has been shown to be restricted to the , lateral septum, dorsal hypothalamus, and medial amygdala (Duxon et al., 1997). There is a paucity of evidence favoring a role for 5-HT2B receptors in modulating dopaminergic activity.

5-HT2C:

Localization. Like the 5-HT2A receptor, 5-HT2C (formerly 5-HT1C) receptor stimulation results in phospholipase C-mediated phosphoinositide hydrolysis and a consequential rise in the intracellular calcium concentration of the host cell (Conn and 22

Sanders-Bush, 1986; Julius et al., 1988; Sanders-Bush et al., 1988; Labrecque et al.,

1995). The 5-HT2C receptor has also been shown to exhibit substantial spontaneous accumulation of inositol phosphates, indicating that 5-HT2C receptors possess a high level of constitutive activity in the absence of agonist stimulation (see Berg et al., 2005 for review).

In-situ hybridization studies have demonstrated that 5-HT2C receptor mRNA is expressed in the VTA (Hoffman and Mezey, 1989; Molineaux et al., 1989; Pompeiano et al., 1994; Wright et al., 1995; Eberle-Wang et al., 1997), both the pars compacta and subdivisions of the SN (Molineaux et al., 1989; Pompeiano et al., 1994; Wright et al., 1995; Eberle-Wang et al., 1997), and in the terminal regions of the nigrostriatal and mesolimbic dopaminergic pathways: the striatum and the NA (Hoffman and Mezey,

1989; Pompeiano et al., 1994; Wright et al., 1995; Eberle-Wang et al., 1997). There is comparatively less evidence for 5-HT2C receptor mRNA in the PFC, however, several studies have shown the presence of 5-HT2C receptor mRNA in the cingulate or anterior (Hoffman and Mezey, 1989; Pompeiano et al., 1994; Wright et al.,

1995). Additionally, a study in showed a similar distribution of 5-HT2C receptor mRNA as has been found in rat brain, including expression in the anterior cingulate cortex (Pasqualetti et al., 1999). Fewer studies have examined the localization of 5-HT2C receptor protein. Several immnunochemistry studies indicate the presence of 5-

HT2C receptors in the striatum (Abramowski et al., 1995; Sharma et al., 1997; Clemett et al., 2000) with the most recent study also showing 5-HT2C receptors in the NA and cingulate cortex as well as the SNpr and SNpc (Clemett et al., 2000).

Recent work demonstrates 5-HT2C immunoreactivity in the VTA and suggests that it is at least partially localized to cell bodies (Bubar et al., 2005; Ji et al., 2006). As for 23

the cellular localization of 5-HT2C receptors in other regions, little is known. In the NA and striatum, the morphology of 5-HT2C mRNA containing cells indicates that they may be efferents, suggesting a localization to GABAergic projection neurons (Eberle-Wang et al., 1997). This study also showed that 5-HT2C mRNA present in the SN and VTA, does not colocalize with hydroxylase (TH) mRNA, a marker for DA neurons. Rather, all cells in the SN that expressed 5-HT2C receptor mRNA also expressed decarboxylase (GAD) mRNA, a marker for GABAergic cells. Physiological data

(discussed below) also provide evidence that 5-HT2C receptors are localized on

GABAergic neurons in the VTA and SN. However, recent work provides anatomical and behavioral support for a localization of 5-HT2C receptors on DA neurons in the VTA (Ji et al., 2006). Although it has not yet been conclusively shown, the possibility that 5-HT2C receptors act as presynaptic heteroreceptors in some regions can not be excluded.

The localization of 5-HT2C receptors in the brain suggests that they are well- positioned to modulate dopaminergic activity in the cell body and/or terminal region of all three major pathways. In agreement, a large body of evidence illustrates that 5-HT2C receptors inhibit DA release in the striatum, NA and PFC and suggests subtle variations between the roles for these receptors in regulating each pathway.

Nigrostriatal Pathway. It is well established that systemic administration of the

5-HT2C receptor inverse agonist SB 206553 increases the firing rate of DA neurons in the

SNpc (DiGiovanni et al., 1999). Although systemic administration of 5-HT2C receptor agonists, including the selective agonist Ro 60-0175, does not significantly decrease basal firing of these neurons (Di Matteo et al., 1999; Di Giovanni et al., 2000), such treatment decreases DA efflux in the striatum (Gobert et al., 2000; De Deurwaerdere et al., 2004;

Alex et al., 2005). Likewise, systemic administration of antagonists (SB 242084) (De 24

Deurwaerdere et al., 2004) and inverse agonists (SB 206553) at 5-HT2C receptors increases DA efflux in this region (De Deurwaerdere and Spampinato, 1999; Di Giovanni et al., 1999; Porras et al., 2002; De Deurwaerdere et al., 2004). These data suggest that 5-

HT2C receptors mediate tonic inhibition of the nigrostriatal pathway either by endogenous

5-HT binding, constitutive activity or a combination of the two. One recent study showed that the 5-HT2C receptor inverse agonist-induced increase in striatal DA was insensitive to the depletion of extracellular 5-HT, suggesting that constitutive activity does indeed play a role in the tonic inhibition (De Deurwaerdere et al., 2004).

There is also evidence that 5-HT2C receptors can modulate the phasic activity of the nigrostriatal pathway. The 5-HT2C inverse agonist SB 206553 has been shown to potentiate cocaine- and morphine-induced increases in striatal DA (Porras et al., 2002;

Navailles et al., 2004). Systemic administration of the 5-HT2C agonist Ro 60-0175 attenuated haloperidol-induced increases in DA in the striatum (Navailles et al., 2004), as well as the nicotine-induced increase in firing rate of SNpc DA neurons (Pierucci et al.,

2004) and striatal DA (Di Matteo et al., 2004).

As most studies to date have employed systemic drug administration, less is known about the location of the relevant receptors. It has been shown that 5-HT2C receptors in the SNpc or SNpr are at least in part responsible for the tonic inhibition of the nigrostriatal system and evidence points to a GABA-mediated effect. Systemic administration of the 5-HT2C receptor agonist mCPP excited SNpr neurons that are presumably GABAergic projection neurons (DiGiovanni et al., 2001). This effect was blocked by pretreatment with the antagonist SB 242084 (DiGiovanni et al., 2001). Also,

Rick et al. showed that a large percentage of (presumably GABAergic) SNpr cells are excited by a 5-HT2C receptor agonist. Interestingly, these effects were TTX-resistant, and 25

therefore the 5-HT2C receptors responsible for these effects are located on the responsive

SNpr neurons (Rick et al., 1995). In addition, we have shown recently that perfusion of the striatum with the 5-HT2C receptor inverse agonist SB206553 increased striatal DA in a concentration-dependent manner (Alex et al., 2005), supporting a role for striatal 5-HT2C receptors in this regulation. Furthermore, systemic administration of the 5-HT2C agonist mCPP decreased striatal DA and this was blocked by intrastriatal infusions of SB 206553.

These results implicate 5-HT2C receptors localized in the striatum in the regulation of nigrostriatal DA.

There is anatomical evidence suggesting that the tonic inhibition of nigrostriatal

DA provided by 5-HT2C receptors is indirect and mediated by the stimulation of

GABAergic cells. Specifically, it can be proposed that the stimulation (or constitutive activity) of 5-HT2C receptors on GABAergic neurons causes an elevation in GABA release in either the SNpr or the striatum that results in inhibition of dopaminergic activity in this pathway. While it is known that 5-HT2C mRNA colocalizes with GAD mRNA in the SNpr and SNpc (Eberle-Wang et al., 1997), the localization of 5-HT2C receptors or receptor mRNA in the striatum remains unknown. The relevant receptors could be located on the cell bodies of striatonigral GABA neurons that have been shown to synapse in the SN on the dopaminergic dendrites of the nigrostriatal neurons (Bolam and Smith, 1990). It is well known that modifications in striatonigral GABA release can produce downstream effects on nigrostriatal dopaminergic activity. Alternately, the relevant 5-HT2C receptors could be located on GABAergic interneurons within the striatum. It has been shown that dopaminergic terminals in the striatum express GABAB but not GABAA receptors (Arias-Montano et al., 1991; Smolders et al., 1995). Additional anatomical studies are required to determine the cell types to which 5-HT2C receptors are 26 localized in each brain region and the circuitry involved in their regulation of nigrostriatal

DA release.

Mesolimbic Pathway: 5-HT2C receptors also regulate the mesolimbic DA pathway. Here too, systemic administration of agonists (most commonly Ro 60-0175) at

5-HT2C receptors decreases DA efflux in the NA (Di Matteo et al., 1999,2000,2004; Di

Giovanni et al., 2000; Gobert et al., 2000; De Deurwaerdere et al., 2004) and decreases the firing rate of VTA DA neurons (Prisco et al., 1994; Prisco and Esposito, 1995; Di

Matteo et al., 1999, 2000; Di Giovanni et al., 2000; Gobert et al., 2000). The inverse agonist SB 206553 (Di Giovanni et al., 1999; Gobert et al., 2000) and the antagonists and SB 242084 (Prisco et al., 1994; Di Matteo et al., 1999 respectively) increase the firing rate of these neurons. Likewise, a corresponding increase in NA DA efflux is seen after systemic administration of inverse agonists (Di Matteo et al., 1998; De

Deurwaerdere and Spampinato, 1999; Di Giovanni et al., 1999; Gobert et al., 2000;

Porras et al., 2002; De Deurwaerdere et al., 2004) and antagonists (Di Matteo et al., 1999;

De Deurwaerdere et al., 2004). As has been shown for the nigrostriatal pathway, the 5-

HT2C receptor inverse agonist-induced increase in NA DA is insensitive to decreases in extracellular 5-HT, suggesting that constitutive activity at these receptors is responsible for the inhibition of DA efflux that is reversed by the drug (De Deurwaerdere et al.,

2004).

There is evidence that 5-HT2C receptors also play a role in modulating phasic mesolimbic activity, as concentrations of 5-HT2C ligands that do not affect basal levels of

DA in the NA can affect stimulated release. Systemic administration of a 5-HT2C inverse agonist or antagonist potentiates the cocaine-induced increase in NA DA (Navailles et al.,

2004). It is possible that the elevated levels of extracellular 5-HT seen after cocaine 27 administration normally provide GABA-mediated negative feedback to the cocaine- stimulated mesolimbic system by acting at 5-HT2C receptors. Thus, antagonism at these receptors would potentiate cocaine-induced increases in NA DA by blocking the 5-HT2C receptor-mediated inhibitory tone (Navailles et al., 2004). In support of this mechanism of action, 5-HT2C receptor knockout mice have been shown to exhibit enhanced cocaine- induced elevations of DA in the NA (Rocha et al., 2002). 5-HT2C antagonists have also been shown to potentiate PCP-induced increases in NA DA, likely by a similar 5-HT- dependant mechanism (Hutson et al., 2000). In addition, the 5-HT2C agonists Ro 60-0175 and MK-212 have been shown to attenuate haloperidol (Navailles et al., 2004) and morphine (Willins and Meltzer, 1998)-induced DA release in the NA respectively. Taken together, these findings suggest a role for 5-HT2C agonists in attenuating the effects of psychostimulants and other drugs of abuse. Interestingly, one recent study suggests that overactive mesolimbic 5-HT2C receptors cause a reduced level of NA DA that is involved in depression. The authors propose that effective putative antidepressants will have 5-

HT2C antagonism, which is known to cause these receptors to internalize over time, neutralizing the imbalance (Dremencov et al., 2005).

As previously mentioned, 5-HT2C receptors have been detected on cell bodies in the VTA by immunochemistry (Bubar et al., 2005) and there is evidence that these receptors regulate mesolimbic DA release. As discussed for the nigrostriatal system, these effects may be mediated by actions on GABA cells in the VTA. Intra-VTA administration of the 5-HT2C inverse agonist SB 206553 has been shown both to attenuate

MDMA-induced increases in VTA GABA and potentiate the concurrent increase in NA

DA (Bankson and Yamamoto, 2004). Likewise, systemic administration of a 5-HT2C agonist has been shown to excite all non-DA, presumably GABAergic cells in the VTA, 28

suggesting that 5-HT2C receptors in this region are localized to GABAergic neurons (Di

Giovanni et al., 2001). In addition, one study demonstrated that local administration of the 5-HT2C receptor antagonist RS 102221 increased NA DA, suggesting that, like the nigrostriatal pathway, the mesolimbic pathway is tonically regulated by 5-HT2C receptors in its terminal region (Dremencov et al., 2005). The cellular localization of these receptors in the NA is, however, unknown. While most work to date suggests that 5-

HT2C receptors tonically inhibit dopaminergic activity in a GABA-mediated manner, a recent study suggests an alternate mechanism. Ji et al. (2006) provide strong correlative evidence that 5-HT2C receptors in the VTA are localized, at least in part, to DA neurons.

Their data indicate that 5-HT2C receptors on VTA DA neurons physically interact with the tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) and that like the systemic administration of a 5-HT2C receptor agonist, disruption of this interaction results in inhibition of the mesolimbic DA pathway. Importantly, disrupting the interaction of PTEN with 5-HT2C receptors mimics the action of 5-HT2C receptor agonists in blocking both the increase in mesolimbic DA activity induced by 9- tetrahydrocannabinol (THC), the psychoactive component of marijuana, and conditioned place preference for both THC and nicotine (Ji et al., 2006). These findings provide further information on the role that 5-HT2C receptors play in mediating the rewarding effects of drugs of abuse and suggest disruption of this protein-protein interaction as a potential treatment for drug .

Mesocortical System. 5-HT2C receptors also appear to tonically inhibit release of

DA from the mesocortical pathway. Systemic administration of 5-HT2C receptor agonists decreases the firing rate of VTA DA neurons, while inverse agonists or antagonists increase the firing rate, as mentioned in the discussion of the mesolimbic pathway. 29

Systemic administration of the 5-HT2C receptor agonist Ro 60-0175 also causes a decrease in DA efflux in the PFC (Millan et al., 1998; Gobert et al., 2000) while inverse agonists (Gobert et al., 2000) and antagonists (Millan et al., 1998; Gobert et al., 2000;

Pozzi et al., 2002) increase DA efflux in the PFC. As for the localization of the relevant receptors, studies suggest that 5-HT2C receptors localized in the PFC do not modulate DA release in this region, either tonically (Pozzi et al., 2002; Alex et al., 2005) or phasically

(Pozzi et al., 2002; Alex et al., 2005; Pehek et al., 2006). We have recently shown that infusions of SB 206553 directly into the PFC did not alter basal, K+-stimulated, or stress- induced cortical DA release (Alex et al., 2005; Pehek et al., 2006). Cortical 5-HT2C receptors do modulate DA-mediated behaviors for intracortical infusions of a 5-HT2C antagonist potentiated the hyperlocomotion induced by a systemic injection of cocaine

(Filip and Cunningham, 2003). However, current evidence suggests that this effect is not mediated by alterations in extracellular DA in the PFC (Alex et al., 2005).

In contrast, it has been shown that administration of the 5-HT2C agonist Ro 60-

0175 in the cell body region, the VTA, completely antagonized stress-induced increases in PFC DA, indicating a role for VTA 5-HT2C receptors in the modulation of PFC DA

(Pozzi et al., 2002). The lack of terminal-region 5-HT2C receptor modulation of the mesocortical pathway represents a significant difference from the nigrostriatal and mesolimbic systems. Elucidating the complete neural circuitry of each pathway will be beneficial to the treatment of disorders that may differentially involve the major dopaminergic pathways.

Summary. 5-HT2C receptors, perhaps because of their high level of constitutive activity, posses a unique ability to tonically regulate DA release from all three major pathways. This tonic inhibition of DA release has been shown to be regulated by 5-HT2C 30 receptors in the terminal regions of the nigrostriatal and mesolimbic pathways, whereas 5-

HT2C receptors in the PFC seem to be incapable of tonically or phasically inhibiting the mesocortical pathway. 5-HT2C receptors in the cell body regions of all three pathways are, however, capable of modulating DA release in stimulated conditions. Thus, characterizing the role of the 5-HT2C receptor in the regulation of dopaminergic transmission may have implications for the treatment of schizophrenia, depression,

Parkinson’s disease, anxiety, and drug abuse.

