The Role Of Sertonin And Vesicular Monoamine Transporters In The Adverse Responses To Methylenedioxymethamphetamine

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Authors Lizarraga-Zazueta, Lucina Eridna

Publisher The University of Arizona.

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THE ROLE OF SERTONIN AND VESICULAR MONOAMINE TRANSPORTERS IN THE ADVERSE

RESPONSES TO METHYLENEDIOXYMETHAMPHETAMINE

by

Lucina Eridna Lizarraga Zazueta

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF AND TOXICOLOGY

In Partial Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

In the Graduate College at

THE UNIVERSITY OF ARIZONA

2014 2

THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE

As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Lucina Eridna Lizarraga Zazueta entitled “The Role of and Vesicular Monoamine Transporters in the Adverse Responses to Methylenedioxymethamphetamine” and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy

______Date: 06/20/2014 Dr. Terrence J. Monks

______Date: 06/20/2014 Dr. Serrine S. Lau

______Date: 06/20/2014 Dr. John W. Regan

______Date: 06/20/2014 Dr. Josephine Lai

______Date: 06/20/2014 Dr. Stephen Wright

Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

______Date: 06/20/2014 Dissertation Director: Dr. Terrence J. Monks 3

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED: Lucina Eridna Lizarraga Zazueta 4

ACKNOWLEDGEMENTS

I would like to begin by thanking the College of Pharmacy at the University of Arizona for giving me the opportunity to attain my goal of pursuing a graduate degree. Also, special thanks to my committee members, Dr. Serrine Lau, Dr. Josephine Lai, Dr. John Regan and Dr. Stephen

Wright, for all their time and advice. In particular, I have to acknowledge my advisor, Dr. Terrence

Monks, for his support and mentorship. His guidance was instrumental in helping me become the critical and independent scientist I am today.

I have to express my most sincere gratitude to all my family, especially to my parents,

Roberto and Maria, and brothers, Roberto, Pablo and Juan. Words cannot describe the love and admiration I have for all of you. You have been and will continue to be my source of strength and inspiration. Thank you to my husband and best friend, Ricardo, for all his love and support. You encourage me every day to pursuit my dreams and face my challenges, helping me to become a better person. I am very thankful for all the joy you bring to my life.

At last, thank you to all my colleagues and friends at the College of Pharmacy. I made great friendships during my journey as a graduate student that I hope to maintain throughout my life. 5

DEDICATION

To my father, Roberto Lizarraga,

Who inspired me to pursuit a career in science

Thank you for all your dedication and guidance 6

TABLE OF CONTENTS

LIST OF FIGURES...... 8 LIST OF ABBREVIATIONS...... 9 ABSTRACT ...... 12 CHAPTER 1: INTRODUCTION ...... 14 1.1 MDMA: THE PROTOTYPE CLUB DRUG...... 14 1.1.1 History ...... 14 1.1.2 Epidemiology...... 15 1.1.3 Chemical Structure, Route of Administration and Dosage ...... 16 1.2 PHARMACOKINETICS OF MDMA...... 19 1.2.1 MDMA Metabolism and Potential Bioactivation ...... 20 1.3 PHARMACOLOGY OF MDMA ...... 27 1.3.1 Mechanism of Action...... 27 1.3.1 Acute Effects and Toxicity...... 32 1.4 MECHANISMS OF ACUTE MDMA TOXICITY, AN EMPHASIS ON HYPERTHERMIA AND THE 5-HT BEHAVIORAL SYNDROME...... 33 1.4.1 Hyperthermia ...... 33 1.4.2 The 5-HT behavioral syndrome...... 42 1.5 MECHANISMS OF LONG-TERM MDMA NEUROTOXICITY...... 45 1.5.1 General Comments on Neurotoxicity ...... 45 1.5.2 MDMA Neurotoxicity in Experimental Animals...... 47 1.5.3 Evidence of Serotonergic Neurotoxicity in Ecstasy Users ...... 51 1.5.4 Molecular Mechanisms of MDMA-Mediated Neurotoxicity ...... 52 1.5.4.1 Oxidative Stress...... 52 1.5.4.2 DA Contributions...... 59 1.5.4.3 The Role of Quinones in MDMA Neurotoxicity ...... 60 1.5.4.4 The Involvement of Serotonin Transporters ...... 66 1.5.4.5 Contributions of Vesicular Transport to MDMA Neurotoxicity ...... 69 1.6 DISSERTATION AIMS ...... 71 CHAPTER 2: MATERIALS AND METHODS ...... 75 2.1 MATERIALS ...... 75 2.1.1 Chemicals and Reagents...... 75 2.1.2 Animals and Dose Regimen...... 76 2.1.2.1 SERT Deficiency in Rats...... 76 2.1.2.2 Pharmacological Inhibition of VMAT2 in Rats ...... 76 2.2 METHODS ...... 77 2.2.1 Confirmation of VMAT2 Inhibition by Ro4-1284 ...... 77 2.2.2 Body Temperature Measurements...... 77 2.2.3 Locomotor Activity Assessment...... 77 2.2.4 Plasma Norepinephrine Extraction...... 78 2.2.5 HPLC-ECD Analysis of Plasma Norepinephrine ...... 79 2.2.6 Stereotaxic Surgery for Cannula Placement...... 79 2.2.7 Microdialysis Probe Implantation...... 80 2.2.8 HPLC-ECD Analysis of Dialysate Monoamine Levels...... 80 2.2.9 Immunohistochemistry...... 80 2.2.10 Isolation from Brain Tissue ...... 82 2.2.11 HPLC-ECD Analysis of Brain Monoamines ...... 82 2.2.12 In Vitro Cell Cultures...... 83 2.2.13 Transient Transfection of hSERT...... 83 7

2.2.14 Cellular Uptake Studies ...... 84 2.2.15 Synthesis and Purification of 5-(GSH)-N-Me-α-MeDA and 5-(NAC)-N-Me-α-MeDA ...... 85 2.2.16 Statistics ...... 86 CHAPTER 3: EFFECTS OF SEROTONIN REUPTAKE TRANSPOTER DEFICIENCY ON THE ACUTE AND LONG- TERM TOXICITIES OF 3,4-(±)-METHYLENEDIOXYMETHAMPHETAMINE...... 87 3.1 INTRODUCTION AND RATIONALE ...... 87 3.2 RESULTS ...... 91 3.2.1 SERT Deficiency Modulates the Thermoregulatory Response to MDMA after Multiple Drug Dosing...... 91 3.2.2 Attenuation of MDMA-Induced Hyperactivity in a Genetic Model of SERT Deficiency ...... 93 3.2.3 Abolishment of Serotonergic Neurotoxicity Mediated by MDMA in SERT Deficient Rats ...... 94 3.2.4 MDMA Treatment Is Insufficient to Induce Microglial Activation in WT and SERT-KO rats...... 96 3.3 DISCUSSION ...... 108 CHAPTER 4: EFFECTS OF VESICULAR 2 INHIBITION IN THE IN VIVO TOXICITIES OF 3,4-(±)-METHYLENEDIOXYMETHAMPHETAMINE...... 116 4.1 INTRODUCTION AND RATIONALE ...... 116 4.2 RESULTS ...... 119 4.2.1 Pharmacological Inhibition of VMAT2 Decreases Brain Monoamine Levels ...... 119 4.2.2 Inhibition of VMAT2 Protects Rats against MDMA-Mediated Hyperthermia ...... 119 4.2.3 Elevation of Peripheral NE Levels with MDMA Treatment Is Unaffected by Inactivation of VMAT2 Function...... 120 4.2.4 Inhibition of VMAT2 Attenuates Induction of Locomotor Activity in MDMA-Treated Rats...... 121 4.2.5 Pharmacological Inactivation of VMAT2 Has Minimal Effects on MDMA-Induced Monoamine Release in Rat Striatum ...... 123 4.2.6 Prevention of MDMA-Mediated Indoleamine Depletion with Acute Antagonism of VMAT2 Function...... 124 4.2.7 Inhibition of VMAT2 Reduces Persistent Serotonergic Nerve Terminal Damage in Rats Exposed to MDMA ...... 125 4.3 DISCUSSION ...... 146 CHAPTER 5: CONCLUDING REMARKS ...... 158 5.1 SUMMARY ...... 158 5.2 FUTURE DIRECTIONS...... 165 APPENDIX A: EFFECTS OF 3,4-(±)-METHYLENEDIOXYMETHAMPHETAMINE AND ITS THIOETHER METABOLITES ON THE IN VITRO FUNCTION OF THE SEROTONIN REUPTAKE TRANSPORTER...... 173 A.1 INTRODUCTION AND RATIONALE...... 173 A.2 RESULTS...... 176 A.2.1 Synthesis and Identification of Thioether MDMA Metabolites ...... 176 A.2.2 N-Me-α-MeDA-Derived Metabolites of MDMA Fail to Modulate 5-HT Uptake into hSERT- Expressing Human Embryonic Kidney Cells ...... 177 A.3 DISCUSSION...... 191 REFERENCES...... 196 8

LIST OF FIGURES

Figure 1-1 - Phenylethylamine Derivatives...... 18 Figure 1-2 - MDMA Metabolism...... 26 Figure 1-3 - Pharmacological Mechanism of Action of MDMA ...... 31 Figure 1-4 - Putative Mechanisms of MDMA-Mediated Thermogenesis...... 41 Figure 1-5 - Primary Sources of ROS in the CNS ...... 58 Figure 1-6 - N-Me-α-MeDA-Derived Quinone Formation and ROS Generation...... 65 Figure 3-1 - MDMA-Induced Hyperthermia Occurs in Both WT and SERT-KO Rats after Repeated Drug Administration ...... 99 Figure 3-2 - MDMA Increases Peak Body Temperature in Wistar Rats Regardless of Genotype...... 100 Figure 3-3 - SERT-KO Rats Display Attenuated ∑ºC x h after Multiple MDMA Doses...... 101 Figure 3-4 - SERT-KO Rats Display Delayed and Attenuated Horizontal Velocity after a Single Dose of MDMA ...... 102 Figure 3-5 - MDMA Causes an Acute Decrease in Vertical Activity in WT Rats and No Changes in SERT-KO Animals...... 103 Figure 3-6 - MDMA-Mediated Indoleamine Depletion is Abolished in SERT-KO Rats Following Multiple Drug Administrations...... 104 Figure 3-7 - MDMA Does Not Affect Striatal Catecholamine Content in WT and SERT-KO Rats...... 105 Figure 3-8 - MDMA Has No Effects on Microglial Size or Morphology in Wistar Rats Irrespective of SERT Genotype ...... 106 Figure 3-9 – MDMA Fails to Cause Any Changes in Cortical Microglial Occupancy in WT or SERT-KO Rats ...... 107 Figure 4-1 - Acute Effects of Ro4-1284 on Striatal Indoleamine and Catecholamine Content ...... 127 Figure 4-2 - Pretreatment with Ro4-1284 Prevents MDMA-Mediated Hyperthermia in Rats...... 129 Figure 4-3 - Ro4-1284 Pretreatment Has No Effect on Acute Induction of Plasma NE Levels after MDMA Administration ...... 130 Figure 4-4 - Effects VMAT2 Inhibition on MDMA-Induced Horizontal Locomotor Activity...... 132 Figure 4-5 - Effects VMAT2 Inhibition on the Stimulation of Vertical Activity with MDMA Treatment ...... 134 Figure 4-6 - Stereotaxic Representation of Microdialysis Probe Implantation into Rat Striatum ...... 135 Figure 4-7 - Dialysate Indoleamine Levels Following Ro4-1284 and MDMA Treatment in Rat Striatum....137 Figure 4-8 - Dialysate Catecholamine Levels Following Ro4-1284 and MDMA Treatment in Rat Striatum139 Figure 4-9 - Ro4-1284 Pretreatment Affords Protection against MDMA-Mediated Serotonin Depletions ...... 140 Figure 4-10 - Ro4-1284 Pretreatment Does Not Inflict Long-Lasting Changes in Catecholamine Content.141 Figure 4-11 - VMAT2 Inhibition Prevents Widespread Reductions in Striatal SERT Staining Produced by MDMA...... 143 Figure 4-12 - Ro4-1284 Pretreatment Attenuates Loss of Serotonergic Nerve Terminal Density in Rat Striatum Following MDMA Dosing...... 145 Figure A-1 - Purification of Thioether Metabolites of N-Me-α-MeDA ...... 180 Figure A-2 - MS/MS Spectrum of 5-(GSH)-N-Me-α-MeDA ...... 182 Figure A-3 - MS/MS Spectrum of 5-(NAC)-N-Me-α-MeDA...... 184 Figure A-4 - Cellular Uptake of 5HT into Various In Vitro Serotonergic Cell Models...... 185 Figure A-5 - Kinetics of 5HT Uptake into HEK Cells Expressing hSERT...... 187 Figure A-6- Kinetics of MDMA, N-Me-α-MeDA, 5-(GSH)-N-Me-α-MeDA and 5-(NAC)-N-Me-α-MeDA- Mediated Modulation of 5-HT Uptake into hSERT-Transfected HEK Cells ...... 189 Figure A-7 - 5-(GSH)-N-Me-α-MeDA and 5-(NAC)-N-Me-α-MeDA Fail to Inhibit 5-HT Uptake into hSERT- Transfected HEK Cells in a Dose-Dependent Manner...... 190 9

LIST OF ABBREVIATIONS

ALDH Aldehyde dehydrogenase

α-MeDA Alpha methyldopamine

AUC Area under the curve aCSF Artificial cerebro-spinal fluid

COMT Catechol-O-methytransferase

CNS Central nervous system

COX Cyclooxygenase

CYP Cytochrome P450

5,7-DHT 5,7-Dihydroxytryptamine

2,3-DHBA 2,3-Dihydroxybenzoic

DOPAC 3,4-Dihydroxyphenylacetic Acid

DTBZ Dihydrotetrabenazine

DA

DAT Dopamine reuptake transporter

EDTA Ethylenediaminetetraacetic acid

GFAP Glial fibrillary protein

γ-GT γ-Glutamyl transpeptidase

5-(GSH)-α-MeDA 5-(glutathion-S-yl)-α-MeDA

2,5-bis-(GSH)-α-MeDA 2,5-bis-(glutathione-S-yl)-α-MeDA

5-(GSH)-N-Me-α-MeDA 5-(glutathion-S-yl)-N-methyl-α-MeDA

2,5-bis-(GSH)-N-Me-α-MeDA 2,5-bis-(glutathion-S-yl)-N-methyl-α-MeDA

GSH Glutathione

5-HIAA 5-Hydroxyindoleacetic acid

HMA 4-Hydroxy-3-methoamphetamine 10

HMMA 4-Hydroxy-3-methoxymethamphetamine icv Intracerebroventricularly ip Intraperitoneally iv Intravenously