Summary and Implications:

This section has summarized the evidence supporting roles for the 5-HT2 receptor subtypes in regulating dopaminergic activity. There is evidence, however, for regulation of dopaminergic activity by nearly all of the 5-HT receptor subtypes. From this collective evidence, commonalities emerge. With the exception of the constitutively active 5-HT2C receptor, the 5-HT receptor subtypes do not appear to tonically modulate DA, as evidenced by the lack of effect of antagonist treatments alone. The 5-HT receptors are nearly all, however, capable of regulating DA activity when 5-HT tone is elevated (e.g. in response to stress or blockade of the 5-HT transporter by cocaine or SSRIs), or when they are stimulated by exogenous agonists. For the receptor subtypes that have mechanisms of regulating DA that are more understood, the effects are often indirect and mediated by complex neuronal circuitry involving other transmitters. For example, 5-HT2A and 5-

HT1A receptors are thought to be localized to pyramidal glutamatergic neurons in the PFC, and to regulate DA through “long-loop” feedback to the VTA. Likewise, there is evidence that 5-HT2C and 5-HT1B receptors in the VTA regulate mesocorticolimbic DA neurons indirectly by influencing GABA release from their host cells. Thus, the complexities of these circuits are significant. Future research must employ a variety of 31 techniques to determine the precise cellular and subcellular localization of each receptor in individual brain regions and the circuitry involved in their regulation of DA release from each pathway. Thus, the goal of this work was to focus on the 5-HT2C receptor, using microdialysis and immunofluorescence studies to examine 5-HT2C receptor- mediated modulation of DA release and the cellular localization of these receptors in the rat brain.

The Unique Importance of the 5-HT2C Receptor:

5-HT2C receptors have received an increasing amount of attention in recent years.

As previously mentioned, these receptors are widely distributed in the brain and are known to tonically inhibit dopaminergic activity, perhaps in part due to their high level of constitutive activity. Polymorphisms in the 5-HT2C receptor gene can cause variations in the level of this constitutive activity (Okada et al., 2004). Recently it has been discovered that 5-HT2C receptors form homodimers in living cells and that this dimerization may be required for normal function of the receptor (Herrick-Davis et al., 2005, 2006). In addition, it is known that 5-HT2C receptors are down-regulated in response to chronic exposure to ligands. Evidence suggests that these receptors are down-regulated in response to agonists, as is the expected compensatory mechanism (Barker and Sanders-

Bush, 1993; Pranzatelli et al., 1993; Schlag et al., 2004) but also down-regulated after chronic exposure to antagonists and inverse agonists (Barker and Sanders-Bush 1993;

Pranzatelli et al., 1993; Labrecque et al., 1995). However, some controversy remains on this topic. Taken together, these studies show that 5-HT2C receptors are dynamically expressed and thus their function can be altered by a number of different mechanisms by treatments and pathologies. 32

It has been shown that 5-HT2C receptors undergo RNA editing in the second intracellular loop of the receptor generating different isoforms of the receptor in a brain- region-specific manner (Burns et al., 1997). These isoforms show different responses to

5-HT and 5-HT agonists due to differences in the strength of coupling to intracellular signaling pathways (Burns et al., 1997; Herrick-Davis et al., 1999; Niswender et al.,

1998, 1999). The isoforms also show differences in constitutive activity in the absence of an agonist (Niswender et al., 1999). Importantly, RNA editing at the 5-HT2C receptor affects the efficacy of lysergic acid diethylamide (LSD) and antipsychotic drugs

(Niswender et al., 2001). In addition to their ability to down-regulate in response to chronic ligand exposure, it has been shown that the editing of 5-HT2C receptor mRNA is able to change, in a compensatory fashion, in response to reduced levels of 5-HT or chronic treatment with an agonist (Gurevich et al., 2002). Specifically, 5-HT depletion causes a decrease in mRNA editing, resulting in increased expression of the most efficiently-coupled form of the 5-HT2C receptor. Likewise, stimulation with an agonist resulted in an increase in mRNA editing, producing inefficiently-coupled receptors and compensating for the chronic stimulation (Gurevich et al., 2002). While in some strains the majority of 5-HT2C receptors are edited, in BALB/c mice, known to be overanxious and reactive to stress, over 70% of the 5-HT2C receptors in the forebrain are non-edited (Englander et al., 2005). Interestingly, these mice also have reduced levels of

5-HT in the forebrain, and thus a need for efficiently-coupled 5-HT2C receptors. In these mice both acute stress and chronic treatment with an SSRI resulted in increases in 5-HT2C receptor mRNA editing, likely in response to elevated levels of 5-HT (Englander et al.,

2005). BALB/c mice also show increased expression of the non-edited form of the 5-

HT2C receptor in the amygdala, a region implicated in the generation of fear and anxiety 33

(Hackler et al., 2006). Thus, it is possible that the overanxious phenotype of BALB/c mice is, at least in part, due to alterations in 5-HT levels and compensatory changes in 5-

HT2C receptor signaling.

Specific patterns of 5-HT2C mRNA editing in the PFC have also been observed in human suicide victims, although the patterns varied between studies (Niswender et al.,

2001; Gurevich et al., 2002). In addition, expression of the non-edited (and most efficiently coupled) form of the 5-HT2C receptor is enhanced in schizophrenia which may result in the potentiation of 5-HT2C-receptor mediated activity (Sodhi et al., 2001).

Lastly, recent evidence suggests that some atypical antipsychotic drugs may act as agonists at some isoforms of the 5-HT2C receptor and antagonists at others, allowing for the possibility of brain-region and activation-state selectivity of stimulation (Shapiro et al., 2003; Zhang et al., 2006).

The administration of psychotherapeutic agents that bind to 5-HT2C receptors is likely to result in a disruption of 5-HT2C receptor-mediated inhibition of dopaminergic activity and a downstream dysregulation of DA release that could manifest itself as benefits and/or side effects of treatment. Typical and atypical antipsychotic drugs bind to a wide array of receptors in the brain including 5-HT2C receptors (Roth et al., 1992). It has been shown that these therapeutics act as inverse agonists at 5-HT2C receptors

(Herrick-Davis et al., 2000; Rauser et al., 2001). This binding results in increases in dopaminergic activity that may be efficacious in the treatment of schizophrenia (Di

Matteo et al., 2002; Navailles et al., 2006). It is known that 5-HT2C receptor agonist administration to the is associated with oral dyskinesia (Eberle-

Wang et al., 1996). Recently, several groups have investigated polymorphisms in the 5-

HT2C receptor gene and their correlation with efficacy of atypical antipsychotic drugs and 34 their side effect profiles and tendency to produce weight gain. The results of these studies are thus far inconclusive (for review see Reynolds et al., 2005) and the specific contributions of 5-HT2C receptor binding to the efficacy and side effects of atypical antipsychotic drugs remain unknown. Consistent with the ability of 5-HT2C ligands to alter DA release in the striatum and affect motor activity, the 5-HT2C antagonist SB

200646A and the inverse agonist SB 206553 have been shown to be beneficial in animal models of Parkinson’s disease (Fox et al., 1998; Fox and Brotchie, 2000). Likewise 5-

HT2C receptor antagonism in the subthalamic nucleus has been predicted to be therapeutic for the hypokinetic state seen in Parkinson’s disease (DeDeurwaerdere and Chesselet,

2000). This treatment can not be optimized until the neural circuitry underlying these benefits is known.

Antidepressants act to elevate the extracellular concentration of 5-HT in the brain, presumably resulting in stimulation of all of 5-HT receptor subtypes. While the specific receptor(s) linked to clinical efficacy are not known, studies in animal models of depression suggest antagonism of 5-HT2C receptor stimulation may play a role in treatment (Dremencov et al 2005). Many SSRIs are antagonists or inverse agonists at 5-HT2C receptors and in many cases this property has been shown to mediate some of their beneficial effects (Clenet et al., 2001; Hietala et al., 2001; Syvalahti et al.,

2006). There is evidence that chronic treatment with SSRIs desensitizes 5-HT2C receptors, a phenomenon which may be responsible for the delay in efficacy of these drugs (Hietala et al., 2001; Yamauchi et al., 2004; Palvimaki et al., 2005). Likewise, it has been shown that in SERT knockout mice 5-HT2C receptor expression is reduced in a brain-region-specific manner (Li et al., 2003) and there is a reduction in 5-HT2C/2A receptor agonist-induced behaviors and intracellular signaling as well (Qu et al., 2005). 35

Taken together, these data suggest that 5-HT2C receptor antagonism may be a vital property of antidepressant medications.

5-HT2C receptors have also been implicated in anxiety. Both 5-HT2C antagonists

(SB 242084) and inverse agonists (SB 206553) possess anxiolytic-like properties in animal models of anxiety (Kennett et al., 1996, 1997; Martin et al., 2002). Studies suggest that the relevant receptors may be located in both the amygdala (Campbell and

Merchant, 2003; de Mello Cruz et al., 2005; Overstreet et al., 2006), and the hippocampus (Alves et al., 2004). 5-HT2C receptor down-regulation following chronic exercise (Broocks et al., 1999), and desensitization in response to SSRI-treatment

(Kennett et al., 1994; Questad et al., 1997) have been proposed as the mechanisms by which these two treatments are anxiolytic.

5-HT2C receptors have also been implicated in feeding behavior. 5-HT2C receptor knockout mice exhibit increased food intake and subsequent obesity (Tecott et al., 1995;

Heisler et al., 1998). Likewise, 5-HT2C agonists have been shown to be hypophagic in several organisms including mice, rats, Siberian hamsters and quails (Fone et al., 1998;

Cedraz-Mercez et al., 2005; Dunlop et al., 2005; Schuhler et al., 2005). Evidence suggests that 5-HT2C receptors in the ventromedial hypothalamic nucleus may be, at least in part, responsible for the decreased food intake associated with 5-HT2C receptor agonists

(Hikiji et al., 2004). These compounds are thus being developed as anti-obesity drugs and have been effective in several animal models of obesity (Dunlop et al., 2005; Sard et al., 2005; Smith et al., 2005; Rosenzweig-Lipson et al., 2006). In particular, it has been shown that obese rats chronically treated with a 5-HT2C agonist showed a lack of tolerance to the hypophagic effects of the drug and thus great potential for the treatment of obesity (Roth and Shapiro, 2001; Rosenzweig-Lipson et al., 2006). 36

5-HT2C receptors may also play a role in the mechanism of action of psychostimulants. Recent studies have shown that systemic administration of the 5-HT2C antagonists SB 242084 and SDZ SER-082 enhances cocaine-induced locomotor activity

(Fletcher et al., 2002; Filip et al., 2004; Liu and Cunningham, 2006), the discriminative stimulus effects of cocaine (Filip et al., 2006) and cocaine self-administration (Fletcher et al., 2002). Likewise, systemic administration of 5-HT2C agonists (MK-212, Ro 60-0175 or mCPP) attenuates cocaine-induced locomotor activity (Grottick et al., 2000; Liu and

Cunningham, 2006), responding for both food and cocaine (Grottick et al., 2000), and the discriminative stimulus effects of cocaine (Callahan and Cunningham, 1995; Frankel and

Cunningham, 2004). Further studies suggest that these effects are mediated, at least in part, by 5-HT2C receptors in the VTA (Fletcher et al., 2004). Additionally, there is evidence that constitutive or agonist-induced activity at 5-HT2C receptors in the PFC is capable of attenuating the hyperlocomotive and discriminative stimulus effects of cocaine

(Filip and Cunningham, 2003).

Taken together, these data suggest a role for the 5-HT2C receptor in the symptoms and/or treatment of a wide range of behaviors and disorders. In particular, 5-HT2C receptors play a unique role in the regulation of DA release that is relevant to schizophrenia, drug abuse, and Parkinson’s disease. Therefore, studies examining the differential control that 5-HT2C receptors exert over the dopaminergic pathways relevant to these conditions are clinically relevant. In addition, with the development of new immunofluorescent tools, research focused on elucidating the localization of these receptors and the circuitry underlying their tonic inhibition of DA activity will be valuable as well. The present work tested the hypothesis that 5-HT2C receptors within the terminal regions of the mesocortical and nigrostriatal pathways are responsible, at least in 37 part, for tonically inhibiting DA release in the PFC and striatum, respectively (Chapter 2).

The results generated a second hypothesis, tested in Chapter 3. It was proposed that the observed 5-HT2C receptor-mediated tonic inhibition of DA release may be GABA- mediated. Thus, it was an aim of this work to test the hypothesis that 5-HT2C receptors are expressed on GABAergic cells in the cortex and the striatum by examining the thus far unidentified cellular localization of 5-HT2C receptors in rat brain. 38

CHAPTER 2:

Summary:

Previous work has demonstrated that dopamine (DA) transmission is regulated by serotonin-2C (5-HT2C) receptors but the site(s) in the brain where these receptors are localized is not known. The present work utilized in vivo microdialysis to investigate the modulation of DA release by 5-HT2C receptors localized in the nerve terminal regions of the mesocortical and nigrostriatal DA pathways. Microdialysis probes implanted in the striatum or the prefrontal cortex (PFC) measured dialysate DA concentrations, while the selective 5-HT2B/2C inverse agonist SB 206553 was given locally by reverse dialysis into these terminal regions. Additionally, the effects of the 5-HT2C agonist mCPP on striatal

DA were measured. Local administration of SB 206553 (0.1-100 µM) into the striatum increased DA efflux in a concentration-dependent manner. Systemic administration of mCPP (1.0 mg/kg i.p.) decreased striatal DA levels and attenuated the SB 206553- induced increase. In contrast, infusion of SB 206553 (0.1-500 µM) by reverse dialysis into the PFC had no significant effect on basal DA efflux in this region. Additionally, high concentrations of SB 206553 had no effect on high potassium (K+)-stimulated DA release in the PFC. These data contribute to a body of evidence indicating that 5-HT2C receptors inhibit nigrostriatal dopaminergic transmission. In addition, the results suggest that the nigrostriatal system is regulated by 5-HT2C receptors localized in the dorsal striatum. Elucidating the mechanisms by which serotonin (5-HT) modulates striatal and prefrontocortical DA concentrations may lead to improvements in the treatment of diverse syndromes such as schizophrenia, Parkinson’s disease, anxiety, drug abuse and/or depression. 39

Introduction:

The nigrostriatal dopamine (DA) pathway is one of four major dopaminergic systems in the brain. The cell bodies of neurons in this pathway reside in the substantia nigra pars compacta (SNpc) and project to the dorsal striatum (caudate-putamen).

Degeneration of these neurons results in the subsequent motor deficits of Parkinson’s disease. While attention has focused on the motor functions of the striatum, recent evidence indicates that this structure may be important for normal cognitive behavior and . For example, Laurelle and colleagues provide evidence for dopaminergic hyperactivity in the striatum of schizophrenic patients and suggest that it underlies the positive symptoms of the disease (Laurelle et al., 1999). In contrast, the mesocortical DA pathway, which originates in the ventral tegmental area (VTA) and terminates in the prefrontal cortex (PFC), has long been associated with the regulation of complex cognitive processes. In this case, hypoactivity of mesocortical DA has been linked to the negative symptoms and impaired cognition associated with schizophrenia (Weinberger et al., 1987).

An accumulating body of evidence has demonstrated important neurochemical differences in the regulation of DA release between the nigrostriatal and mesocortical pathways (see Wolf et al., 1987 for review). One class of drugs that differentially modulate DA release in the PFC and the striatum are the atypical antipsychotic agents

(Kuroki et al., 1999; Moghaddam and Bunney, 1990; Pehek and Yamamoto, 1994).

While these drugs bind to many different receptor subtypes, evidence indicates that their ability to alter DA release may result, in part, from their actions as antagonists and/or inverse agonists at serotonin (5-HT) receptors (Pehek et al., 2001, Kuroki et al., 1999).

The cell bodies and the terminal regions of the nigrostriatal and mesocortical 40 pathways are innervated by 5-HT neurons originating in the raphe nucleus (Azmitia and

Segal 1978; Herve et al., 1987). In vivo microdialysis studies have shown that serotonergic ligands alter DA release (e.g. Bowers et al., 2000; Galloway et al., 1993;

Pehek, 1996). In particular, systemic administration of inverse agonists at the 5-HT2C receptor subtype increases DA efflux in both the striatum (De Deurwaerdere and

Spampinato, 1999; Di Giovanni et al., 1999; Porras et al., 2002) and PFC (Gobert et al.,

2000; Millan et al., 1998; Pozzi et al., 2002). Receptor binding, in-situ hybridization and immunohistochemical studies have demonstrated that 5-HT2C receptors are expressed in specific brain regions (Abramowski et al., 1995; Clemett et al., 2000; Eberle-Wang et al.,

1997; Molineaux et al., 1989; Pasqualetti et al., 1999; Pompeiano et al., 1994). These areas include those containing mesocortical and nigrostriatal cell bodies: the VTA

(Eberle-Wang et al., 1997; Molineaux et al., 1989; Pompeiano et al., 1994), and the SNpc

(Abramowski et al., 1995; Eberle-Wang et al., 1997; Molineaux et al., 1989; Pasqualetti et al., 1999; Pompeiano et al., 1994), respectively. 5-HT2C receptors are also present in the terminal regions: the striatum (Abramowski et al., 1995; Clemett et al., 2000; Eberle-

Wang et al., 1997; Pasqualetti et al., 1999; Pompeiano et al., 1994) and the PFC

(Pasqualetti et al., 1999; Pompeiano et al., 1994). Thus, 5-HT2C receptors could modulate

DA transmission at the level of the cell bodies or nerve terminals.

Previous microdialysis studies demonstrating that systemically administered 5-

HT2C inverse agonists and antagonists increase DA release (see above) indicate that 5-

HT2C receptors inhibit dopaminergic neurons comprising the nigrostriatal and mesocortical pathways. However, because systemic injections block all 5-HT2C receptors, the anatomical localization of the relevant receptor population(s) remains unknown. To address this, the present experiments used SB 206553 to examine the specific 41

contributions of 5-HT2C receptors in DA nerve terminal areas. SB 206553 has been characterized as an inverse agonist at these receptors (Berg et al., 1999). The affinity of this ligand is at least 100-fold greater for 5-HT2B (pKI 8.9) and 5-HT2C (pKI 7.9) receptors than for any other receptor (Kennett et al., 1996). SB 206553 was infused, by reverse dialysis, into either the striatum or PFC and changes in dialysate DA in these regions were measured. Additionally, the 5-HT2C agonist mCPP was administered systemically, alone and in combination with intrastriatal SB 206553, while striatal DA was quantified.

Lastly, the ability of SB 206553 to alter high potassium (K+)-stimulated DA release in the

PFC was tested. It was hypothesized that local administration of the inverse agonist would increase extracellular DA in the striatum and PFC and potentiate high K+- stimulated DA release in the PFC, providing evidence that DA terminals in the PFC express 5-HT2C receptors. It was also predicted that administration of mCPP would decrease striatal DA levels and attenuate the SB 206553-induced increase, providing evidence that the effects are 5-HT2C receptor-mediated. These results would indicate that

5-HT2C receptors in the terminal regions mediate the increases in extracellular DA observed in response to the systemic administration of 5-HT2C ligands.

Materials and Methods:

Animals and Surgery:

The animals used in this study were male Sprague-Dawley rats (Harlan, Indianapolis, IN, or Zivic-Miller, Zelienople, PA) weighing from 200-400 g at the time of surgery. Rats were housed in pairs in a temperature-controlled room with free access to food and water on a 12hour/12hour light-dark schedule. Prior to surgery, rats were anesthetized with a (131.25 mg/kg) and (11.25 mg/kg) cocktail that was injected i.m.

Subjects were then mounted in a stereotaxic frame. Measurements from bregma were 42 subsequently used to drill a hole through the skull above the anterior striatum (AP 1.2,

ML 3.0) or the PFC (AP 3.0, ML 0.7) according to the atlas of Paxinos and Watson

(1998). 21 gauge stainless steel guide cannulae were then chronically implanted to sit directly on the brain surface without disturbing the brain matter. The guide cannulae were secured in position with QuickTite super glue gel at the skull/cannulae junctions and with three skull screws covered with cranio-plastic cement. Each rat was placed on a heating pad during the initial recovery to maintain a body temperature of 37°C. After awakening from anesthesia, the animals were placed in individual cages where they remained for the 3-7 day period between surgery and microdialysis experiments. All animal use procedures were in strict accordance with the NIH Guide for the Care and

Use of Laboratory Animals and were approved by the local animal care committee.

Microdialysis:

Microdialysis data were collected using probes of a concentric flow design (for details see

Yamamoto and Pehek, 1990). PFC probes were constructed to dialyze the dorsally located anterior cingulate cortex, the prelimbic cortex, and the ventrally located infralimbic subregion. Striatal probes dialyzed the lateral aspects of the anterior striatum.

The active dialyzing surface of the membrane (Spectra/Por Hollow, MW cutoff = 13,000, diameter = 200 m) was 5 mm for the PFC and 3 mm for the striatum. 18 to 24 hours before the start of the experiments, probes were slowly inserted through the guide cannulae into the of awake rats and secured in place with QuickTite super glue gel. The animals were then placed in clear plexiglass test chambers and tethered to counterbalance arms that allowed relatively free movement. Food and water were available ad libitum in these test chambers until the start of the experiment. 43

A micro-infusion pump (Pump 22, Harvard Apparatus, South Natick, MA) and liquid swivels (Instech, Plymouth Meeting, PA) were used to perfuse artificial cerebrospinal fluid (aCSF) through the probes at a constant rate. Unless otherwise noted the aCSF used was modified from commercially available Dulbecco’s phosphate buffered saline (137 mM NaCL, 2.7 mM Kcl, 0.5 mM MgCl2, 1.5 mM KH2PO4, 8.1 mM NA2HPO4, pH: 7.4) by the addition of CaCl2 (1.2 mM) and glucose (10 mM) and was pumped at a rate of 1

L/min. Dialysate samples were collected every 30 minutes until basal DA concentrations were stable (approximately 180 minutes). At this time, drug was administered either by a systemic injection, or by manually switching tubing connections to allow drug diluted in aCSF to pump through the probes. These tubing switches were performed rapidly to maintain constant flow rates and collection volumes. Sample collections continued every 30 minutes for another 3 to 7 hours (see Procedures for individual experiments). Each rat was used for only one microdialysis experiment. At the conclusion of each experiment probe placements were verified histologically. Only data from animals whose probe placements were verified to be in the desired brain region were included in the study.

Drugs:

The free base form of SB 206553, N-3-Pyridinyl-3,5-dihydro-5-methylbenzo(1,2-b:4,5-b') dipyrrole-1(2H)carboxamide, (kindly donated by SmithKline Beecham Pharmaceuticals,

Harlow, UK) was used in all experiments. For reverse dialysis into the striatum, SB

206553 was dissolved in 5 L glacial acetic acid (GAA) and 1 mL deionized water to create a 10 mM stock solution. This solution was then diluted with aCSF to the appropriate micromolar concentration. (0.1-500 M). The pH of the final solutions was 44

7.4. For systemic mCPP administration, mCPP (m-chlorophenylpiperazine) hydrochloride (Tocris, Bristol, UK) was dissolved in deionized water to 1 mg/ml and injected at a volume of 1 ml/kg body weight (i.p.). This dose refers to the salt and was chosen based on previous studies (e.g. Di Giovanni et al., 2000). The pH of the solution was approximately 5.5. Vehicle consisted of deionized water adjusted to the same pH as the drug solution (1 ml/kg i.p.).

Chromatography:

High Performance Liquid Chromatography (HPLC) and electrochemical detection were used to isolate and measure DA in each dialysate sample. Twenty-microliter dialysis samples were injected onto an Ultracarb column (PhenomenexTM, Torrance, CA, 3 μM particle size, ODS 20, 100 x 2 mm). The mobile phase consisted of 32 mM citric acid, 54 mM sodium acetate, 0.074 mM EDTA, 0.215 mM octylsulfonic acid, and 3% methanol

(vol/vol), pH 4.2. When this mobile phase did not provide a clear separation of DA from its metabolite DOPAC and the 5-HT metabolite 5-hydroxyindoleacetic acid, the pH of the mobile phase and the concentration of octylsulfonic acid were adjusted in order to achieve optimal separation. Either a BAS LC-4C or an Antec Intro electrochemical detector with a glassy carbon electrode, maintained at a potential of +0.60 V relative to an

Ag/AgCl reference electrode, was employed. The limit of detection for DA was 0.1 pg/20 µL.

Procedures:

Effects of intrastriatal SB 206553 on striatal DA:

This experiment tested the concentration-dependence of reverse dialysis with SB 206553 into the striatum. There were 3 groups: low concentrations, higher concentrations, and control (aCSF without drug). For the first group, a 0.1 µM concentration was infused for 45

120 minutes followed by a perfusion with a 1.0 µM concentration for 60 minutes. Drug- free aCSF was then infused for 60 minutes before the animals were sacrificed. The second group was similar but 10 µM was infused for 120 min followed by 100 µM for 60 min. These groups were compared to the control group.

Effects of mCPP on the 10 M intrastriatal SB 206553 DA increase:

This experiment tested the ability of the 5-HT2C agonist mCPP to block the effects of SB

206553. There were 4 groups tested in this experiment: mCPP + drug-free aCSF,

VehiclemCPP + drug-free aCSF, mCPP + 10 M SB 206553, and VehiclemCPP + 10 M SB

206553. mCPP (1 mg/kg) or vehicle was given systemically after baselines stabilized.

One hour after the mCPP or vehicle injection, SB 206553 or drug-free aCSF was infused into the brain through the microdialysis probe for 90 minutes. This was followed by perfusion with drug-free aCSF for an additional 90 minutes. The flow rate for this experiment was 1.5 L/min.

Effects of mCPP on the 100 M intrastriatal SB 206553 DA increase:

This experiment tested the ability of mCPP to block the effects of a higher concentration of SB 206553. Two new groups were added: VehiclemCPP + 100 M SB 206553, and mCPP (1 mg/kg) + 100 M SB 206553 in place of the 10 M SB 206553 groups. Other than the change in the concentration of SB 206553, the experimental procedure was identical to the preceding experiment.

Effects of 0.1, 1.0, and 10 M intracortical SB 206553 on PFC DA:

This experiment tested the ability of lower concentrations of SB 206553, infused intracortically, to affect DA release in the PFC. Three concentrations of SB206553 were 46 infused into the PFC in increasing order (0.1, 1.0 and 10 µM) for 120 minutes each.

Drug-free aCSF was then infused through the probe for 60 minutes before the termination of the experiment.

Effects of 100 and 500 M intracortical SB 206553 on PFC DA:

This experiment tested the ability of higher concentrations of SB 206553, infused intracortically, to affect DA release in the PFC. For this experiment and the experiment that follows, the basal perfusion medium used was a Krebs-Ringer buffer (137 mM NaCl,

3 mM KCl, 1.2 mM MgSO4, 0.4 mM KH2PO4, 1.2 mM CaCl2, and 10 mM glucose; pH:

7.4). This buffer was used so that the potassium concentration of the medium could be altered as needed. The 10 mM stock solution of SB 206553 was made as detailed in the drugs section and was diluted with this Krebs-Ringer buffer to 100 or 500 µM (pH 7.4).

Either the 100 or 500 µM solution was infused into the PFC for 60 minutes. Drug-free aCSF was then infused through the probe for 3 hours before the termination of the experiment.

Effects of intracortical SB 206553 on high K+-stimulated DA release in the PFC:

This experiment tested the ability of 100 and 500 M SB 206553, infused intracortically, to alter high K+-stimulated DA release in the PFC. The high K+ buffer was made by increasing the KCl to 80 mM and decreasing the NaCl to 60 mM in order to maintain the osmolarity of the solution. One group of rats was perfused with this high K+ buffer for 30 minutes after baseline samples were collected with the normal Krebs-Ringer buffer. Two additional groups of rats received pretreatments with SB 206553 (100 or 500 µM) beginning 30 minutes before the initiation of high K+ buffer perfusion. The SB 206553 perfusion lasted 60 minutes, and therefore was terminated at the conclusion of the 30 minute high K+ buffer perfusion period. Normal drug-free Krebs-Ringer buffer was then 47 infused for 3.5 hours before the experiment was terminated.

Data Analysis:

Data were expressed as a percentage of the average of the last three pre-drug baseline samples. Statistical analyses were performed using repeated measures ANOVAs. For two-way ANOVAs, time was the repeated measures factor and drug condition was the independent factor. For one-way ANOVAs, time was the repeated factor. Post-hoc comparisons utilized Dunnett’s test for comparing treatment means with a control value.

Results:

Effects of intrastriatal SB 206553 on striatal DA:

Infusions of 0.1 and 1.0 µM SB 206553 significantly increased dialysate DA in the striatum relative to drug-free aCSF controls [2-way ANOVA: significant time by drug interaction F(8,84) = 3.860, p < 0.001; see Fig. 1a]. Post-hoc tests demonstrated a significant increase in DA following both the 0.1 µM and 1.0 µM concentrations (see

Figure 1a). During the 0.1 M infusion, dialysate DA reached a maximum of 168% of baseline 120 minutes after the start of drug infusion. Once the concentration was increased to 1.0 M DA continued to rise to a maximum of 175% of baseline. There was no significant difference between the DA increase in response to 1.0 and 10 M.

Switching to non-drug aCSF resulted in a decrease in dialysate DA.

Infusions of 10 and 100 µM SB 206553 significantly increased dialysate DA in the striatum relative to drug-free aCSF controls [2-way ANOVA: significant time by drug interaction F(8,88) = 6.640, p < 0.001; see Fig. 1b]. Post-hoc tests demonstrated a significant increase in DA following either the 10 µM or 100 µM concentrations (see

Figure 1b). During the 10 M infusion, dialysate DA reached a maximum of 142% of 48 baseline 60 minutes after the start of drug infusion and was maintained at elevated levels until the concentration was increased to 100 M. Following this change in concentration

DA continued to rise to a maximum of 212% of baseline. Switching to non-drug aCSF resulted in a decrease in dialysate DA that approached basal concentrations after one hour. The basal level of dialysate DA averaged from the three separate intrastriatal groups in Figure 1 was 6.38 +/- 1.05 pg/20 l (n = 19).

Effects of mCPP on the 10 M intrastriatal SB 206553 DA increase:

There were four groups tested in this experiment: mCPP + drug-free aCSF, VehiclemCPP +

drug-free aCSF, mCPP + 10 M SB 206553, and VehiclemCPP + 10 M SB 206553. mCPP was administered systemically (1 mg/kg i.p.) while SB 206553 was administered by reverse dialysis into the striatum. A two-way ANOVA comparing all four drug treatments revealed a significant interaction between drug treatment and time [F(24,176)

= 1.829, p = 0.014; see Fig. 2a). Treatment with mCPP resulted in a small but significant decrease in striatal DA [1-way ANOVA: F(8,40) = 2.398, p = 0.032; see Fig. 2]. For the

VehiclemCPP + drug-free aCSF group there was no significant change in striatal DA.

Similarly to the first experiment, infusion of 10 M SB 206553 significantly increased dialysate DA in the striatum [1-way ANOVA: F(8,56) = 2.316, p = 0.032; see Fig. 2a].

The maximal increase was 143% of baseline. Pretreatment with systemic mCPP attenuated this increase in striatal DA [1-way ANOVA on the mCPP + 10 M SB 206553 group showed no significant effect of time: F(8,32) = 1.252, p = 0.302; see Fig. 2a].

Effects of mCPP on the 100 M intrastriatal SB 206553 DA increase:

Two new groups were added: VehiclemCPP + 100 M SB 206553, and mCPP (1 mg/kg) +

100 M SB 206553. These groups were compared to the mCPP + drug-free aCSF and 49

VehiclemCPP + drug-free aCSF groups from the preceding experiment. A two-way

ANOVA on all four treatments groups showed a significant time by drug interaction [F

(24,144) = 11.197, p < 0.001] as shown in Figure 2b. A comparison of Figures 2a and 2b illustrates the concentration-dependence of the SB 206553-induced increase in striatal

DA. The infusion of 100 M SB 206553 significantly increased dialysate DA to a maximum of 345% [1-way ANOVA: [F(8,32) = 13.939, p < 0.001; see Fig. 2b].

Pretreatment with mCPP (1 mg/kg) slightly attenuated this increase in striatal DA.

However, this attenuation was not significant as infusions of 100 µM SB 206553 still significantly increased dialysate DA following mCPP administration [1-way ANOVA: [F

(8,24) = 9.869, p < 0.001]; see Fig 2b]. The average basal level of dialysate DA for the 6 groups of rats that comprise figures 2a and 2b was 3.98 +/- 0.29 pg/20 l (n = 35).

Effects of 0.1, 1.0, and 10 M intracortical SB 206553 on PFC DA:

Figure 3 shows the dialysate DA concentrations after infusion of three increasing concentrations of SB 206553 (0.1, 1.0 and 10 M). No significant increase from basal

DA levels was seen in response to any concentration of SB 206553 [1-way ANOVA: [F

(14,70) = 0.922, p = 0.540; see Fig. 3]. The basal level of dialysate DA for the intracortical group was 0.52 +/- 0.06 pg/20 l (n = 6).