Iba1 Ionized calcium-binding adapter molecule 1

MDA 3,4-(±)-Methylenedioxyamphetamine

MDMA 3,4-(±)-Methylenedioxymethamphetamine

MPTP 1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine

MAO Monoamine oxidase

5-(NAC)-α-MeDA 5-(N-acetylcystein-S-yl)-α-MeDA

2,5-bis-(NAC)-α-MeDA 2,5-Bis-(N-acetylcystein-S-yl)-α-MeDA

5-(NAC)-N-Me-α-MeDA 5-(N-acetylcystein-S-yl)-N-methyl-α-MeDA

2,5-bis-(NAC)-N-Me-α-MeDA 2,5-Bis-(N-acetylcystein-S-yl)-N-me-α-MeDA

N-Me-α-MeDA N-methyl-α-methyldopamine

NAC N-acetyl cysteine

NOX NADPH oxidase

NIDA National Institute on Drug Abuse

NADPH Nicotinamide adenine dinucleotide phosphate

NE Norepinephrine

NET Norepinephrine reuptake transporter

6-OHDA 6-Hydroxydopamine po Per oral

PTP Permeability transition pore

PDN Postnatal day

PSH Prostaglandin H synthase

ROS Reactive oxygen species 11

RNS Reactive nitrogen species

SSRI Selective serotonin

5-HT Serotonin

SERT Serotonin reuptake transporter

SERT-KO SERT-knockout

SD Sprague-Dawley

SNS Sympathetic nervous system sc Subcutaneously

SULT Sulfotransferase

TH Tyrosine hydroxylase

TPH Tryptophan hydroxylase

UCP Uncoupling protein

UGT Glucuronyltransferase

VMAT2 Vesicular monoamine transporter 2 12

ABSTRACT

3,4-(±)-Methylenedioxymethamphetamine (MDMA, Ecstasy) is a widely abused derivative with potent stimulant properties. The neuropharmacological effects of

MDMA are biphasic in nature. MDMA initially causes synaptic monoamine release, primarily of serotonin (5-HT), producing hyperthermia and hyperactivity (5-HT syndrome). Conversely, the long-term effects of MDMA manifest as a prolonged depletion in 5-HT, and structural damage to serotonergic nerve terminals. Monoamine transporter systems at the plasma membrane and storage vesicles of 5-HT neurons have been implicated in MDMA toxicity. Nonetheless, many mechanistic questions remain regarding the precise role of uptake transporters in MDMA neurotoxicity. The present study was designed to address the importance of the serotonin reuptake transporter (SERT) and the vesicular monoamine transporter 2 (VMAT2) to the physiological, behavioral and neurotoxic responses to MDMA.

SERT functions as a primary regulator of 5-HT homeostasis, mediating the reuptake of 5-

HT from the synaptic space following its release during neurotransmission. SERT is a molecular target site for MDMA and many antidepressant agents such as the selective serotonin reuptake inhibitor (SSRI) class. Pharmacological inhibition of SERT protects against MDMA-induced serotonergic neurotoxicity. Thus, the effects of MDMA are in part mediated by an ability to interact with and inhibit SERT. Using a SERT-knockout (SERT-KO) rat model, we determined that

SERT deficiency modulated the acute toxicities of MDMA, such as hyperthermia and hyperactivity, whilst completely preventing long-term depletions in tissue 5-HT levels, indicating the abolishment of neurotoxicity. 13

Disruption of vesicular monoamine storage via interaction with VMAT2 has also been implicated in MDMA neurotoxicity. VMAT2 participates in the transport of monoamine , in particular 5-HT and dopamine (DA), into intra-neuronal storage vesicles. As such, VMAT2 is critical in maintaining neuronal health by preventing neurotransmitter oxidation within the cytosol. Pharmacological inhibition of VMAT2 with Ro4-1284 reduced MDMA-induced hyperactivity and averted hyperthermia along with persistent serotonergic deficits.

Overall, our results corroborate the hypothesis that SERT and VMAT2 are critical to the in vivo effects of MDMA. Furthermore, given that VMAT2 inhibition diminished the behavioral response to MDMA in rats, pharmacological manipulation of this transporter could be used in the treatment of MDMA abuse and overdose. 14

CHAPTER 1: INTRODUCTION

1.1 MDMA: THE PROTOTYPE CLUB DRUG

MDMA is a ring substituted amphetamine derivative, structurally related to both stimulant and hallucinogenic compounds, hence, it has been termed the prototype empathogen.

Methylenedioxyamphetamines or empathogens act primarily on the serotonergic system through the modulation of 5-HT release and reuptake inhibition (Johnson et al., 1986; Steele et al., 1987).

In contrast, stimulants have profound effects on the dopaminergic system, while hallucinogenic drugs act directly on 5-HT2A binding sites. This mode of action provides MDMA, and

MDMA-like drugs, with unique psychoactive properties that make them “the drug of choice” at raves and dance clubs. These club drugs induce social interaction and sensory stimulation, creating a sense of closeness to others and inducing feelings of empathy and euphoria (Downing,

1986). The popularity of these substances is enhanced by their low cost relative to other recreational drugs, and convenient packaging, usually in the form of small pills. Club drugs are often ingested with alcohol or combined with other drugs, elevating the risk of toxicity and overdose (Gahlinger, 2004).

1.1.1 History

MDMA was synthesized in 1912 by the German company, Merck, under the name of

“methylsafrylamin” (Freudenmann et al., 2006). The company filed a patent on the synthesis of

MDMA, describing it as an intermediate for therapeutically active compounds. The next set of

MDMA records at Merck are from 1927, when Dr Max Oberin expressed interest in the chemical similarities of MDMA to ephetonine and adrenaline (Freudenmann et al., 2006). He began synthesizing MDMA as a hydrochloride salt instead of a free base in order to perform the first 15

pharmacological tests on this compound. Toxicological examination of MDMA in experimental animal models was initiated in 1953 by the U.S. Army under a chemical warfare program aimed at evaluating the toxicity of mescaline-like compounds (Karch, 2011). Another milestone in MDMA history came in 1976, when American chemist Shulgin and psychologist Leo Zeff began experimenting with MDMA as an adjuvant to psychotherapy, becoming the first scientists to describe the psychoactive effects of this drug in humans (Stolarff, 1997). Zeff introduced the drug to the psychotherapeutic community all throughout the U.S. under the name of “Adam,” to expresses a state of returning to primal innocence. The use of MDMA for therapeutic purposes quickly rose in popularity because it could comfort patients and increase their self-esteem, facilitating communication during therapy (Grinspoon and Bakalar, 1986). Consumption of MDMA for recreational purposes was popularized in the 1980s in San Francisco, where it was marketed as a “club drug” and given the name of “ecstasy” (Beck and Morgan, 1986). In 1985, MDMA was categorized as a schedule I drug by the Drug Enforcement Administration (DEA) because of its high abuse potential, lack of medical use and neurotoxic profile (Karch, 2011). Despite restrictions in MDMA production and consumption, this substance remains the number one “party drug” sold at raves and dance clubs worldwide.

1.1.2 Epidemiology

MDMA abuse is widespread. The United Nations estimated the prevalence of MDMA in

2012 to be 0.2-0.6% of the global population between the ages of 15-64; however, higher rates were found in both North America (0.9%) and Western and Central Europe (0.8%) (UNODC, 2012).