Effects of 100 and 500 M intracortical SB 206553 on PFC DA:

There also was no significant increase in PFC DA in response to higher concentrations

(100 and 500 M) of SB 206553 as shown in Figure 4a. The average basal level of dialysate DA for the 2 groups of rats that comprise Figure 4a was 0.75 +/- 0.08 pg/20 l

(n = 14).

Effects of intracortical SB 206553 on high K+-stimulated DA release in the PFC: 50

PFC DA was increased to a maximum of 292% of baseline in response to perfusion with high K+ buffer [1-way ANOVA shows significant effect of time F(9,81) = 8.839, p <

0.001; see Fig. 4b]. Pretreatment with 100 or 500 M SB 206553 had no significant effect on high K+-stimulated DA release in the PFC, as illustrated in Figure 4b [1-way

ANOVAs continue to show significant effects of time F(9,45) = 3.197, p = 0.005 and F

(9,54) = 3.616, p = 0.001 respectively]. The average basal level of dialysate DA for the 4 groups of rats that comprise Figure 4b was 0.62 +/- 0.05 pg/20 l (n = 31).

Discussion:

These data illustrate that local administration of the 5-HT2C inverse agonist SB

206553 into the striatum increases striatal DA in a concentration-dependent manner. This increase was attenuated by the systemic administration of the 5-HT2C agonist mCPP.

These results are the first to demonstrate that 5-HT2C receptors localized in the striatum may normally serve to inhibit DA release in the nigrostriatal pathway. They indicate that the effects of systemically administered SB 206553 may be mediated, at least in part, by actions on striatal 5-HT2C receptors.

Previous work has shown that systemic administration of SB 206553 also increases DA release in the PFC (Gobert et al., 2000; Millan et al., 1998). However, in the present work, intracortical administration of this ligand in concentrations ranging from 0.1 to 500 µM did not alter basal or high K+-stimulated DA release. These results indicate that the effects of systemic SB 206553 are not due to actions on 5-HT2C receptors localized in the PFC.

The present increase in striatal DA after local administration of SB 206553 is consistent with and extends previous work employing the systemic administration of 5- 51

HT2C ligands. A recent electrophysiological study demonstrated that treatment with SB

206553 (systemically) increases the firing rate of DA neurons in the SNpc (DiGiovanni et al., 1999). In addition, dialysis studies have shown that systemic administration of SB

206553 results in increased striatal DA concentrations (De Deurwaerdere and

Spampinato, 1999; DiGiovanni et al., 1999; Gobert et al., 2000) whereas injections of the

5-HT2C agonist Ro 60-0175 decreases dialysate DA in this region (Gobert et al., 2000).

Several studies using a variety of approaches from receptor binding to immunocytochemisty and in situ hybridization have determined that 5-HT2C receptors are present in the striatum (Abramowski et al., 1995; Clemet et al., 2000; Eberle-Wang et al.,

1997; Pasqualetti et al., 1999; Pompeiano et al., 1994). The present experiments demonstrate that these receptors regulate DA release. Intrastriatal administration of SB

206553 increased DA efflux significantly at concentrations from 0.1 to 100 M (Fig. 1 and 2). This result is inconsistent with one study that found a 30% reduction in striatal

DA in response to 1 M intrastriatal SB 206553 (Lucas and Spampinato, 2000). The discrepancy between these results may reflect differences in experimental procedures. In particular, differences in probe placements could be responsible. In this study probes were implanted in the anterolateral striatum whereas the previous study employed striatal placements that were both more posterior and more medial. Other previous work illustrates the heterogeneity of the dopaminergic system in the striatum (Yamamoto and

Pehek, 1990). In addition, 5-HT2C mRNA has been shown to vary between striatal subregions (Eberle-Wang et al., 1997). he present finding that local administration of

SB 206553 into the striatum resulted in a concentration-dependent increase in striatal DA suggests that the nigrostriatal DA system is regulated by 5-HT2C receptors in the 52 anterolateral striatum. The question of whether or not these neurons are also regulated by

5-HT2C receptors at the level of the cell bodies remains unanswered. Infusing SB 206553 into the SNpc by reverse dialysis could resolve this matter.

It is worth noting that SB 206553 binds to the 5-HT2B receptor with approximately the same affinity as for the 5-HT2C receptor (Kennett et al., 1996). While this is a limitation of the present data, evidence suggests that 5-HT2B receptors are located primarily in the stomach fundus and in heart valves (Duxon et al., 1997; Setola et al.,

2003). The limited number of these receptors in the brain has been shown to be restricted to the cerebellum, lateral septum, dorsal hypothalamus, and medial amygdala (Duxon et al., 1997). Therefore, in the brain regions examined in this study, the effects of SB

206553 should not be due to actions at 5-HT2B receptors. In addition, a recent study has shown that the effects on striatal DA of systemic administration of the inverse agonist SB

206553 can be reversed by pretreatment with the selective 5-HT2C antagonist SB 242084

(De Deurwaerdere et al., 2004).

In order to provide additional evidence for 5-HT2C regulation of nigrostriatal neurons, one aim of the present research was to examine the effects of systemic administration of the 5-HT2C agonist mCPP on DA release in the striatum, alone and in combination with SB 206553. mCPP is a widely used agonist with high affinity for the

5-HT2C receptor (pKI = 6.9) (Bonhaus et al., 1997) and moderate affinities for 5-HT1A, 5-

HT1B, and 5-HT2A receptors and the 5-HT transporter (Matsumoto et al., 1992; Bonhaus et al., 1997; Owens et al., 1997). Despite this limitation, mCPP has been shown to act in a

5-HT2C specific manner in many studies. Systemically administered mCPP has been shown to decrease dialysate DA in the nucleus accumbens and decrease the firing rate of

DA cell bodies in both the VTA and the SNpc (Di Giovanni et al., 2000). These effects 53

were attenuated by the selective 5-HT2C antagonist SB 242084, indicating that they were due to actions of mCPP at 5-HT2C receptors (Di Giovanni et al., 2000). The data illustrated in Figures 2A and 2b are consistent with and extend this previous work.

Systemic administration of mCPP alone resulted in a small but significant decrease in striatal DA, providing further evidence that stimulation of 5-HT2C receptors inhibits dopaminergic activity. Additionally, pretreatment with mCPP attenuated the 10 M SB

206553-induced increases in striatal DA. These results suggest that the two drugs, when given in combination, compete for 5-HT2C receptors and support the hypothesis that stimulation of these receptors inhibits the nigrostriatal DA system.

Several recent studies have shown that treatment with SB 206553 (Gobert et al.,

2000) or SB 242084 (Gobert et al., 2000; Millan et al., 1998; Pozzi et al., 2002) increases

PFC DA efflux while administration of the selective 5-HT2C agonist Ro 60-0175 decreases DA levels in this region (Gobert et al., 2000; Millan et al., 1998). Anatomical studies have demonstrated the presence of 5-HT2C receptors in the PFC (Pasqualetti et al.,

1999; Pompeiano et al., 1994). However, in the present study, intracortical infusions of

SB 206553 over a wide concentration-range (0.1-500 M) did not alter basal or high K+- stimulated DA efflux (Figures 3 and 4). Thus, unlike the nigrostriatal system, these results do not support the hypothesis that cortical 5-HT2C receptors regulate the release of

DA from the mesocortical system. They also are consistent with recent work demonstrating that intracortical infusions of the agonist Ro 60-0175 (Pozzi et al., 2002), or the antagonist SB 242084 (Francesc Artigas, personal communication), did not affect

PFC DA. However, infusions of the 5-HT2C antagonist RS 102221 into the PFC potentiated the hyperlocomotion induced by a systemic injection of cocaine (Filip and 54

Cunningham, 2003). Thus, further work must be performed in order to more fully assess the role of cortical 5-HT2C receptors in the regulation of mesocortical DA function. There is evidence that 5-HT2C receptors localized in the VTA regulate mesocortical DA release

(Pozzi et al., 2002).

The cellular localization of 5-HT2C receptors in the striatum has yet to be determined. Thus, the circuitry underlying the regulation of DA release by these receptors remains unknown. Previous work indicates that 5-HT2C receptors are not localized presynaptically on nigrostriatal DA terminals, for lesions of DA neurons do not alter 5-HT2 receptor binding in the striatum or other DA-rich brain areas (Leysen et al.,

1982). Relevant anatomical evidence does demonstrate that 5-HT2C receptors are localized on GABAergic, but not dopaminergic, neurons in the midbrain SNpc and VTA

(Eberle-Wang et al., 1997). Thus, it is possible that 5-HT2C receptors are localized to

GABAergic interneurons or striatonigral projection neurons in the striatum.

The present findings in the striatum are consistent with behavioral data examining the effects of 5-HT2C ligands on DA-mediated behaviors. Administration of 5-HT2C agonists decreases locomotion (Kennett et al., 2000) and attenuates cocaine-induced hyperactivity (Filip and Cunningham, 2003; Grottick et al., 2000), suggesting an inhibition of dopaminergic function. Treatment with 5-HT2C antagonists potentiates cocaine-induced hyperlocomotion (Filip and Cunningham, 2003) and blocks the ability of

5-HT2C agonists to attenuate this hyperlocomotion (Grottick et al., 2000). Additionally,

5-HT2C knock-out mice show increased hyperlocomotion in response to cocaine (Rocha et al., 2002). In support of these behavioral data, one recent study demonstrates that both

SB 206553 and the 5-HT2C antagonist SB 242084 potentiate the cocaine-induced increase in dialysate DA in both the striatum and the nucleus accumbens (Navailles et al., 2004). 55

In addition to alterations in responses to cocaine, 5-HT2C knock-out mice show increased obesity (Heisler et al., 1998) and susceptibility to seizures (Applegate and

Tecott, 1998), suggesting that 5-HT2C receptors play a role in the control of feeding behavior and in modulating neuronal network excitability. 5-HT2C receptors have also been implicated in anxiety. Both 5-HT2C antagonists and inverse agonists possess anxiolytic-like properties in animal models of anxiety (Kennett et al., 1996, 1997). 5-

HT2C receptor down-regulation following chronic exercise (Broocks et al., 1999) and desensitization in response to SSRI-treatment (Questad et al., 1997) have been proposed as the mechanisms by which these two treatments are anxiolytic. Thus, 5-HT2C receptors have been implicated in the regulation of a wide range of behaviors/syndromes.

Studies suggest that serotonergic ligands may have therapeutic potential. Recent work suggests that a combination of the DA-releaser phentermine (Phen) and the 5-HT- releaser (Fen) may be an effective treatment for drug abuse (Brauer et al.,

1996; Rea et al., 1998). Phen has been shown to have a profile of effects similar to d- amphetamine, both in enhancing mood and a high potential for abuse (Brauer et al.,

1996). Combining Phen with Fen, however, reduces the potential for abuse (Brauer et al.,

1996; Rea et al., 1998). Since 5-HT2C agonists can substitute for Fen in drug- discrimination studies, it is thought that these receptors mediate the effects of the drug

(McCreary et al., 2003). Thus, Fen may decrease the abuse potential of Phen by stimulating 5-HT2C receptors that reduce DA release. Additionally, 5-HT2C antagonists have been shown to be beneficial in animal models of Parkinson’s disease (Fox et al.,

1998; Fox and Brotchie, 2000). This evidence is consistent with the present finding that administration of SB 206553 results in increased DA release in the striatum. Thus, elucidating the role of 5-HT2C receptors in the regulation of DA systems may be critically 56 important to the development of new therapeutic agents.

In summary, the present work indicates that the nigrostriatal DA system is under by 5-HT2C receptors localized within the terminal region, the striatum.

Knowledge of the anatomical localization of 5-HT2C receptors regulating DA release will aid in the elucidation of the circuitry underlying this regulation. In turn, knowledge of the underlying circuitry may have implications for the understanding and treatment of numerous syndromes such as schizophrenia, depression, Parkinson’s disease, drug abuse and anxiety. 57

Figure 1: 58

Figure 1: A. Effects of local administration of 0.1 and 1.0 µM SB 206553 on dialysate DA concentrations in the striatum. For the drug group, 0.1 µM SB 206553 was perfused intrastriatally for 120 minutes followed by 60 minutes of 1.0 µM. Drug- free aCSF was then delivered for the final 60 minutes of the experiment. The control group received drug-free aCSF for the duration of the experiment. Data are the means +/-

SEM of 7 animals for each group. * p < 0.05 relative to pre-drug baseline. B. Effects of local administration of 10 and 100 µM SB 206553 on dialysate DA concentrations in the striatum. For the drug group, 10 µM SB 206553 was perfused intrastriatally for 120 minutes followed by 60 minutes of 100 µM. Drug-free aCSF was then delivered for the final 60 minutes of the experiment. The control group received drug-free aCSF for the duration of the experiment. Data are the means +/- SEM of 5-7 animals. * p < 0.05 relative to pre-drug baseline. 59

Figure 2: 60

Figure 2: A. Effects of systemic administration of mCPP (1 mg/kg i.p.) on the 10 µM

SB 206553-induced increase in dialysate DA in the striatum. mCPP or vehicle were injected at time zero as indicated by the arrow. SB 206553 or drug-free aCSF (control) was perfused intrastriatally one hour later for 90 minutes as indicated by the bar. Data are means +/- SEM of 5-8 animals per group. * p < 0.05 relative to pre-drug baseline. B.

Effects of systemic administration of mCPP (1 mg/kg i.p.) on the 100 µM SB 206553- induced increase in dialysate DA in the striatum. mCPP or vehicle were injected at time zero as indicated by the arrow. SB 206553 or drug-free aCSF (control) was perfused one hour later for 90 minutes as indicated by the bar. Data are means +/- SEM of 4-8 animals per group. * p < 0.05 relative to pre-drug baseline. 61

Figure 3:

Figure 3: Effects of local administration of 0.1, 1.0, and 10 µM SB 206553 on dialysate DA concentrations in the PFC. 0.1 µM SB 206553 was perfused intracortically for 120 minutes followed by 120 minutes of 1.0 µM and then 120 minutes of 10 µM. Drug-free aCSF was then delivered for the final 60 minutes of the experiment.

Data are the means +/- SEM of 6 animals. 62

Figure 4: 63

Figure 4: A. Effects of local administration of 100 and 500 µM SB 206553 on dialysate DA concentrations in the PFC. 100 or 500 µM SB 206553 was perfused intracortically for 60 minutes followed by 180 minutes of drug-free aCSF. Data are the means +/- SEM of 7 animals for each group. B. Effects of local administration of 100 and 500 µM SB 206553 on high (80 mM) K+-induced increases in dialysate DA concentrations in the PFC. High K+ solution was infused for 30 minutes as indicated by the solid bar. SB 206553 was infused for 60 minutes as indicated by the dashed bar.

Data are the means +/- SEM of 3-6 animals per group. * p < 0.05 relative to pre-drug baseline. 64

CHAPTER 3:

Summary:

Previous work suggests that serotonin-2C (5-HT2C) receptors are widely distributed in the rodent brain in areas including the hippocampus, cortex, striatum and choroid plexus.

These receptors have been implicated in the pathology and/or treatment of anxiety, obesity, schizophrenia, depression and drug abuse and have been shown to tonically inhibit dopamine (DA) release from all three major dopaminergic pathways. In addition,

5-HT2C receptors exhibit a uniquely high level of constitutive activity that may be responsible for their regulatory role under basal conditions. Due to a lack of specific reagents, little has been done to conclusively describe the cellular localization of 5-HT2C receptors in the brain. The present work employed immunofluorescent methods to examine this localization in Sprague-Dawley rats. In both the cortex and hippocampus,

5-HT2C receptors show a large degree of colocalization with 5-HT2A receptors on pyramidal neurons. In addition, 5-HT2C receptors do not colocalize with parvalbumin in either region, suggesting a complete absence of expression on this type of interneuron.