Teenagers and young adults are particularly vulnerable to MDMA abuse. In 2010, 96% (2.5million) of the total past-year MDMA users in the U.S. and 80% (2.0 million) in Europe were between the ages of 14-34 (UNODC, 2012). Earlier epidemiological studies indicated that MDMA use in the U.S. 16

increased substantially between 1989 to 1995, from 0.5% to 2.4%, among college students and from 0.7% to 1.6% among young adults (Johnston et al., 2008). No data was collected from younger age groups to avoid attracting attention to this club drug. In 1996, when the survey was given to students in secondary school, the annual rates for MDMA consumption among 10th and

12th graders (4.6%) were surprisingly much higher than those of college students (2.8%). From

1998 to 2001, MDMA prevalence spiked in all age groups, almost doubling for three consecutive years in the 12th grader population (Johnston et al., 2008).

MDMA abuse was on a steady decline from 2000-2009, which reflected a parallel decreasing trend in substance availability worldwide, and the increase in perceived risk among users as a result of an anti-ecstasy campaign launched in 2002 by the U.S. government (Johnston et al., 2008). In the last few years, the global MDMA market has shown a slight recovery. The amount of MDMA confiscated at the U.S./Canadian border increased from 1.9 million ecstasy tablets in 2006 to 3.9 million tablets in 2010, according to the National Drug Intelligence Center

(NDIC, 2011). Similarly, MDMA prevalence increased in 2013 among 10th and 12th graders, despite showing a marginal decrease among the general U.S. population (Johnston et al., 2013). A growing concern of MDMA use among teenagers comes from its association with other drugs of abuse, such as alcohol, cannabis, heroin and other (Pedersen and Skrondal, 1999). There is also a greater risk for polydrug MDMA users to engage in high risk sexual behavior (Banta-Green et al., 2005; Klitzman et al., 2002) and a higher incidence of psychiatric symptoms when compared to “non-drug” users or “other drug” users (Keyes et al., 2008; Montoya AG et al., 2002).

1.1.3 Chemical Structure, Route of Administration and Dosage

MDMA is a ring-substituted analog of amphetamine and . It possesses a methylenedioxy (-O-CH2-O-) group attached to positions 3 and 4 of the aromatic ring in the 17

amphetamine molecule. This ring substitution makes MDMA structurally similar to naturally occurring hallucinogenic compounds such as mescaline, which is derived from peyote cacti from

North America (Kalant, 2001). The pharmacological effects of MDMA and MDMA-like drugs are a combination of those of amphetamines and hallucinogens (Green et al., 1995). Substances in this group are referred to as empathogens or “designer drugs.” Empathogens structurally and functionally resemble monoamine neurotransmitters, inducing many of the biological actions of

5-HT, DA and norepinephrine (NE) (Kalant, 2001). The chemical structures of MDMA and phenylethylamine-related compounds are detailed in Figure 1-1.

MDMA and related compounds are synthetic amines that can be manufactured as free bases or salts of a variety of acids. MDMA salt is soluble and quite stable; therefore, it is almost exclusively sold in the form of tablets for oral consumption (Kalant, 2001; Tanner-Smith, 2006).

Ecstasy tablets are often colored and marked with attractive designs or logos. In 2005, a comprehensive study reported on the pharmacological content of ecstasy pills between 1999 and

2005 in the U.S. They found that 39% of the tablets contained MDMA only, 46% contained no

MDMA, and 15% consisted of mixtures of MDMA and other substances. The most common substances that appeared in contaminated pills included other amphetamines such as 3,4- methylenedioxyamphetaimne (MDA) and methamphetamine, in addition to, legal and inexpensive compounds such as caffeine, dextromethorphan and pseudo-ephedrine (Tanner-

Smith, 2006). On average, the dose of MDMA in an ecstasy tablet ranges from 50 to 150 mg

(Kirsch, 1986). MDMA users typically consumed 1-2 tablets in combination with alcohol or other drugs at “raves” or “dance parties” (Siegel, 1986) but doses of up to 10 tablets have been reported, usually associated with high incidence of toxicity (Dar and McBrien, 1996; Walubo and

Seger, 1999). 18

Figure 1-1 - Phenylethylamine Derivatives

Chemical structures of phenylethylamine derivatives structurally related to MDMA and other amphetamines (including amphetamine, methamphetamine, and MDA). The structure of phenylethylamine is highlighted at the center of this figure. Amphetamines possess a phenylethylamine core, sharing structural and functional similarities to monoamine neurotransmitters such as serotonin, dopamine and norepinephrine, and hallucinogenic compounds such as mescaline. Adapted from (Capela et al., 2009). 19

1.2 PHARMACOKINETICS OF MDMA

MDMA and other substituted amphetamines are weak bases, with a pKa of approximately

9.9 and a low molecular weight (~200 g/mol). These compounds are capable of crossing cell membranes and lipid bio-layers by simple diffusion, thereby accumulating in nonconventional biological matrices such as saliva, hair, nails and sweat (de la Torre et al., 2004a). MDMA exhibits good oral bioavailability, low plasma protein binding (34% estimated in dog plasma) (Garrett et al., 1991), and an apparent volume of distribution of 435 L (de la Torre et al., 2004b). Following oral administration of 75 mg of MDMA, maximal concentrations in the plasma (Cmax = 130.9 µg/L) are reached at 1.8 h, with an elimination half-life of 7.7 h in healthy volunteers (de la Torre et al.,

2000). Moreover, dose-dependent increases in plasma MDMA concentrations display non-linear pharmacokinetics, possibly due to a saturation of MDMA metabolism, or an interaction of MDMA metabolites with enzymes involved in the MDMA metabolic pathway (de la Torre et al., 2000).

The methylenedioxy group of MDMA is thought to form metabolite-enzyme complexes to cause the quasi-irreversible inactivation of cytochrome P450s (CYPs), in particular of CYP2D6 (Delaforge et al., 1999).

In agreement with human studies, the pharmacokinetics of MDMA follows a non-linear pattern in rats, and the breakdown of MDMA to MDA is linear up to doses of 10 mg/kg. Moreover, concentrations of MDMA in rat brain are high relative to plasma and other tissues (Chu et al.,

1996). This discrepancy is attributed to the chemical properties of MDMA that facilitate passive diffusion across lipid membranes, in addition to the interaction of MDMA with SERT that further enables the accumulation of this drug inside pre-synaptic 5-HT terminals (Capela et al., 2009).

SERT-mediated transport of MDMA into 5-HT neurons is part of the pharmacological mechanism of MDMA, and is discussed in detail in section 1.3.1. Furthermore, MDMA pharmacokinetics in 20

rats is profoundly influenced by polymorphisms, primarily among CYP isoenzymes, resulting in sex and strain differences in MDMA disposition and toxicity.

CYP2D1 has been linked to strain differences in MDMA pharmacokinetics. Rat CYP2D1 is the orthologue of human CYP2D6, and is one of the enzymes involved in the demethylenation of

MDMA (Kumagai et al., 1994). Dark Agouti female rats are deficient in CYP2D1 activity and are often used as a model for the human CYP2D poor metabolizer phenotype (Colado et al., 1995).

Liver microsomes isolated from female Dark Agouti rats have only 10% of the total CYP activity of

Sprague Dawley (SD) rats (Kumagai et al., 1994), which is consistent with an elevation in brain

MDMA concentrations with low drug doses (5 and 10 mg/kg) compared to both male and female

SD rats. At high MDMA doses (20 and 40 mg/kg), brain MDMA concentrations are comparable to those of SD rats, indicating activation of alternative metabolic pathways (Chu et al., 1996).

Additionally, Dark Agouti females are more susceptible to MDMA-induced lethality, perhaps due to increases in MDMA plasma levels. In contrast, Dark Agouti female rats also show attenuation of MDMA-mediated 5-HT depletion both acutely (Chu et al., 1996) and 7 days after drug treatment (Herndon JM, Monks TJ et al., unpublished observations). These findings support a role for CYP2D-derived metabolites in the development of 5-HT deficits, suggesting that metabolism is important to the neurotoxic effects of MDMA.

1.2.1 MDMA Metabolism and Potential Bioactivation

MDMA is extensively metabolized in the liver in both humans and rats, and MDMA metabolism is known to directly influence its long-term neurotoxicity (Esteban et al., 2001;

Gollamudi et al., 1989). Two major pathways have been identified for the metabolism of MDMA in-vivo (Figure 1.2). The first involves CYP-mediated O-demethylenation to 3,4- dihydroxymethamphetamine (N-Me-α-MeDA, HHMA), which predominantly occurs in humans, 21

and displays biphasic kinetics. CYP2D6 is the only low Km enzyme detected in human liver microsomes for the demethylenation of MDMA; however, other isozymes with higher Km values, such as CYP1A2 and to a lesser extent CYP2B6 and CYP3A4, have substantial contributions to the total microsomal activity at high MDMA concentrations (200µM) (Kreth et al., 2000). In rats, O- demethylenation of MDMA is primarily mediated by CYP2D1 at low Km values, and by CYP2B1 at high Km values.

MDMA is also N-demethylated to MDA, a process that exhibits monophasic kinetics (Kreth et al., 2000). This metabolic route is minor in humans as MDA reaches only 8-9% of MDMA plasma concentrations, and MDA urinary recovery is only 1.5% of the dose administered, compared to

17.7% for N-Me-α-MeDA (de la Torre et al., 2004b). Moreover, in human liver microsomes, the

Vmax for N-demethylation of MDMA is lower (7-fold) than for the corresponding O- demethylenation. CYP2B6 is the main enzyme involved in the N-demethylation of MDMA to MDA in humans, followed by CYP1A2 and CYP3A4 (Kreth et al., 2000). In contrast, MDA is the major

MDMA metabolite in rats; it accumulates in the brain dose-dependently and in parallel with plasma levels, reaching concentrations similar or even greater than those of MDMA (Chu et al.,

1996). N-Demethylation of MDMA in rats is catalyzed by CYP1A2 and CYP2D1 (Hirt et al., 2010).

In mice, MDMA is the main component observed in plasma and brain. Urinary excretion of the parent compound, within 24 h of drug administration, is more prominent in mice compared to rats, and perhaps related to a greater resistance to MDMA-induced neurotoxicity (Lim et al.,

1992).

Subsequent to N-demethylation, MDMA and MDA follow parallel metabolic pathways that result in the formation of catechol intermediates (Figure 1.2). N-Me-α-Me-DA is derived from

O-demethylenation of MDMA and 3,4-dihydroxyamphetamine (α-MeDA, HHA) is formed from the 22

O-demethylenation of MDA. N-Me-α-Me-DA and α-MeDA can undergo further metabolism by a number of phase II metabolic enzymes that lead to their excretion from the body, including O- methylation by catechol-O-methyltransferase (COMT), glucuronidation by glucuronyltransferases

(UGTs) and sulfation by sulfotransferases (SULTs). Analysis of phase II MDMA metabolites in human urine revealed that the major proportion of urinary recovery occurred within 24 h of drug administration (Schwaninger et al., 2011a). All metabolites of MDMA were recovered as glucuronide and sulfate conjugates; however, N-Me-α-Me-DA metabolites were primarily identified compared to α-MeDA metabolites. Most of a low MDMA dose (1 mg/kg, po) was recovered as 4-hydroxy-3-methoxymethamphetamine (HMMA) sulfate (13%), whereas, more parent compound was recovered after a higher MDMA dose (1.7 mg/kg, po), indicative of non- linear pharmacokinetics. Overall, sulfate conjugates were present in urine at higher concentrations than glucuronides (Schwaninger et al., 2011b), which correlates with in-vitro data, demonstrating that N-Me-α-Me-DA sulfation is 2-10 times higher than glucuronidation

(Schwaninger et al., 2011b). Moreover, catecholamine MDMA metabolites act as both substrates and inhibitors of COMT and SULT, inhibiting their own metabolism and that of other endogenous compounds, such as DA and estradiol (Meyer and Maurer, 2009; Schwaninger et al., 2011a).

Similarly, phase II MDMA metabolites are primarily excreted as sulfate and glucuronide (>85%) conjugates in rat and mice urine, with HMMA and 4-hydroxy-3-methoxyamphetamine (HMA) identified as the predominant metabolites in both species (Lim et al., 1992).

The unstable catecholamine metabolites can also undergo oxidation to their corresponding ortho-quinones, and subsequent reaction with glutathione (GSH) (Figure 1-2) via adduction of the cysteinyl sulfhydryl group, and with other thiol containing compounds (Lim and

Foltz, 1988; Patel et al., 1991). Conjugation of N-Me-α-Me-DA and α-MeDA by GSH results in their 23

elimination from the body via the mercapturic acid pathway, initiated by γ-glutamyl transpeptidase (γ-GT). The corresponding cysteine metabolites interact with N-acetylcysteine transferase to yield N-acetylcysteine (NAC) conjugates (Monks and Lau, 1998). GSH conjugates form in the liver, but are readily distributed into different organs. Systemic formation of GSH conjugates is followed by uptake into the brain across the blood-brain barrier via a carrier- mediated mechanism that is also capable of transporting GSH (Kannan et al., 1990). Indeed, uptake of 5-(GSH)-α-Me[3H]DA into the brain is significantly reduced with GSH treatment, indicating that these two compounds share a common transporter (Miller et al., 1996). Phase I and II enzymes, including CYP2D1, COMT and γ-GT, are present in the brain and appear to have significant activity (Hanigan and Frierson, 1996; Mannisto and Kaakkola, 1999; Tyndale et al.,

1999), thus, localized MDMA metabolism is thought to occurred within the brain.

GSH and NAC conjugates of MDMA accumulate in rat brain. Jones et al. (2005) reported concentrations of thioether MDMA metabolites in striatal dialysate after a neurotoxic MDMA dose (20 mg/kg, sc) that were comparable to those obtained with intracerebroventricular (icv) administration of 5-(GSH)-α-MeDA. Furthermore, thioether N-Me-α-MeDA conjugates accumulated in rat striatum after repeated MDMA administration, with mercapturic acid metabolites showing a slow rate of elimination (Erives et al., 2008). The area under the curve

(AUC) between the first and last dose increased 33-50% for 5-(GSH)-N-Me-α-MeDA and 2,5-bis-

(GSH)-N-Me-α-MeDA and 35-85% for 5-(NAC)-N-Me-α-MeDA and 2,5-bis-(NAC)-N-Me-α-MeDA, respectively (Erives et al., 2008). Accumulation of these reactive metabolites at such high concentrations in the brain raises concerns about their potential to cause neurotoxicity.