Lastly, 5-HT2C receptors do not colocalize with glutamic acid decarboxylase (GAD), a marker for neurons containing -aminobutyric acid (GABA) in the striatum, hippocampus, or cortex. Taken together, these data will aid in the elucidation of the mechanism by which 5-HT2C receptors tonically inhibit DA release, and allow for improvements in the development of new therapeutics for a variety of disorders and conditions. In particular, the finding that 5-HT2C and 5-HT2A receptors are colocalized in the cortex and hippocampus is potentially significant for the design of new antipsychotic drugs. 65

Introduction:

In recent years, serotonin (5-HT) has been implicated in a wide variety of pathologies and treatments for clinical diseases and disorders. 5-HT cell bodies, residing in the raphe nuclei, project to forebrain areas regulating a multiplicity of functions/behaviors. For example, research has shown a role for 5-HT in the control of sleep (Gannon and Millan, 2006), mood (see Kroeze and Roth, 1998; Stockmeier, 2003 for review), feeding (Cedraz-Mercez et al., 2005; Schuhler et al., 2005), taste (Huang et al., 2006), stress ( see Leonard, 2005), aggression (Hendricks et al., 2003), and sexual behavior (Rodriguez et al., 1984; Giuliano and Clement, 2005). Studies suggest that 5-

HT binding sites may be targets for the treatment of depression (for review see Briley and

Moret, 1993; Kroeze and Roth, 1998), migraine (for review see Goadsby, 2005), anorexia nervosa (Kaye et al., 2005), schizophrenia (Meltzer, 1999), anxiety (DeVane et al., 2005), Alzheimer’s disease (McEntee and Crook, 1991), drug abuse (Pandey and

Pandey, 1996; McMahon and Cunningham, 2001; Fletcher et al., 2002; Filip et al., 2006), and obesity (Tecott et al., 1995; Dunlop et al., 2005; Smith et al., 2005; Rosenzweig-

Lipson et al., 2006).

How is it that one can affect such an eclectic group of functions and behaviors? Perhaps the answer has little to do with the transmitter itself. There are

14 different subtypes of 5-HT receptors with different expression patterns in the periphery as well as in the brain. Of the serotonin receptor subtypes all but the 5-HT3 receptor (a cation channel) are G-protein-coupled. Despite this structural similarity, the 5-HT receptor subtypes vary in their cellular localizations, cell signaling pathways and mechanisms of action (see Barnes and Sharp, 1999 for review). Thus, the diverse array of 66 functions under serotonergic control is likely due to the complexity and diversity of its targets.

Recent research has focused on identifying the specific targets (receptor subtype

(s)) of endogenous 5-HT involved in the regulation of these functions. Once identified, these receptors can be targeted by therapeutic agents that aim to correct serotonergic dysfunction. This research has highlighted 5-HT3 antagonists as anti-emetics (Bermudez et al., 1988), 5-HT2C antagonists as anxiolytics (Kennett et al., 1996,1997) and 5-HT1B receptor agonists as the leading treatment for migraines (see Goadsby, 2005), to name a few clinically relevant examples where targeting a single receptor is beneficial. In some arenas, studies have shown a role for nearly every 5-HT receptor subtype. One such function is the regulation of dopaminergic activity (see Alex and Pehek, 2006). The 5-

HT2A and 5-HT2C receptor subtypes appear to be of particular importance to the treatment of dopaminergic diseases and disorders such as Parkinson’s disease, schizophrenia and drug abuse. In addition, both the 5-HT2A and the 5-HT2C receptor subtypes have been implicated in a broad array of clinical applications extending beyond their control of dopaminergic activity.

The 5-HT2A receptor is a Gq coupled receptor. Agonist stimulation can result in the stimulation of both phospholipase C and phospholipaseA2 leading to the production of inositol phosphates and the release of arachadonic acid, respectively (Berg et al.,

1998). A role for 5-HT2A receptors is postulated in a growing number of conditions. For instance, recent studies suggest that 5-HT2A receptors mediate the neurochemical effects of stress (Pehek et al., 2006), and may be involved in associative learning and working memory (Romano et al., 2006 and Williams et al., 2002, respectively). 5-HT2A receptors also may be involved in the treatment of anxiety (Weisstaub et al., 2006) and the 67 mechanism of action of antidepressants (Marek et al., 2005; Syvalahti et al., 2006;

Yamauchi et al., 2006). Perhaps most importantly, 5-HT2A receptor agonists, such as

LSD, are known to be hallucinogenic and 5-HT2A receptor antagonism is a critical property of atypical antipsychotic medications used to treat schizophrenia (Meltzer, 1999;

Li et al., 2005). In particular, 5-HT2A receptor blockade is thought to be responsible for the reduction of extrapyramidal side effects and negative symptoms associated with this new generation of antipsychotic medication.

Like the 5-HT2A receptor, the 5-HT2C receptor is Gq coupled to phospholipases C and A2 (Berg et al., 1998). 5-HT2C receptors have received a lot of attention in recent years due to their high level of constitutive activity in the absence of agonist stimulation

(see Berg et al., 2005 for review). In addition, 5-HT2C receptors undergo RNA editing that generates different isoforms of the receptor, which differ in their levels of constitutive activity (Burns et al., 1997; Niswender et al., 1999). Studies suggest a possible role for altered 5-HT2C mRNA editing in suicide victims as well as in schizophrenia (Niswender et al., 2001; Sodhi et al., 2001; Gurevich et al., 2002). In addition, studies implicate 5-HT2C receptors as a possible target for the treatment of schizophrenia (Sodhi et al., 2001; Li et al., 2005; Navailles et al., 2006; Zhang et al.,

2006), obesity (Tecott et al., 1995; Cedraz-Mercez et al., 2005; Dunlop et al., 2005;

Schuhler et al., 2005; Smith et al., 2005; Rosenzweig-Lipson et al., 2006), epilepsy

(Tecott et al., 1995; see Isaac, 2005 for review), anxiety (Kennett et al., 1996, 1997; de

Mello Cruz et al., 2005), depression (Dremencov et al., 2005; Palvimaki et al., 2005), drug abuse (Willins and Meltzer, 1998; Rocha et al., 2002; Navailles et al., 2004; Filip et al., 2006), and Parkinson’s disease (Fox et al., 1998; Fox and Brotchie, 2000). 68

Among the brain regions innervated by serotonergic axons are posterior cingulate cortex, frontal cortex, hippocampus, and striatum (Azmitia and Segal, 1978). 5-HT2C receptors are thought to be expressed in a variety of brain regions in rats, monkeys, and humans, including the cingulate cortex (Clemett et al., 2000), the hippocampus (Pandey et al., 2006) and the striatum (Abramowski et al., 1995; Sharma et al., 1997; Clemett et al., 2000; Pandey et al., 2006), and are expressed at high levels in the choroid plexus

(Pandey et al., 2006). As for their cellular localization in these regions, little is known.

5-HT2A receptors are localized to fewer regions of the brain but are indeed expressed in posterior cingulate cortex (Jakab and Goldman-Rakic, 1998), frontal cortex (Cornea-

Hebert et al., 1999), hippocampus (Cornea-Hebert et al., 1999; Luttgen et al., 2004), and striatum (Cornea-Hebert et al., 1999; Bubser et al., 2001). It is known that in the prefrontal cortex 5-HT2A receptors are predominately expressed on pyramidal neurons but also found on interneurons (Willins et al., 1997; Jakab and Goldman-Rakic, 1998, 2000;

Cornea-Hebert et al., 1999; Miner et al., 2003; Santana et al., 2004). However, as for 5-

HT2C receptors, much is left to learn about their cellular localizations in most brain regions.

The aim of this work was to better characterize the expression pattern of 5-HT2C receptors in the rat brain. Specifically, these studies aimed to compare 5-HT2C receptor expression with the well-characterized expression of 5-HT2A receptors by testing the hypothesis that 5-HT2C receptors colocalize with 5-HT2A receptors in the cortex and the hippocampus and that like 5-HT2A receptors, 5-HT2C receptors are expressed in parvalbumin-containing interneurons in these regions. In addition to qualitatively examining the expression patterns, the colocalization of 5-HT2C receptors with both parvalbumin-containing cells and cells that express 5-HT2A receptors were quantified in 69 these regions. Studies examining the localization of these receptors have been hindered by a lack of specific antibodies to the 5-HT2C receptor subtype. Therefore, a second, equally important aim of this work was to test the specificity of the commercially available 5-HT2C receptor antibody used. Western blots and immunofluorescence experiments using cell lines that stably express either 5-HT2A or 5-HT2C receptors were conducted in order to ensure the specificity of the antibody for 5-HT2C receptors.

Methods:

Animals and Fixation:

The animals used in these studies were male Sprague-Dawley rats (Harlan, Indianapolis,

IN) weighing from 200-400 g at the time of sacrifice. Rats were housed in pairs in a temperature-controlled room with free access to food and water on a 12hour/12hour light- dark schedule. Prior to perfusion, rats were deeply anesthetized with sodium pentobarbitol (100 mg/2 ml/rat i.p.). The rats were first perfused with ice cold PBS (~180 mls/rat). When the liver cleared of blood and the fluid outflow from the heart was clear, the rat was perfused with 4% parformaldehyde (Sigma, St. Louis, MO) in PBS (200 mls/rat), also ice cold. For the data displayed in Figure 10 the perfusate also included

0.2% gluteraldehyde. The brains were then removed and post-fixed in the fixative for 24 hours. The brains were then cryoprotected in a 20% sucrose solution for 24 hours and then flash-frozen and stored at -80 °C. All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local animal care committee.

Immunohistochemistry:

The brains were sliced on a cryostat at -19 °C. Immunohistochemistry was performed on free-floating sections (30 µm). The sections first underwent a peroxidase reaction (15 70 minutes), and then were permeabilized with 0.3% triton for 15 minutes. The sections were then blocked for at least an hour in a PBS solution containing 5% milk, 4% normal goat serum and 0.3% triton. The primary antibody was diluted in this blocking buffer and the sections were incubated in primary antibody for 2 hours at room temperature and overnight at 4 °C. The 5-HT2C receptor primary antibody was obtained from Santa Cruz

(Santa Cruz, California) (D-12 mouse) and used at a dilution of 1:100. The 5-HT2A receptor primary antibody (rabbit) has been called AB51 in previous publications (Willins et al., 1997) and has been extensively studied. This antibody was used at a dilution of

1:500. The GAD 65/67 antibody used was a rabbit antibody (Chemicon, Temecula, CA) and was used at a dilution of 1:2000. The parvalbumin primary antibody (rabbit) (Swant,

Bellinzona, Switzerland) was used at a dilution of 1:2000. After primary antibody incubation 5 10 minute washes in .3% triton (in PBS, 1 ml/well) were completed. After the 5th wash secondary antibody solution was added (made in blocking buffer). The secondary antibodies used were Alexa Fluor 488 goat-anti-mouse at a dilution of 1:200, and Alexa Fluor 594 goat-anti-rabbit at a dilution of 1:200 (Molecular Probes, Carlsbad,

California). The sections incubated in secondary antibody solution for an hour at room temperature and then the sections were washed 5 times. The first four washes were in .3% triton and the last wash was PBS. The slices were then removed with a transfer pipette and mounted onto slides using anti-fade mounting media (Kirkegaard & Perry

Laboratories, Gaithersburg, Maryland). Immunofluorescence experiments in cultured cells were also conducted. PO1C and GF62 cells are NIH-3T3 mouse fibroblasts that show stable expression of 5-HT2C and 5-HT2A receptors respectively. Cells were grown on coverslips and stained using the same procedure as for the free-floating sections.

Western Blots: 71

PO1C and GF62 cells were pelleted by centrifugation and then the pellets were lysed in 1 mL of Hepes buffer including CHAPS and protease inhibitors to prepare lysates. Lysates were normalized for protein content. Half of the lysates for each cell type were incubated with Wheat Germ Agglutinin (WGA)/lectin beads for 2 hours at 4 °C. SDS sample buffer was then added to lysates and beads and they were allowed to incubate at 67 °C for 5 minutes. The beads were then spun down and 30 µL from the top of the samples was loaded onto the gel. The protein was then transferred to a nitrocellulose membrane overnight. The membranes were then incubated in blocking buffer (tris buffered saline

(TBS), 0.1% Tween, 5% milk) for one hour and then incubated for 2 hours in the primary antibody solution (Santa Cruz D-12 goat-anti-mouse 5-HT2C receptor antibody, Santa

Cruz, California 1:500). The membranes were then washed several times, incubated in horseradish-peroxidase secondary antibody, and washed again several times. Finally, the membranes were incubated in the western blot substrate and developed.

Microscopy and Quantification:

Sections were examined on a Zeiss LSM 410 Confocal Microscope (Carl Zeiss,

Thornwood, NY) and images were obtained with the associated software. For each quantification experiment, 3 rats were used and 2 sections were examined per rat. The parvalbumin-positive and 5-HT2C receptor-positive cells were counted from 32 fields of view per cortical slice and 20 fields of view per hippocampal slice. The 5-HT2A receptor- positive and 5-HT2C receptor-positive cells were counted from 24 fields of view per cortical slice and 26 fields of view per hippocampal slice. Figures 4 and 7 illustrate the rostro-caudal levels of the sections examined in cortex and striatum (0.7 mm from bregma) as well as hippocampus (-3.8 mm from bregma) (Paxinos and Watson, 1998). In each case single-channel black and white images were used for cell-counting and 72 expressing cells were marked. After expressing cells were marked from each channel independently, the overlay was examined and coexpressing cells were counted.

Results:

5-HT2C Receptor Antibody Detects 5-HT2C Receptors in POIC, but not GF62 Cell Lysates by Western Blotting and Immunofluorescence:

Figure 1 shows that the Santa Cruz D-12 goat anti-mouse primary 5-HT2C receptor antibody specifically recognizes 5-HT2C and not 5-HT2A receptors in cell lysates. An appropriate 50 kDa band was observed when the cells used were PO1C cells, a cell line known to stably express 5-HT2C and not 5-HT2A receptors. Likewise, no band was observed when the cells used were GF62 cells, a cell line known to stably express 5-HT2A and not 5-HT2C receptors. Gq, known to be present in both cell lines, was included as a loading control. Likewise, immunofluorescence experiments showed that the 5-HT2C receptor antibody recognized receptors in PO1C, but not GF62 cells as shown in Figure 2.

5-HT2C Receptors are Expressed at High Levels in Rat Choroid Plexus:

5-HT2C receptors were detected at high levels in several brain regions including the cortex, hippocampus, striatum, and choroid plexus. As previously mentioned, the choroid plexus is a unique brain region in that 5-HT2C receptor expression is dense and the expression of the other 5-HT receptor subtypes is virtually nonexistent. As shown in

Figure 3, 5-HT2C receptors were expressed at high levels in rat choroid plexus, while 5-

HT2A receptors were not expressed in the same sections (data not shown).

5-HT2C Receptors are not Expressed in Parvalbumin-Containing Cells in Cortex and

Hippocampus:

Parvalbumin expression and 5-HT2C receptor expression were investigated in the regions of cortex illustrated by Figure 4. The regions examined included the cingulate, frontal 73 and parietal cortex, according to the atlas of Paxinos and Watson (1998). Figure 5 shows representative low and higher power images from cortex. 5-HT2C receptor staining showed negligible colocalization with parvalbumin in this region. This colocalization is quantified in Table 1. There were no significant differences between the regions of cortex studied. As shown in Figure 6, a similar lack of colocalization was observed in the pyramidal cell layer of the hippocampus (viewed at low and high power). The quantification of these results is also included in Table 1 and the rostro-caudal level of hippocampus examined is illustrated by Figure 7.

5-HT2C Receptors and 5-HT2A Receptors Colocalize in Cortical and Hippocampal

Neurons:

5-HT2A and 5-HT2C receptor expression were investigated in the regions of cortex illustrated by Figure 4. The regions examined included the cingulate and frontal cortex, according to the atlas of Paxinos and Watson (Paxinos and Watson, 1998). Figure 8 shows representative low and higher power images from cortex. 5-HT2C receptor staining showed a high degree of colocalization with 5-HT2A receptor staining in both regions of cortex. For the cingulate cortex 56.8% +/- 3.4% of 5-HT2A receptor expressing cells were also 5-HT2C receptor-positive, while 72.2% +/- 2.1% of 5-HT2C receptor expressing cells were also 5-HT2A receptor-positive. Similarly, in the frontal cortex 61.1% +/- 2.0% of 5-

HT2A receptor expressing cells were 5-HT2C receptor-positive, while 69.2% +/- 2.5% of 5-

HT2C receptor expressing cells were 5-HT2A receptor-positive. This colocalization is quantified in Table 1. As shown in Figure 9, a similar degree of colocalization was observed in the pyramidal cell layer of the hippocampus (viewed at low and high power).

In this region, 65.6% +/- 2.4% of 5-HT2A receptor expressing cells were 5-HT2C receptor- 74

positive, while 76.6% +/- 1.6% of 5-HT2C receptor expressing cells were 5-HT2A receptor- positive. These results are also quantified in Table 1.