Importantly, 5-(NAC)-N-Me-α-MeDA and 5-(NAC)-α-MeDA have also been detected in human urine with a recreational dose of MDMA (1.5 mg/kg) (Perfetti et al., 2009). 24

Although conjugation of N-Me-α-Me-DA and α-MeDA ortho-quinones with GSH is a detoxication pathway that facilitates their excretion, these metabolites are highly electrophilic, and can readily interact with nucleophiles, forming covalent bonds with macromolecules, which may contribute to toxicity. The potential bioactivation of quinol/quinone eletrophiles, following

GSH conjugation is well documented (Kleiner et al., 1998; Monks, 1995). Moreover, quinone thioethers possess the ability to redox cycle, generating reactive oxygen species (ROS) and forming protein adducts (Kleiner et al., 1998). Superoxide-mediated oxidation of N-Me-α-Me-DA and α-MeDA and subsequent thiol adduction demonstrated the redox potential of MDMA metabolites, and their ability to form covalent adducts (Hiramatsu et al., 1990). The contribution of metabolic bioactivation of thioether metabolites to MDMA neurotoxicity and their possible mechanisms of toxicity are discussed in more detailed in section 1.5.4.3. 25 26

Figure 1-2 - MDMA Metabolism

The metabolic scheme displays the major pathways of MDMA metabolism in humans and rats. MDMA is N-demethylated to MDA (1), followed by O-demethylenation to form α-MeDA (3), both reactions are carried out by CYP isoenzymes in the liver. This constitutes the major metabolic pathway of MDMA in rats. In humans, MDMA is preferentially O-demethylenated to form N-Me- α-MeDA (3), a reaction that is also mediated by CYP enzymes. α-MeDA and N-Me-α-MeDA undergo O-methylation carried out by COMT, forming catechol intermediates, HMMA and HMA (4 and 5), respectively. α-MeDA, N-Me-α-MeDA, HMMA and HMA are conjugated by a variety of phase II reactions, primarily sulfation and glucuronidation mediated by SULT and UDGPT enzymes. These conjugation reactions lead to the excretion of MDMA metabolites via the urine and feces. Alternatively, CYP-formed metabolites of MDMA can oxidize to their corresponding ortho- quinones, which can lead to the formation of glutathione adducts (6 and 7). Furthermore, glutathione conjugates undergo metabolism via the mercapturic acid pathway to yield N- acetylcysteine conjugates (8 and 9). Thioether metabolites of MDMA are thought to be the ultimate toxicants in the mechanism of MDMA neurotoxicity.

Abbreviations: CYP, cytochrome P450; COMT, catechol-O-methyltransferase; HMMA, 4-hydroxy- 3-methoxyamphetamine; HMA, 4-hydroxy-3-methoxyamphetamine; SULT, sulfotransferase; UDPGT, uridine diphosphate glucuronyltransferase. 27

1.3 PHARMACOLOGY OF MDMA

1.3.1 Mechanism of Action

MDMA has profound effects on both the peripheral and central nervous systems, primarily through modulation of monoaminergic neurotransmitter systems. In-vitro studies have shown that MDMA acts as an indirect monoaminergic , causing the release of [3H]-5-HT, and to a lesser extent of [3H]-DA and [3H]-NE, from rat hippocampal and caudate nucleus brain slices (Fitzgerald and Reid, 1993; Johnson et al., 1986). In contrast, amphetamine is more efficient at stimulating the release of [3H]-DA than [3H]-5-HT and [3H]-NE (Fitzgerald and Reid, 1993).

MDMA-mediated release of [3H]-5-HT and [3H]-DA from rat synaptosomes is partially inhibited in the presence of omega-agatoxin-IVA, a P-type voltage operated Ca2+ channel blocker, suggesting that monoamine release is Ca2+ dependent (Crespi et al., 1997). However, a different study using rat brain slices found that MDMA-mediated monoamine release was unaltered by Ca2+ (Johnson et al., 1986), similar to the Ca2+-independent mechanism of amphetamine-mediated monoamine release (Arnold et al., 1977). Such discrepancies could be attributed to differences in experimental design and in-vitro models utilized. Moreover, synaptosomal [3H]-5-HT release induced by MDMA and other substituted amphetamines, such as methamphetamine, p-chloroamphetamine and flenfluramine, is blocked by 5-HT uptake inhibitors, fluoxetine and , suggesting a role for reuptake transporters in the mechanism of action of these drugs (Berger et al., 1992).

MDMA, and other psychostimulants, interact with monoamine reuptake transporters, inhibiting the function of SERT, the DA transporter (DAT) and the NE transporter (NET). Studies in transfected intestine 407 cells revealed that MDMA has greater potency at inhibiting SERT than

DAT, which is in direct contrast to the pharmacological mechanism of other amphetamines (Han and Gu, 2006). The potency of MDMA for the different monoamine transporters from mouse 28

cDNAs is: SERT (KI = 0.64µM), NET (KI = 1.75µM) and DAT (KI = 4.87µM) (Han and Gu, 2006).

Similarly, MDMA inhibits SERT (KI = 0.24µM) in rat synaptosomes more potently than NET (KI =

0.46µM) and DAT (KI = 1.57µM) (Rothman et al., 2001). Data suggest that the addition of a methylenedioxy group to form MDMA increases the potency of this drug at SERT, whilst reducing its affinity towards NET and DAT, in contrast to amphetamine and methamphetamine (Han and

Gu, 2006). Differences in monoamine transporter affinity among amphetamine derivatives is thought to account for differences in their mechanism of action and toxicity.

Microdialysis experiments in live animals reveal similar increases in the release of biogenic amines upon MDMA administration. In awake rats, MDMA (1-20 mg/kg, iv) induces a dose-dependent increase in dialysate 5-HT concentrations in the striatum and medial prefrontal cortex within 30 min of drug administration (Baumann et al., 2005; Gudelsky and Nash, 1996).

Moreover, elevations in 5-HT are blocked by fluoxetine, supporting a role for SERT in MDMA- mediated 5-HT release (Gudelsky and Nash, 1996). MDMA also induces the release of DA in the caudate and nucleus accumbens (Yamamoto and Spanos, 1988). Elevations in 5-HT synthesis with concomitant carbidopa and L-5-hydroxytryptophan treatment potentiates the efflux of 5-HT and

DA produced by MDMA, increasing concentrations of these monoamines to a greater extent than either treatment alone. These results suggest a stimulatory role of 5-HT in MDMA-mediated DA release (Gudelsky and Nash, 1996). Additional studies demonstrated that elevations in extracellular DA levels induced by MDMA in the striatum are affected by 5-HT neurotransmission

(Koch and Galloway, 1997), primarily via stimulation of 5-HT2A receptors (Schmidt et al., 1992).

Correspondingly, impulse-driven release of DA by MDMA is attenuated by infusion of tetrodoxin, a voltage-gated Na+ channel blocker (Yamamoto et al., 1995), and by treatment with a 5-HT reuptake inhibitor (Koch and Galloway, 1997). Furthermore, MDMA-mediated DA release is 29

dampened by increases in GABA neurotransmission in the ventral tegmental area via the activation of 5-HT2C receptors (Bankson and Yamamoto, 2004). Thus, increased 5-HT release and neurotransmission appear to have a primary role in MDMA-evoked DA release.

5-HT release also modulates the stimulation of NE levels following MDMA administration, as evident by the attenuation of NE-related cardiovascular side-effects in humans via 5-HT reuptake inhibition with citalopram (Liechti et al., 2000a). The release of NE is critical to the acute physiological effects of MDMA (Hysek et al., 2011) and other psychostimulants (Rothman et al.,

2001). Although central release of NE has yet to be demonstrated, MDMA is a potent NE agonist in-vitro, and plasma NE concentrations are markedly elevated in humans (Hysek et al., 2012) and rats (Sprague et al., 2005) upon MDMA exposure. Interestingly, MDMA has a higher binding affinity and greater potency at inhibiting the human NET compared to SERT and DAT (Han and Gu,

2006), which contrast with data obtained with rodent transporters (Han and Gu, 2006; Verrico et al., 2007). The consequences of the more potent interaction of MDMA with NET compared to other uptake transporters in humans are currently unknown.

Amphetamines may enter nerve terminals via interaction with monoamine transporters, inducing carrier-mediated release of biogenic amines (Schuldiner et al., 1993). In the case of

MDMA, 5-HT release involves exit of neurotransmitter molecules down their concentration gradient via reversal of SERT function by the disruption of vesicular monoamine storage, which causes 5-HT to accumulate in the cytosol (Capela et al., 2009). The mechanism of monoamine release induced by amphetamines consists of binding to uptake transporters and causing an inward current that allows for the rapid concentration of Na+ intracellularly, thereby enhancing carrier-mediated efflux of neurotransmitter molecules (Khoshbouei et al., 2003). Similarly, disruption of vesicular 5-HT storage by MDMA occurs through a carrier-mediated exchange 30

mechanism that involves VMAT2. MDMA is thought to enter storage vesicles via VMAT2 interaction, depleting neurotransmitter concentrations by reversal of transporter function.

Additionally, MDMA enhances cytosolic 5-HT concentrations available for SERT-mediated efflux by blocking degradation via monoamine oxidases (MAO) (Leonardi and Azmitia, 1994). A summary of the main putative events that transpire during acute MDMA toxicity is illustrated in Figure 1-3. 31

Figure 1-3 - Pharmacological Mechanism of Action of MDMA

MDMA enters pre-synaptic 5-HT nerve terminals via SERT activity. Once in the cytoplasm, MDMA interacts with VMAT2 and causes depletion of vesicular 5-HT stores via a carrier-mediated exchange mechanism. MDMA is also capable of inhibiting MAOs, preventing the degradation of 5-HT. These two events result in an increase in free cytosolic 5-HT that is available for SERT- mediated transport into the synaptic cleft. Excess 5-HT release results in the activation of 5-HT receptors located in post-synaptic neurons involved in the physiological and behavioral response to MDMA. 32

1.3.1 Acute Effects and Toxicity

MDMA’s popularity derives from its mood-enhancing properties that are summarized in the 3 Es: energy, empathy and euphoria (Hall and Henry, 2006). Recreational MDMA doses (0.8-

1.9 mg/kg, po) produce a wide range of acute psychological changes, generally of a positive nature

(Downing, 1986; Solowij et al., 1992; Vollenweider et al., 1998). MDMA-naive healthy volunteers report experiencing a state of enhanced mood and well-being that includes a sense of peacefulness with enhanced insight, euphoria, heightened openness, increased responsiveness to emotions, and a sense of closeness to others (Vollenweider et al., 1998). MDMA can also affect cognition, frequently increasing alertness, confusion, impaired judgment and short-term memory loss, whilst inducing disturbances in sleep and appetite (Baylen and Rosenberg, 2006). Altered sensory perception is a prevalent subjective effect of this drug, causing changes in visual perception, sound alterations, enhanced sense of touch and hallucinations (Baylen and

Rosenberg, 2006). Although rare, increased anxiety (Peroutka et al., 1988), dysphoric and psychotic reactions (Hermle et al., 1993), and risky sexual behavior, such as unprotected sex,

(Banta-Green et al., 2005) have been documented with MDMA consumption. Moreover, MDMA has similar reinforcing effects to other dopaminergic in healthy moderate MDMA users

(Tancer and Johanson, 2003).

MDMA also induces acute physical effects that resemble those of other amphetamines.

The most frequent somatic effects are jaw clenching (bruxism), nausea/vomiting, muscle aches, headache, sweating, fatigue, numbness, anorexia, and motor restlessness (Baylen and Rosenberg,

2006; Vollenweider et al., 1998). Moderate increases in systolic and diastolic blood pressure are common with MDMA consumption, and can pose a risk to individuals with latent cardiovascular problems (Vollenweider et al., 1998). Hyperthermia is the most recurrent adverse reaction of 33

MDMA acute toxicity, and can be life-threading, preceding intravascular coagulation, rhabdomyolysis, multiple organ failure, and acute renal failure (Hall and Henry, 2006). Other lethal intoxications associated with MDMA in humans include arrythmias, 5-HT syndrome, coagulopathy, hyponatremia, acidosis, pulmonary congestion, cerebral infarction and sudden death (Liechti et al., 2005; McCann et al., 1996).

The prevalence, frequency and intensity of the acute symptoms associated with MDMA are dependent upon three main factors: time between ingestion and assessment, dose level, and gender (Baylen and Rosenberg, 2006). These acute effects appear as early as 30-60 min and reach their peak around 75-120 min subsequent to drug ingestion, enduring for 2-12 h. Negative effects are reported with greater frequency and intensity the day after MDMA ingestion. Higher MDMA doses correlate with more intense and long-lasting experiences, with lower doses reducing the frequency of negative symptoms. Lastly, women report greater intensity of undesirable effects than males, including physical illness, depression, anxiety, thought disorders, and perceptual changes (Baylen and Rosenberg, 2006).

1.4 MECHANISMS OF ACUTE MDMA TOXICITY, AN EMPHASIS ON HYPERTHERMIA AND THE 5-

HT BEHAVIORAL SYNDROME

1.4.1 Hyperthermia

MDMA-mediated hyperthermia in humans has been long associated with its recreational use, and fatalities involving increases in body temperature above 40ºC have been reported.

(Chadwick et al., 1991; Halpern et al., 2011). Although marked elevations in body temperature are absent in human subjects after low-dose MDMA treatment (0.25-1.0 mg/kg), a trend to increasing temperature with MDMA dosage was observed (Grob et al., 1996). Higher doses of 34

MDMA (1.5-1.7 mg/kg) produce significant increases in body temperature of up +0.4ºC between

60-120 min post-administration (Liechti et al., 2000b; Vollenweider et al., 1998). Ambient temperature has a minimal effect on MDMA-mediated hyperthermia in human volunteers

(Tancer et al., 2003), which is in direct contrast to the thermoregulatory response to MDMA in rodents. Furthermore, the thermoregulatory effects of MDMA have been extensively studied in recreational settings, including raves and week-long dance parties. These studies report higher body temperatures of +1.2-1.8ºC in the MDMA users compared to controls, with subjects consuming up to 6 ecstasy tablets in a weekend (Morefield et al., 2009; Parrott et al., 2007;

Parrott and Young, 2005). One study in particular revealed a positive correlation between elevated body temperatures and plasma MDMA levels (Irvine et al., 2006). Subjective thermal effects were also recorded, where subjects described ‘feeling hot’ or experiencing ‘hot and cold flashes’ (Cohen, 1995; Parrott, 2006).

Similar to human studies, MDMA causes a dose-dependent increase in body temperature in experimental animals, including rats, mice and nonhuman primates, leading to hyperthermia.

Rats display an elevation in body temperature of +1-2ºC, 40-60 min post-MDMA administration, under normal ambient temperatures (20-22ºC) (Broening et al., 1995; O'Shea et al., 1998), whereas, mice exhibit a greater variability in the MDMA thermogenic response under these conditions, based upon differences in the strain of mouse and drug dose (Green et al., 2003).