5-HT2C Receptors do not Colocalize with the GABAergic Marker Glutamic Acid

Decarboxylase (GAD65/67 in the Striatum:

5-HT2C receptor expression and GAD65/67 expression were co-examined in slices of rat striatum. As illustrated by the representative section shown in Figure 10, the staining of the two antibodies demonstrated a lack of colocalization of 5-HT2C receptors with

GAD65/67 in this region. While the GAD65/67 staining was preferentially localized to the gray matter in the striatum, the 5-HT2C receptor staining was most intense in the axon tracts that traverse the region and are shown here in coronal crosssection.

Discussion:

These data illustrate that 5-HT2C receptors are expressed in rat hippocampus, striatum and areas of cortex including frontal cortex and posterior cingulate cortex. In addition this work shows that 5-HT2C receptors in these cortical regions and in the hippocampus are not expressed in parvalbumin-containing interneurons. Likewise, in the striatum the data show a lack of colocalization of 5-HT2C receptors with a marker for

GABAergic cells. In fact, 5-HT2C receptors were shown to be localized primarily to tracts traversing the striatum, rather than to the medium spiny neurons or interneurons that make up the gray matter of the region. Importantly, these studies are the first to show that 5-HT2A receptors and 5-HT2C receptors show a high degree of colocalization in both cortex and hippocampus. Presumably pyramidal neurons in these regions express both 5-HT2 receptor subtypes. As both the 5-HT2A and the 5-HT2C receptor have great potential as targets for the treatment of multiple disease and disorders, 75

and in many cases putative therapeutics are not selective for one 5-HT2 receptor subtype, these data may prove important to drug design.

The cortex contains different subpopulations of GABAergic interneurons that can be characterized by the calcium-binding proteins that they express (Gabbot et al., 1997).

These cell populations are distinct (Gabbot et al., 1997; Gonzalez-Burgos et al., 2005) and may play different roles in regulating the activity of neighboring pyramidal neurons and thus cortical function. Thus it is critical to determine the 5-HT2 receptor expression patterns in the various cortical cell types in order to elucidate the role that they play in regulating cortical function.

It has previously been shown that 5-HT2A receptors are localized to pyramidal neurons in the rat and primate cortex (Willins et al., 1997; Jakab and Goldman-Rakic,

1998, 2000; Cornea-Hebert et al., 1999; Miner et al, 2003; Santana et al., 2004). In the prefrontal cortex specifically it has been shown that a high percentage of pyramidal neurons and a low percentage of GABAergic cells express mRNA for 5-HT2A receptors

(Willins et al., 1997; Santana et al., 2004). Likewise in the primate cortex 5-HT2A receptors have been detected in virtually all pyramidal neurons, as well as specific populations of interneurons (Jakab and Goldman-Rakic, 1998). It is also known that 5-

HT2A receptors and 5-HT1A receptors are highly colocalized, thus there is a precedent for the simultaneous serotonergic regulation of these neurons by actions at multiple receptor subtypes (Araneda and Andrade, 1991; Martin-Ruiz et al., 2001; Santana et al., 2004).

Santana et al. report that 5-HT2A and 5-HT1A receptors are 80% colocalized, suggesting slightly more overlap than that seen here for 5-HT2A/5-HT2C receptors (cingulate: 57% of

5-HT2A-positive cells are 5-HT2C-positive, 72% of 5-HT2C-positive cells are 5-HT2A- positive; frontal: 61% of 5-HT2A-positive cells are 5-HT2C-positive, 69% of 5-HT2C- 76

positive cells are 5-HT2A-positive – as detailed in Table 1). In contrast, it has been shown that 5-HT3 receptors and 5-HT2A receptors are expressed exclusively in different populations of cortical neurons, suggesting that 5-HT acts through complex mechanisms to regulate cortical function (Jakab and Goldman-Rakic, 2000).

In contrast to the 5-HT2A receptor, there is a body of evidence that suggests that 5-

HT2C receptors are, for the most part, localized to GABAergic neurons. This hypothesis is based, in part, on data showing that 5-HT2C receptors provide a tonic inhibitory control of dopaminergic activity in several brain areas (Di Matteo et al., 2001; Alex et al., 2005).

Studies suggest that this tonic inhibition may be GABA-mediated, supporting a localization of 5-HT2C receptors to GABAergic cells (Rick et al., 1995; Di Giovanni et al.,

2001; Bankson and Yamamoto, 2004). In addition, 5-HT2C receptor knockout mice suffer from seizures as a result of the removal of 5-HT2C receptor mediated control of neuronal network excitability (Tecott et al., 1995). Lastly, there is anatomical evidence suggesting the presence of 5-HT2C receptors in GABAergic cells. It has been shown that cells that express GAD mRNA, but not those that express mRNA for the dopaminergic marker tyrosine hydroxylase (TH), also express 5-HT2C receptors in the substantia nigra and the ventral tegmental area (Eberle-Wang et al., 1997). Likewise, in the raphe nuclei

5-HT2C receptor mRNA colocalized with GAD mRNA and not with mRNA for the 5-HT transporter, a marker for serotonergic neurons (Serrats et al., 2005). In addition, it has previously been shown that some GABA interneurons in the prefrontal cortex express mRNA for 5-HT2C receptors (Vysokanov et al., 1998). However, in support of the present finding that 5-HT2C receptors colocalize with 5-HT2A receptors (presumably in pyramidal neurons) and not with parvalbumin, studies have also shown that some 77

pyramidal neurons in the prefrontal cortex contain 5-HT2C receptor mRNA (Vysokanov et al., 1998; Carr et al., 2002).

It is known that 5-HT-producing cells in the raphe nuclei send projections to the hippocampus, and specifically that the pyramidal layer comes into contact with these fibers from the raphe (Bjarkam et al., 2003). Interestingly, this serotonergic innervation is more dense in the CA3 region, than in the CA1 region (Bjarkam et al., 2003). This anatomy correlates well with the pattern of expression of 5-HT2C receptors found in these studies. As shown in Figure 9, 5-HT2C receptor staining was the most intense in the CA3 region. However, 5-HT2A receptor expression was consistently higher in the CA1 region

(as compared to 5-HT2C receptor expression), resulting in a more even distribution of 5-

HT2A receptors throughout the pyramidal layer. This finding is in agreement with previous results (Cornea-Hebert et al., 1999). While 5-HT2C receptors and 5-HT2A receptors were here shown to exhibit a high degree of colocalization in the pyramidal cell layer of the hippocampus, there were differences in their distributions within the neurons.

5-HT2C immunoreactivity was almost exclusively seen in the cell , while 5-HT2A receptor immunoreactivity could be clearly seen in the dendrites as well as the soma.

This pattern of 5-HT2A receptor expression is in agreement with findings from a previous study in which an inhibitor of axonal-dendritic transport was used in order to prevent the transport of 5-HT2A receptor protein to the dendrites, thus concentrating the immunoreactivity in the somas (Luttgen et al., 2004). Cornea-Hebert et al. also found strong 5-HT2A receptor staining in the dendrites of pyramidal neurons in rat hippocampus

(Cornea-Hebert et al., 1999).

This study reports that 5-HT2C receptors are not localized to parvalbumin- containing interneurons in the hippocampus or the regions of cortex examined. This 78

illustrates a difference between 5-HT2 receptor subtypes. It has been shown that 5-HT2A receptors are expressed in parvalbumin-positive cells in rat prefrontal cortex (Willins et al., 1997) and hippocampus (Luttgen et al., 2004) as well as primate cortex (Jakab and

Goldman-Rakic, 2000). However, in support of the absence of 5-HT2C receptors in this subset of hippocampal interneurons, it has been reported that 5-HT projections to the hippocampus do not synapse onto parvalbumin-containing neurons (Freund et al., 1990;

Miettinen and Freund, 1992a,b). The present data suggest that in the hippocampus, and the frontal and posterior cingulate cortex, 5-HT2C receptors are largely localized to pyramidal neurons and are not localized to the subset of GABAergic interneurons that contain parvalbumin. These data do not exclude the possibility that 5-HT2C receptors are expressed by other subsets of GABAergic interneurons in these regions. Future studies should address this question.

There is evidence that the posterior cingulate cortex plays a role in memory retrieval and the pathology of Alzheimer’s disease (Nyberg et al., 1996; Cabeza et al.,

1997 and Valla et al., 2001, respectively). In particular, this region shows the largest and earliest decrement in energy metabolism in the Alzheimer’s brain (Valla et al., 2001). It has also been shown that this region is under 5-HT2C receptor-mediated serotonergic control. Stimulation of 5-HT2C receptors with systemic administration of the 5-HT2C receptor agonist mCPP can affect bloodflow in the posterior cingulate cortex. (Kuhn et al., 2004) It has also been shown that a polymorphism in the 5-HT2C receptor gene can reverse the direction of this effect on bloodflow (Kuhn et al., 2004). This finding suggests that the 5-HT2C receptor polymorphism results in 5-HT2C receptors, within the posterior cingulate cortex or elsewhere, that have an alteration in their responsiveness to agonist stimulation (Kuhn et al., 2004). It appears as though the posterior cingulate 79 cortex is one area in which this change in responsiveness can produce a measurable change. It remains to be seen, however, whether these effects are mediated by receptors within the posterior cingulate cortex.

It is known that the hippocampus plays an important role in learning and memory

(Huang et al., 2006). It has been shown in both the rat and the tree shrew that serotonergic projections to the hippocampus undergo degradation with aging (Nishimura et al., 1998; Keuker et al., 2005). In particular, there is decreased serotonergic innervation in the region where these data show high levels of expression of 5-HT2A receptors on the dendrites of neurons from the pyramidal layer (Keuker et al., 2005).

Thus, it is possible that decreased stimulation of hippocampal 5-HT2A receptors by endogenous 5-HT is involved in memory deficits associated with aging.

A number of studies suggest that 5-HT2A and 5-HT2C receptors exert opposite effects on measures of behavior and neurotransmitter release, while coupling to the same intracellular signaling pathways to produce the same effects in their host cells

(depolarization upon stimulation). In particular, 5-HT2A and 5-HT2C receptors are thought to play opposite roles in the modulation of dopaminergic activity (for review see

Alex and Pehek, 2006). For example, in a recent study 5-HT2A receptor blockade attenuated the discriminative stimulus properties of cocaine, while 5-HT2C receptor blockade produced the opposite effect (Filip et al., 2006). However, there is evidence that the two receptor subtypes play a similar role in the regulation of some behaviors. 5-

HT2A receptor knockout mice show decreased anxiety (Weisstaub et al., 2006) and it is well known that 5-HT2C receptor antagonists and inverse agonists are anxiolytic (Kennett et al., 1996, 1997). It is possible that these effects are mediated by 5-HT2A/C receptors that are here shown to be colocalized to pyramidal neurons in the cortex and/or 80

hippocampus. It is known that 5-HT2A and 5-HT2C receptors are dynamically expressed and can internalize or change responsiveness after chronic ligand exposure (Willins et al.,

1998; Devlin et al., 2004). Importantly, a body of evidence is accumulating that suggests that desensitization or downregulation of 5-HT2A and/or 5-HT2C receptors occurs in response to chronic treatment with antidepressants and correlates with their efficacy

(Gray and Roth, 2001; Qu et al., 2005; Schechter et al., 2005; Syvalahti et al., 2006;

Yamauchi et al., 2006). These studies suggest that these changes may be involved in the mechanism of action of antidepressants and responsible for their therapeutic benefits.

Thus, studies examining the regional and cellular localization of these receptor subtypes are critical to the understanding and treatment of mood disorders such as depression and anxiety.

In conclusion, these data show that 5-HT2C receptors are not expressed in parvalbumin-containing GABAergic interneurons in rat hippocampus and regions of cortex including frontal and posterior cingulate. Importantly, this study is also the first to show that 5-HT2C receptors and 5-HT2A receptors show a high degree of colocalization, presumably on pyramidal neurons, in these regions. Given the known differences in constitutive activity and 5-HT binding affinity for the two receptor subtypes, these results suggest that pyramidal neurons are finely-tuned by 5-HT (Berg et al., 2005). Perhaps they are under 5-HT2C receptor control in tonic conditions and 5-HT2A receptor control only when extracellular 5-HT levels are elevated. It has, in fact, been shown that cortical pyramidal neurons are held in balance by opposing actions of 5-HT at 5-HT1A and 5-HT2A receptors on these cells (Arandea and Andrade 1991; Martin-Ruiz et al., 2001).

Therefore, drugs that bind selectively to one 5-HT receptor subtype expressed in these neurons disrupt normal cortical activity. It remains to be seen whether these data could 81 lead to manipulations of cortical activity that serve to correct natural imbalances seen in clinical disorders and diseases such as depression, anxiety, schizophrenia and

Alzheimer’s disease. 82

Figure 1: A western blot using cultured cells illustrates the specificity of the 5-HT2C receptor antibody by showing that the antibody detected 5-HT2C receptors in PO1C cell lysates (which stably express 5-HT2C, but not 5-HT2A receptors) but not in GF62 cells (which stably express 5-HT2A but not 5-HT2C receptors). 83 84

Figure 3: 5-HT2C receptor antibody recognizes 5-HT2C receptors in the choroid plexus of the rat. Magnification – 40x. 85

Figure 4: Regions of the rat cortex examined for colocalization of 5- HT2C receptors with parvalbumin and 5-HT2A receptors, from the atlas of Paxinos and Watson (1998). 86 87 88

Figure 7: Rostro-caudal level of hippocampus examined for colocalization of 5-HT2C receptors with parvalbumin and 5-HT2A receptors, from the atlas of Paxinos and Watson (1998). 89 90 91 92 93

CHAPTER 4:

The data presented here are the first to show that 5-HT2C receptors within the striatum tonically regulate DA release from the nigrostriatal pathway. In addition, the data suggest that 5-HT2C receptors localized within the PFC may not control release of

DA from the mesocortical pathway under basal conditions. Because the 5-HT2C receptor and its ability to modulate DA release have been implicated in an increasing number of disorders and diseases in recent years, this discovery of brain region-specific control of dopaminergic pathways is highly significant. In order to determine the mechanism of action of this regulation these studies examined the cellular localization of 5-HT2C receptors in the rat brain. Importantly, it was determined that 5-HT2C receptors in the cortex and hippocampus colocalize with 5-HT2A receptors, which have been shown to be expressed largely in pyramidal neurons in these regions. In addition, the data show that

5-HT2C receptors do not colocalize with parvalbumin in the cortex or hippocampus, or with a marker for GABAergic cells in the striatum. This section will discuss the implications of these data, putative neural circuitry, alternate explanations, and future studies that should be completed to address the questions that these data have generated.

Using Microdialysis to Measure Changes in DA Release:

An important strength of microdialysis is that it enables the investigator to study neurochemical changes in the brains of freely-moving animals. While this approach is often used to study neurochemical changes in response to drugs, it can also be used to measure changes in response to behavior such as feeding or stress (Church et al., 1987;

Pehek et al., 2006). It has been shown that the changes in dialysate levels of DA are 94 calcium-dependent (Westerink et al., 1988) and TTX-sensitive (Nomikos et al., 1990).

Probe implantation does result in brain damage in a small surrounding area and an immediate increase in dialysate DA levels. It has been shown that this increase in dialysate DA is not calcium or TTX-sensitive and thus does not represent release. Most likely it is caused by physical disruption of nerve terminals (Westerink and De Vries,

1988). 24 hours after probe implantation, however, dialysate DA levels were calcium and

TTX-sensitive suggesting that at this time the probe could measure DA release

(Westerink and De Vries, 1988). Thus, in these data, the experiments were conducted 18-

24 hours after probe implantation in order to minimize the effects of the damage. In addition, baseline samples were collected for 2-3 hours, until dialysate levels of DA were stable. While some investigators measure and report the recovery of their microdialysis probes in vitro (typically probes recover 10-20% of the DA concentration in the local area), it has been shown that this figure means little, as the limiting factor for diffusion is the tissue, not the probe membrane (Bungay et al., 1989). Also, it has been shown that for reverse dialysis much higher concentrations are needed than would be expected based on in vitro binding affinities. In fact, a dual microdialysis study provides evidence that in order to get diffusion of the administered drug in a 1 mm radius around the probe, concentrations of 1-10 mM are needed (Westerink and De Vries, 2001). Lastly, it has been shown that microdialysis does not disrupt the electrical properties of relatively nearby cells and thus can be combined with intracellular recording techniques in anesthetized animals (West et al., 2002). Taken together, these control experiments show that microdialysis is an appropriate technique for measuring changes in DA release and that the concentrations of drugs used in the reverse dialysis experiments presented here are within reason for examining a local effect. There are, however, limitations to 95 microdialysis. Perhaps most importantly samples are collected every 20-30 minutes and thus the temporal resolution is poor. Rapid, short-lived effects on DA may therefore be impossible to detect. It must also be noted that changes in dialysate DA levels could result from changes in release or changes in reuptake (Chefer et al., 2005). By comparing each animal’s dialysate DA levels after drug administration to their pre-drug baseline levels, one can gather information on the direction and magnitude of the effect of the drug on extracellular DA. Thus, microdialysis is a valuable tool for studying the ability of 5-

HT2C receptor subtypes to modulate dopaminergic activity.

Differential Regulation of the Nigrostriatal and Mesocortical Pathways:

The finding that the nigrostriatal pathway, but not the mesocortical pathway, is regulated by 5-HT2C receptors within its terminal region introduces some intriguing questions. It is known that there are 5-HT2C receptors present in both the striatum and the

PFC (Pasqualetti et al., 1999; Pompeiano et al., 1994). It is possible that the 5-HT2C receptors in the PFC act in the same manner as those in the striatum but produce a lower lever of tonic inhibition that is below our limits of detection. This would be consistent with immunofluorescence studies that illustrate that the level of 5-HT2C receptor expression is much lower in the PFC than in the striatum (data not shown). 5-HT2C receptors in the PFC may be so sparsely spread throughout the region that SB 206553, when administered by reverse dialysis at relatively conservative concentrations, is incapable of blocking the receptors effectively. It is possible that microinjections of SB

206553 into the PFC would produce a more widespread blockade of the 5-HT2C receptors in the region and it is critical to test whether effects on DA release are seen in this case. 96

Alternately, the 5-HT2C receptors in the PFC could have no role in regulating mesocortical DA release and be localized in such a way as to have no ability to affect mesocortical cell firing or release from local terminals. In support of this, data were presented here showing that 5-HT2C receptors in the PFC also do not modulate local stimulated release. This finding is consistent with work examining the serotonergic modulation of stress-induced DA release in which there is no role for 5-HT2C receptors in the PFC (Pehek et al., 2006).

It is known that the mesocortical pathway is tonically inhibited by 5-HT2C receptors, as the systemic administration of 5-HT2C inverse agonists produces a robust increase in PFC DA (Gobert et al., 2000; Millan et al., 1998; Pozzi et al., 2002). The present data suggest that the relevant receptors are not located in the PFC. Future studies should be aimed at determining whether or not there is a role for 5-HT2C receptors in the cell body regions of the nigrostriatal and mesocortical pathways. Thus, the 5-HT2C inverse agonist SB 206553 should be applied locally by reverse dialysis or microinjections to the

VTA and the SNpc and DA concurrently measured in the PFC and dorsolateral striatum, respectively.

Tonic Inhibition of DA by 5-HT2C Receptors may be GABA-Mediated:

Because the 5-HT2C receptor is known to couple positively to phospholipase C, resulting in an excitation of the host cell, the finding that systemic (or in the case of the nigrostriatal pathway – local) administration of an inverse agonist results in an increase in

DA release points to an indirect action. As previously detailed, these findings suggest that in both DA pathways examined, 5-HT2C receptors tonically inhibit release. From these data questions arise surrounding the mechanism of action and the cellular localization of the relevant receptors. Some immunohistochemical and 97

electrophysiological data have provided evidence that 5-HT2C receptors in the midbrain may be localized to GABAergic cells (Rick et al., 1995; Eberle-Wang et al., 1997; Di

Giovanni et al., 2001). Thus, it was hypothesized that 5-HT2C receptors may exert their effects on DA release indirectly through changes in the GABA release. This has not, however, been conclusively shown and the microdialysis data presented here provided strong to examine the cellular localization of 5-HT2C receptors and test the hypothesis that 5-HT2C receptors, in the striatum in particular, are localized to

GABAergic neurons. In particular, it was thought that the relevant 5-HT2C receptors in the striatum could be located on the cell bodies of striatonigral GABA neurons that have been shown to synapse in the SN on the dopaminergic dendrites of the nigrostriatal neurons (Bolam and Smith, 1990). In this case, the tonic inhibition of nigrostriatal DA by

5-HT2C receptors in the striatum would involve GABAergic feedback to the cell body region. Alternately, if 5-HT2C receptors in the striatum were indeed found to be expressed in GABAergic cell bodies, they could be local interneurons that mediate the tonic inhibition by releasing GABA locally on DA terminals. Thus, the second half of the experiments presented here were conducted in order to examine the cellular localization of 5-HT2C receptors in the brain in order to elucidate the circuitry responsible for the tonic inhibition of DA release illustrated by the microdialysis work.

While several studies have examined the regional localization of 5-HT2C receptors throughout the brain, few have produced data on the cellular or subcellular localization of these receptors. In addition, there has been widespread skepticism in the field about any immunohistochemical data due to a lack of antibodies that are specific for the 5-HT2C 98 receptors. The difficulty in creating a specific antibody is due to the extensive similarities in sequence of the 5-HT2 receptor subtypes (Backstrom and Sanders-Bush, 1997). Thus, the data presented here illustrating the specificity of the Santa Cruz antibody for the 5-

HT2C receptor versus the 5-HT2A receptor, via western blot and immunofluorescence experiments in cultured cells, represent an important contribution to the field. It is likely that with the availability of a specific antibody, the detailed characterization of the expression patterns of 5-HT2C receptors will be a growing area of research with vast implications for the development of new therapeutics.

The Heterogeneous Composition of the Striatum:

The striatum is a complex brain region that is compartmentalized in several different ways. In the rat brain approximately 90% of the cell bodies within the striatum are GABAergic projection cells called medium spiny neurons (see Parent and Hazrati,

1995 for review). The remaining 10% is made up of GABAergic and cholinergic populations of interneurons (see Kawagucki et al., 1995 for review). Thus, the medium spiny neurons act as the efferents of the striatum. These neurons project to subcortical structures including the SNpc and SNpr, where they provide feedback to the nigrostriatal

DA cell bodies (Bolam and Smith, 1990; Caille et al., 1996). Within the striatum, medium spiny neurons possess spiny dendrites and axons that, in addition to carrying information out of the striatum, send collaterals, within the striatum, to interneurons and other medium spiny neurons (see Parent and Hazrati, 1995; Bolam et al., 2000 for review). In addition, the striatum is divided into two compartments named the striosomes or “patches” (named after their appearance in coronal section) and the matrix that surrounds them. Both are gray matter regions and medium spiny neurons and interneurons are found in both compartments, thus GAD staining is seen in both regions 99

(Levesque et al., 2004). It has been shown, however, that µ-opiate receptors and are selectively expressed in the patches and matrix respectively and are thus known as compartmental markers (Graybiel and Ragsdale, 1978; Levesque et al.,

2004). In addition, it is known that dopaminergic nigrostriatal neurons project to both the matrix and the striosomes, while GABAergic nigrostriatal neurons project only to the matrix (Gerfen et al., 1987). One study has shown that typical and atypical antipsychotic drugs differentially activate neurons in the patch and the matrix suggesting one functional outcome of these anatomical differences (Bubser and Deutch, 2002).

The rat striatum is traversed by fiber bundles from the that can be viewed in saggital section as stripes, which thus provide the pattern for which the region is named (see Levesque et al., 2004). It is known that cortical cells send projections to the ipsilateral subcortical structures through the internal capsule (Richards et al., 1997).

The corticostriatal neurons are glutamatergic and are thought to project a map onto the striatum (see Parent and Hazrati, 1995 for review). Studies suggest that there may be laminar differences in projections to the patch and matrix (Gerfen, 1989). In addition, there are regional differences. For example, the corticostriatal neurons of the PFC have been shown to project selectively to the matrix (Selemon et al., 1994). It is known that corticostriatal terminals synapse with spines on the dendrites of medium spiny neurons, directly stimulating the efferent pathway of the striatum (see Bolam et al., 2000). More recently it has been shown that these projections also stimulate striatal interneurons, thus providing feed-forward inhibition to the efferent pathway (Ramanathan et al., 2002;

Mallet et al., 2005). The medium spiny neurons are silent (inactive) most of the time, and 100 unresponsive to single excitatory signals from cortex (Wilson and Kawaguchi, 1996;

Stern et al., 1997). In fact, these neurons are activated only in response to excitation by multiple cortical inputs (Wilson and Kawaguchi, 1996; Stern et al., 1997). Much remains to be elucidated regarding the complex circuitry of the striatum in order to determine how local 5-HT2C receptors are regulating DA release in this region.

Elucidating the Circuitry Involved in Control of DA by Striatal 5-HT2C Receptors:

In terms of putative localizations for the relevant 5-HT2C receptors, there are a finite number of possibilities. 5-HT2C receptors could be expressed in the cell bodies and/or dendrites of medium spiny neurons, GABA interneurons or cholinergic interneurons. In addition, because medium spiny neurons frequently have axon collaterals that terminate within the striatum, striatal 5-HT2C receptors could be located on axon terminals of the same three striatal cell types. It is also possible, however, that the relevant 5-HT2C receptors are located on axon terminals from neurons that project to the striatum from other regions of the brain. This group of neurons includes glutamatergic neurons from the cortex or the thalamus, serotonergic neurons from the raphe nuclei,

GABAergic neurons from the globus pallidus and the dopaminergic axons themselves.

The work presented here tested the possibility that 5-HT2C receptors colocalize with a marker for GABAergic cells in the striatum, which, if true, would suggest that striatal 5-

HT2C receptors tonically inhibit nigrostriatal DA release in a GABA-mediated manner.

Visualizing striatal cell bodies using the GABAergic cell marker GAD proved difficult, likely due to the density of dendrites and axon terminals that were GAD-positive in this region. It is known that including gluteraldehyde in the fixative is useful in the detection of GABA and GAD by immunohistochemical methods (Dr. Ariel Deutch, personal communication). In fact, upon the inclusion of 0.2% gluteraldehyde in the 101

fixative solution, both the GAD staining and the 5-HT2C receptor staining became more specific and it was clear that the two were not colocalized in the striatum. These data suggest that 5-HT2C receptors are expressed on axons that traverse the striatum, some of which may be corticostriatal projection neurons from the cortex. Putative circuitry is illustrated by Figures 1 (in basal conditions) and Figure 2 (after administration of SB

206553). As shown in these figures, 5-HT2C receptors in the striatum may exert their effects on nigrostriatal DA release by stimulating glutamatergic axons that activate medium spiny neurons. These striatal efferents synapse with and inhibit the cell bodies of the nigrostriatal neurons that release DA in the striatum. The terminals of these

GABAergic projection neurons express D1-type DA receptors that are thought to stimulate GABA release in the SN upon DA binding. Recent studies show that local administration of D1 agonists causes an increase in GABA release that correlates with changes in motor behavior (Trevitt et al., 2002). These changes are presumably due to alterations in striatal dopamine, as this region is known to be involved in motor function.

Additionally, these D1-agonist-induced changes in motor activity can be blocked by coadministration of the GABAA antagonist bicuculline and mimicked by the GABAA agonist muscimol, suggesting that the behavioral effects are indeed mediated by GABA

(Trevitt et al., 2002). Likewise, previous studies have shown that infusion, by reverse dialysis, of GABA agonists and antagonists into the SN can produce significant effects on dialysate DA in the striatum (Santiago and Westerink, 1992). Thus, evidence exists to support the hypothesis that modifications in striatonigral GABA release can produce downstream effects on nigrostriatal dopaminergic activity. 5-HT2A receptors in the 102 striatum are primarily localized to the terminals of afferents from the cortex and the globus pallidus (Bubser et al., 2001). Thus, there is a precedent for the expression of presynaptic 5-HT2 heteroreceptors in the striatum. It is possible, however, that the inclusion of gluteraldehyde in the fixative produced an interaction of the 5-HT2C receptor antibody specifically with white matter and that the observed localization of 5-HT2C receptors in fiber bundles of the internal capsule is, at least in part, an artifact. Future experiments are therefore required to test the proposed circuitry and to exclude any possibility that these data were affected by the preparation. Among the future studies that are suggested, some are further immunohistochemistry studies to confirm the localization of 5-HT2C receptors in the striatum and to examine expression in other areas. Due to the difficulty in visualizing GABAergic cell bodies in the striatum using an antibody to

GAD, combined in situ hybridization/immunohistochemistry should be done to examine colocalization of GAD mRNA and 5-HT2C receptor protein. In addition, double in situ hybridization of GAD and 5-HT2C receptor mRNAs could be attempted. If 5-HT2C receptors are expressed predominately on terminals in the striatum, 5-HT2C receptor protein and mRNA will show very different expression patterns in the brain. In this case it would be likely that striatal levels of 5-HT2C receptor mRNA would be much lower than the levels of 5-HT2C receptor protein. However, the possibility cannot be excluded that medium spiny neurons express 5-HT2C receptors at their terminals in the SNpc, SNpr or globus pallidus. If this is the case, medium spiny neurons would be positive for 5-

HT2C mRNA in double in situ hybridization experiments. Regardless, these studies would augment the data presented in this work.

Another way to examine whether or not 5-HT2C receptors are expressed in the various types of striatal neurons is to use markers for these neurons other than GAD. For 103

instance, the putative colocalization of 5-HT2C receptor protein with choline acetyltransferase, a marker for cholinergic interneurons, should be examined in the striatum. The GABAergic interneurons are known to also express either the calcium binding protein parvalbumin, the calcium binding protein calretinin, or the peptide somatostatin (Kawaguchi et al., 1995). Future studies should examine whether or not 5-

HT2C receptor protein colocalizes with each of these marker proteins in the striatum. The results would indicate whether 5-HT2C receptors are present in none, some, or all of the striatal GABAergic interneurons. Likewise, medium spiny neurons can be divided into two projections that selectively express cotransmitters. The striatonigral neurons contain substance P or dynorphin in addition to GABA, while the projections to the globus pallidus contain enkephalin (Kawaguchi et al., 1995). The data presented here suggest that 5-HT2C receptor expression would not colocalize with any makers for interneurons, or with markers for medium spiny neurons, because the majority of 5-HT2C receptor staining seen was localized to the white matter of the region.

Methods other than immunofluorescence studies would also be valuable in elucidating the circuitry involved. For example, retrograde tracing studies could be useful in determining the location of the cell bodies that send 5-HT2C receptor-positive projections to the striatum. It is also important to determine whether the 5-HT2C receptor-mediated inhibition of DA release in the striatum requires feedback from other regions of the brain.

For example, if 5-HT2C receptors are exerting this control by constitutively activating, or mediating the excitatory effect of endogenous 5-HT on, cortical terminals in the striatum and thus augmenting the inhibition of nigrostriatal cells by medium spiny neurons, 104 feedback to the SNpc is critical to their actions. This possibility could be tested in striatal slices, where feedback to extrinsic structures is not a factor. Specifically, future studies should test the ability of SB 206553, applied to striatal slices, to enhance DA release from the slices. If the disinhibition seen in the work presented here employing microdialysis persists in this experimental system, then the 5-HT2C receptors involved are not only located within the striatum, but regulate DA locally as well.

Lastly, further microdialysis experiments could be used to test the hypothesis that the tonic inhibition is GABA-mediated. If 5-HT2C receptors are localized to striatal

GABAergic neurons, they may reside on striatal GABAergic interneurons, striatonigral projection neurons or in both locations. To test these possibilities, dual probe microdialysis could be utilized to correlate changes in striatal DA with changes in GABA in both the striatum and the SN after SB 206553 is infused into the striatum. If the increase in striatal DA (presented here) is accompanied by a decrease in striatal GABA, then the relevant 5-HT2C receptors could be located on GABAergic interneurons, or on medium spiny neurons known to have axon collaterals that terminate in the striatum. If only a decrease in GABA in the SN is found, the relevant 5-HT2C receptors are likely to be on medium spiny cell bodies in the striatum. To augment these studies, GABA receptor antagonists could be administered in conjunction with a 5-HT2C receptor inverse agonist to block the any putative GABA-mediation. If the increase in striatal DA presented here is not achieved in the presence of these antagonists, it is likely that the effect is not GABA-mediated. In addition, the GABA antagonists can be delivered by reverse dialysis in order to block GABA-mediation in a region-specific manner in order to more precisely examine the circuitry involved. From the work presented here, it is expected that these experiments would confirm that the tonic inhibition of nigrostriatal 105

DA by 5-HT2C receptors is not GABA-mediated. Additional experiments examining the localization of 5-HT2C receptors on the terminals of glutamatergic projection neurons would then be critical. Double in situ hybridization experiments examining the putative colocalization of 5-HT2C receptor mRNA with markers for these neurons on their cell body regions in the thalamus and cortex should be attempted. However, by process of elimination, it can be concluded that corticostriatal axon terminals are the most likely location for the striatal 5-HT2C receptors capable of modulating DA release (see Figures

1 and 2 for putative circuitry involved in this modulation). Immunofluorescence experiments showed no colocalization of 5-HT2C receptor expression with GAD 65/67 or TH expression in the striatum or the SN (data not shown). These data suggest that 5-

HT2C receptors are not localized to GABAergic cell bodies or terminals in the striatum, nor dopaminergic terminals projecting from the SN. In addition, it has been shown that in the SNpc 5-HT2C receptor mRNA does not colocalize with a marker for dopaminergic cells (Eberle-Wang et al., 1997), thus it is unlikely that the 5-HT2C receptors in the dorsolateral striatum are localized to dopaminergic terminals. In addition, it has been shown that in the dorsal and medial raphe nuclei 5-HT2C receptor mRNA does not colocalize with 5-HT transporter mRNA (Serrats et al., 2005), suggesting that it is also unlikely that these receptors are localized to serotonergic terminals in the striatum.

Therefore, it is most likely that the relevant 5-HT2C receptors are localized on glutamatergic projections to the striatum. These projections most likely come form the cortex (not the thalamus) where 5-HT2C receptors were shown here to be abundantly expressed. 106

Cellular Localization of 5-HT2C Receptors in the Cortex and Hippocampus:

In addition to the finding that 5-HT2C receptors showed no colocalization with

GAD in the striatum, the data presented here shows that in the cortex and the hippocampus 5-HT2C receptors are not expressed in the subset of GABAergic neurons that express parvalbumin. However, the possibility remains that some GABAergic cells

(including calretinin or calbindin expressing subsets) do express 5-HT2C receptors in these regions. Thus, future studies should examine the colocalization of 5-HT2C receptor protein with calretinin and calbindin in these regions.

Importantly, these studies did reveal high levels of colocalization of 5-HT2C receptors with 5-HT2A receptors in the cortex and hippocampus. The regions of cortex examined express higher levels of 5-HT2C receptors than are seen in the PFC (data not shown). However, both 5-HT2A and 5-HT2C receptor expression can be observed in the

PFC and it is intriguing to consider the possibility that these receptors are highly colocalized there as well. 5-HT2A receptors in the PFC have been shown to be involved in the regulation of stimulated DA release from the mesocortical pathway. In particular,

5-HT2A receptor blockade in the PFC attenuates the increase in PFC DA seen in response to both systemic 5-HT2A receptor agonist administration and to stress (Pehek et al., 2006).

In addition, it has been shown that 5-HT2A receptors in the PFC are expressed on glutamatergic neurons that project to the VTA, likely providing positive feedback to the mesocortical system (Carr and Sesack, 2000). In fact, systemic administration of a 5-

HT2A receptor agonist not only increases PFC DA but increases VTA glutamate, an effect that is blocked by the blockade of PFC 5-HT2A receptors (Pehek et al., 2006). As previously mentioned, it has been shown, here and elsewhere, that systemic 5-HT2C receptor inverse agonism profoundly increases DA in the PFC but that 5-HT2C receptors 107 within the PFC do not regulate basal or stimulated DA release from the mesocortical system. If 5-HT2A and 5-HT2C receptors are, for the most part, expressed in the same pyramidal neurons in the PFC as we have shown them to be in other cortical regions, these findings would be difficult to explain. It is possible that the 20-30% of 5-HT2C or 5-

HT2A receptor-expressing cells that are NOT colocalized are well-positioned interneurons that provide an additional layer of complexity to the circuitry. In addition, differing levels of constitutive activity and receptor expression between the two 5-HT2 receptor subtypes could play a role. It may be that glutamatergic neurons in the PFC experience competing effects of 5-HT2A and 5-HT2C receptors. Perhaps 5-HT2C receptors are more important tonically because of their higher level of constitutive activity, but 5-HT2A receptors are more important phasically because they are more highly expressed in PFC

(data not shown). However, the data presented here suggest that if 5-HT2C receptor constitutive activity and/or stimulation by endogenous 5-HT tonically inhibits mesocortical DA release through glutamatergic projections or otherwise, the removal of this inhibition produces an immeasurable change in PFC DA. Thus, it is likely that 5-

HT2C receptors in the PFC do not regulate DA release in this region tonically or under the stimulated conditions that have thus far been tested. Immunohistochemical studies examining the cellular localization of 5-HT2C receptors in the PFC are critical to the understanding of this noticeable lack of involvement of 5-HT2C receptors.

Additional studies would also be useful in the regions of cortex examined. By correlation it is hypothesized that the 5-HT2A/5-HT2C receptor expressing cells in the cortex are pyramidal neurons. This hypothesis should be tested by examining the 108

colocalization of 5-HT2C and 5-HT2A receptor proteins with glutamate and/or Emx1, markers of cortical pyramidal neurons (Chan et al., 2001). In addition, anterograde tracing studies could be used to determine whether the 5-HT2A/5-HT2C receptor expressing

(presumably pyramidal) neurons in the cortex project to the striatum. From the results presented here showing a lack of colocalization of GAD staining with the 5-HT2C receptor expression in the striatum and the work done by others ruling out 5-HT2C receptor expression on dopaminergic and serotonergic terminals in the region it is hypothesized that these some or all of these neurons are corticostriatal. The percentage of non- colocalized cells may involve selective 5-HT2A or 5-HT2C receptor expression to subsets of interneurons. For example, it has been shown that some 5-HT2A receptors are expressed in parvalbumin neurons (Willins et al., 1997), while these data show no expression of 5-HT2C receptors in these cells. Immunofluorescence studies examining putative 5-HT2 receptor expression in calretinin and calbindin expressing interneurons could be helpful in resolving the identity of the non-colocalized cells. However, it is possible that 5-HT2C receptors are only expressed in pyramidal neurons in the cortex and hippocampus. In this case, the non-colocalized cells could be examples of pyramidal neurons under the control of one, but not the other, 5-HT2 receptor and it would be interesting to look for patterns in where these neurons project.

Lastly, it must be mentioned that there is evidence that suggests that the pattern of

5-HT2A receptor staining is highly dependent on the antibody used. One study showed that the antibody used here (AB51) as well as one commercially available antibody (from

Pharmingen) preferentially stained pyramidal neurons in the amygdala, whereas a second commercially available antibody (from Calbiochem) mainly stained interneurons in this region (McDonald et al., 2006). Likewise, in the cortex, AB51 and the Pharmingen 109 antibody stained pyramidal cell bodies and apical dendrites while the Calbiochem antibody did not recognize 5-HT2A receptors on cell somas, and more intensely stained apical dedrites in layer 3 (Alexander McDonald, Personal Communication). Therefore, it would be beneficial to verify the colocalization of 5-HT2A receptors with 5-HT2C receptors in the cortex and hippocampus using additional specific 5-HT2 subtype receptor antibodies as they become available.

The finding that 5-HT2A receptors and 5-HT2C receptors are colocalized in the cortex and hippocampus is significant. It is known that 5-HT2A receptors colocalize with

5-HT1A receptors in pyramidal neurons and that stimulation of these two receptor subtypes has opposing effects on these cells (Santana et al., 2004). However, 5-HT2A and

5-HT2C receptors have their primary effects on second messengers in common (Gq- mediated activation of phospholipase C). Therefore, it appears redundant for the majority of pyramidal neurons that express one of these two receptors to express the other as well.

Despite their structural similarity and commonalities in coupling to intracellular signaling pathways important differences exist. In addition to their primary coupling to phospholipase C, 5-HT2 receptor stimulation also activates phospholipase A signaling. 5-

HT2C receptors have been shown to express a higher level of constitutive activity than 5-

HT2A receptors when activity was measured by phospholipase C activation. However, 5-

HT2A receptors have been shown to express higher levels of constitutive stimulation of the phospholipase A pathway (see Berg et al., 2005). In addition, it is possible that the two receptors are desensitized differently in response to chronic ligand exposure. A great deal of data has been accumulated on this subject, but still uncertainties remain. It has 110

been shown in vivo and in vitro that both 5-HT2A and 5-HT2C receptors are desensitized after chronic agonist treatment (see Gray and Roth, 2001; Van Oekelen et al., 2003 for review). This response is thought to be compensatory. There is also data examining the response of these receptors to chronic antagonist treatment. In vivo data suggests that both

5-HT2A receptors and 5-HT2C receptors desensitize after chronic blockade, however in vitro studies have produced inconsistent results (see Gray and Roth, 2001; Van Oekelen et al., 2003 for review). In addition, in vivo studies examining changes in receptor binding typically examine 5-HT2A receptors in the frontal cortex and 5-HT2C receptors in the choroid plexus. Thus, given these data showing that 5-HT2A and 5-HT2C receptors are expressed in the same cortical cells, future studies examining desensitization of these receptors in the cortex in response to chronic treatment of antagonists and inverse agonists are warranted. It is also known that 5-HT2 receptors express a PDZ ligand at their intracellular terminus and interact with specific sets of PDZ-binding proteins (Xia et al., 2003; Becamel et al., 2004). It has been hypothesized that these interactions may regulate receptor trafficking, including cell surface expression and internalization, in addition to independently affecting signaling of the receptors through effector pathways

(see Gavarini et al., 2004 for review). Thus, differences in constitutive activity, relative contributions of second messenger pathways, and response to chronic ligand exposure are all mechanisms by which 5-HT2A receptors and 5-HT2C receptors in the same pyramidal neurons may provide different inputs to these cells in basal and/or stimulated conditions.

Clinical Implications:

These data have implications for the treatment of schizophrenia, Parkinson’s disease and depression. Studies have shown that in schizophrenia hypoactivity of the mesocortical pathway is linked to impaired cognition and anhedonia, while hyperactivity 111 of the mesolimbic pathway is thought to be involved in the hallucinations associated with the disease. In addition, extrapyramidal side effects of antipsychotic drugs are thought to be linked to disregulation of DA in the striatum. Thus it is thought that DA pathways may be differentially disregulated in the schizophrenic brain. Accumulating information about differential regulation of the DA pathways in the healthy brain may reveal ways in which the DA pathways can be manipulated by therapeutic drugs, in order to treat the impairments seen in schizophrenia while minimizing the side effects. In particular, elucidating the brain-region specific regulation of DA by receptor subtypes to which antipsychotic drugs are known to bind, including the 5-HT2C receptor, will aid in the development of new drugs with the optimal mix of binding affinities for this myriad of receptors. More specifically, it has been shown that suicide patients, and schizophrenics may have altered patterns of RNA editing of the 5-HT2C receptor – which may result in brain-region-specific differences in the levels of constitutive activity of these receptors and thus their tonic inhibition of DA (Niswender et al., 2001; Sodhi et al., 2001;

Gurevich et al., 2002). In addition, it has been suggested that schizophrenic patients have lower levels of 5-HT2A receptor expression in the PFC than control subjects, which may increase the relative contribution of 5-HT2C receptor signaling in PFC pyramidal neurons (Burnet et al., 1996). Lastly, it has been shown that some atypical antipsychotic drugs may act as agonists at some isoforms of the 5-HT2C receptor and antagonists at others, allowing for the possibility of brain-region and activation-state selectivity of stimulation (Zhang et al., 2006). Thus, the data presented here, showing differential 112

regulation of two DA pathways by 5-HT2C receptors is highly relevant to the development of new strategies for the treatment of schizophrenia.

The finding that 5-HT2C inverse agonism increases DA release in the striatum may have implications for the treatment of Parkinson’s disease. It has been shown that 5-HT2C receptor blockade attenuates haloperidol-induced catalepsy, perhaps by facilitating nigrostriatal DA release (Balsara et al., 2005). There is evidence that atypical antipsychotic drugs may lack the extrapyramidal side effects seen with typical antipsychotic drugs because of their ability to act as antagonists or inverse agonists at 5-

HT2C receptors (Reavill et al., 1999; Herrick-Davis et al., 2000; Rauser et al., 2001).

Further, the evidence presented here that suggests that 5-HT2C receptors may affect striatal

DA by regulating glutamate release from the corticostriatal pathway could be useful in the development of new therapeutics to treat the disease. Clearly more experiments will be required to further examine this putative circuitry and test a role for drugs with 5-HT2C inverse agonist properties as treatments for Parkinson’s disease.

It has been suggested that 5-HT2C and 5-HT2A receptors may be involved in the efficacy of antidepressant drugs. Some studies suggest that direct stimulation of 5-HT2C receptors is effective in animal models of depression (Cryan and Lucki, 2000; and see

Schechter et al., 2005 for review). Other work suggests that 5-HT2C or 5-HT2A receptor antagonism can supplement the efficacy of some antidepressant drugs (Millan et al., 2005 and Marek et al., 2005 respectively). Lastly, a growing body of evidence suggests that chronic SSRI treatment results in desensitization of 5-HT2A and possibly 5-HT2C receptors and that this effect may be critical to their efficacy in treating the symptoms of depression

(Palvimaki et al., 2005; Qu et al., 2005; Syvalahti et al., 2006; Yamauchi et al., 2006).

Thus, these data, illustrating the colocalization of 5-HT2A and 5-HT2C receptors in the 113 same neurons in the cortex and hippocampus, are relevant to the development of antidepressant treatments. Studies examining the function of these cortical and hippocampal 5-HT2 receptors in response to chronic treatment with the same antidepressant drugs may aid our understanding of the mechanism of action of these agents and the delay in efficacy observed after the initiation of treatment.

Summary:

The data presented here expose a differential regulation of two major dopaminergic pathways that is highly relevant clinically, to the treatment of schizophrenia, as well as to our understanding of the interactions of 5-HT and DA in the brain. In addition, these data uncovered the regional localization of the 5-HT2C receptors responsible for tonically inhibiting nigrostriatal DA release. Further, evidence was provided suggesting that these effects are not GABA-mediated but may show a role for corticostriatal projections in regulating DA release in the striatum. Lastly, these data show that two of the most prominent targets of therapeutics for mood disorders, schizophrenia and drug abuse are colocalized in the cortex and the hippocampus. The studies presented here also raised further questions that should be investigated in future studies. These data show the strength of using multiple techniques to characterize the role of a receptor subtype. By combining microdialysis and immunofluorescence the 5-

HT2C receptor was shown to mediate neurochemical changes in freely-moving animals and then the location of the 5-HT2C receptors relevant to this in vivo effect was studied at length. Taken together, these data suggest that 5-HT2C receptors may tonically inhibit nigrostriatal DA release via complex circuitry involving both corticostriatal and 114 striatonigral projection neurons. Given the importance of nigrostriatal DA release to normal motor function, and Parkinson’s disease and the increasingly widespread use for drugs with affinity for 5-HT2C receptors in the treatment of obesity, depression, anxiety and schizophrenia, these studies make a critical contribution to the field. 115

CORTEX

GLU

STRIATUM

GABA

SUBSTANTIA NIGRA DA

= 5-HT2C receptors

= GABA

RAPHE 5-HT 5-HT = Glutamate NUCLEI = Dopamine

Figure 1: Putative circuitry of the regulation of nigrostriatal DA by 5-HT2C receptors under basal conditions. The relevant 5-HT2C receptors may be localized to corticostriatal axon terminals where they augment glutamate release that increases the activity of medium spiny GABAergic neurons that project to the substantia nigra. Increased release of GABA in the substantia nigra would decrease nigrostriatal DA cellular activity and subsequent DA release in the striatum. 116

CORTEX

GLU

STRIATUM

GABA

SUBSTANTIA NIGRA DA

= 5-HT2C receptors

= GABA

RAPHE 5-HT 5-HT = Glutamate NUCLEI = Dopamine

= SB 206553 (5-HT2C receptor inverse agonist)

Figure 2: Putative circuitry of the regulation of nigrostriatal DA by 5-HT2C receptors in the presence of SB 206553. In the proposed model, the drug would act at 5-HT2C receptors to decrease glutamate release in the striatum, decreasing GABA in the substantia nigra, and subsequently increasing DA release in the striatum (which was measured in these studies. 117

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