Thus, repeated MDMA treatment (10 mg/kg, 3x at 3h intervals) produced hypothermia in Swiss-

Webster mice, but a single MDMA dose (30 mg/kg) resulted in a pronounced hyperthermic effect

(O'Shea et al., 2001). Similarly, NIH/Swiss mice displayed significant hyperthermia following multiple MDMA doses (25 mg/kg, 3x at 3h intervals) (Colado et al., 2001). 35

Ambient temperature plays a pivotal role in MDMA-mediated thermogenesis in both rats and mice. Holtzman rats exposed to high MDMA doses (20 mg/kg) experienced hyperthermia at ambient temperatures above 28ºC and hypothermia at ambient temperatures below 22ºC

(Malberg and Seiden, 1998). In C57BL/6J mice, similar MDMA doses produced hyperthermia at room temperatures below 22 ºC and hypothermia at 15ºC conditions (Miller and O'Callaghan,

1994). Conversely, rhesus monkeys displayed a similar hyperthermic response to MDMA (0.56-

2.4 mg/kg) over a wide range of ambient temperatures (18 - 30ºC) (Von Huben et al., 2007), suggestive of a more tightly controlled thermoregulatory response in primates, as in the case of humans.

The thermogenic effects elicited by MDMA in humans and experimental animals are orchestrated by activation of the sympathetic nervous system (SNS) and stimulation of the hypothalamic-pituitary-thyroid axis (Mills et al., 2004). The influence of ambient temperature on

MDMA-induced hyperthermia in rodents indicates that a central deregulatory mechanism is involved in this process, presumably mediated by excess monoamine release (Gordon et al.,

1991). Thermoregulation occurs within the hypothalamus, and is directly controlled by neurotransmitter pathways, primarily involving 5-HT, DA, and NE (Cox and Lee, 1980; Mallick et al., 2002; Rothwell, 1994). Direct administration of 5-HT into the anterior hypothalamus increases body temperature (Gordon et al., 1991), whereas, DA microinjection yields the opposite effect (Clark and Lipton, 1985). MDMA behaves as an indirect monoamine agonist, causing the release of neurotransmitters in several regions of the brain, including thermoregulatory centers

(Gudelsky and Nash, 1996; Mechan et al., 2002; Shioda et al., 2008). Furthermore, increases in c-fos expression in the supraoptic and median optic nucleus of the hypothalamus upon MDMA treatment, indicates activation of the hypothalamic axis (Stephenson et al., 1999). 36

Much like MDMA, other potent 5-HT releasing compounds, such as p- chloroamphetamine, are capable of inducing hyperthermia in rats (Colado et al., 1993).

Moreover, persistent indoleamine (5-HT and 5-hydroxyindoleacetic acid, 5-HIAA) deficits and morphological damage to 5-HT neurons occur in the hypothalamus with MDMA neurotoxicity

(Commins et al., 1987; O'Hearn et al., 1988) along with long-term defects in thermoregulation

(Mechan et al., 2001). Altogether, these findings support the hypothesis that 5-HT is a key mediator of the MDMA thermoregulatory response. The role of central 5-HT release in MDMA- induced hyperthermia has been further established by pharmacological manipulation of post- synaptic 5-HT receptors. A selective 5-HT2 receptor antagonist, MDL11939, blocks MDMA- mediated hyperthermia (Mechan et al., 2002; Schmidt and Taylor, 1990), and pretreatment with other non-selective 5-HT2A, antagonists (ketanserin, risperidone or R96544) produces a similar effect. In contrast, antagonism of 5-HT1A, 5-HT2B/2C and 5-HT2C receptors fails to attenuate MDMA- induced hyperthermia (Shioda et al., 2008). Thus, it can assumed that 5-HT2A activation is particularly important to the mechanism of MDMA-mediated thermoregulation.

Other studies argue that 5-HT release plays only a minor role in MDMA-induced hyperthermia, highlighting the fact that non-selective 5-HT2A antagonists show substantial inhibitory activity at other receptors (Mechan et al., 2002). Indeed, inhibition of dopaminergic receptors with selective D1 antagonists reverses MDMA-mediated hyperthermia, leading to the conclusion that enhanced DA release is involved in such process via modulation of D1 receptors

(Mechan et al., 2002). DA release is involved in the thermogenic response to methamphetamine

(Bronstein and Hong, 1995), therefore, it is not surprising that blockade of D1 receptors has profound effects on MDMA-induced hyperthermia. 37

Although the precise role that 5-HT and DA play in MDMA thermoregulation remains a topic of much debate, it is evident that alterations in the levels of these biogenic amines are central to this process. Part of the controversy surrounding these studies arises from the promiscuity of pharmacological inhibitors that show activity at multiple post-synaptic receptors.

Chapter 3 examines the involvement of SERT in MDMA-mediated hyperthermia in SERT-deficient rats. Characterization of the SERT-KO rat model showed that neurochemical changes in these animals were limited to the serotonergic neurotransmitter system (Homberg et al., 2007), thus providing a unique tool to study the effects of disturbed 5-HT neurotransmission in MDMA thermoregulation. Similarly, contributions of vesicular monoamine stores to the hyperthermic response of MDMA are evaluated in Chapter 4 via inhibition of the VMAT2 protein.

Activation of the SNS presumably follows central neurotransmitter release in the hypothalamus during MDMA thermogenesis (Mills et al., 2004). Thermoregulation in warm- blooded animals, including humans, requires a balance between heat generation and dissipation

(Mills et al., 2004). Heat production occurs primarily in skeletal muscle in humans (Lin and

Klingenberg, 1980) but, in rodents, brown adipose tissue plays a major role (Smith and Horwitz,

1969). Both of these tissues have the capability of prominent heat production through the activation of uncoupling proteins (UCP) that induce proton leakage into the inner mitochondrial membrane, thus, “uncoupling” mitochondrial respiration from ATP synthesis (Brand et al., 1994;

Rolfe and Brand, 1996). During mitochondrial respiration, the inner membrane electrochemical gradient created from reduction/oxidation (redox) reactions of electrons is used for the oxidative phosphorylation of ADP to ATP. Alternatively, this free energy is released by proton leakage to generate heat during periods of excessive supply of energy to prevent ROS accumulation from mitochondrial redox reactions (St-Pierre et al., 2002). The process of generating heat via UCP 38

activation to regulate body temperature is referred to as facultative thermogenesis, and it is rapidly induced in response to external stimuli such as cold ambient temperatures or excess food intake (Himms-Hagen et al., 1978).

MDMA-mediated hyperthermia resembles facultative thermogenesis as both mechanisms involve input from the hypothalamus and the SNS (Mills et al., 2004). UCP3 is expressed at high levels in skeletal muscle cells, therefore, its induction is important in driving thermogenesis. MDMA is known to raise temperature in skeletal muscle (Sprague et al., 2003a), and following MDMA challenge, mice deficient in UCP3 (UCP3-/) are resistant to hyperthermia and acute lethality compared to their wildtype counterparts (Mills et al., 2003). Moreover, MDMA increases skeletal muscle lysis as a consequence of excessive coupling, resulting in rhabdomyolysis, and renal failure as evidenced by elevations in blood urea nitrogen and serum creatine kinase in rats and humans (Cunningham, 1997; Sprague et al., 2004). These studies indicate that MDMA is perhaps capable of activating UCPs and uncoupling oxidative phosphorylation to initiate thermogenesis. Despite inducing ATP depletion in brain and skeletal muscle tissue (Brown and Yamamoto, 2003; Rusyniak et al., 2005), the direct effects of MDMA on mitochondrial respiration are only minor compared to the established uncoupling agent, dinitrophenol, which emphasizes the participation of additional mechanisms in MDMA thermogenesis (Rusyniak et al., 2004). Indeed, body temperature regulation also involves cutaneous vascular control in order to mediate heat loss. MDMA increases both sympathetic nerve activity and vasoconstriction during acute hyperthermia, thereby reducing blood flow to the pinna in rabbits, and to the tail in rats (Blessing et al., 2003; Pedersen and Blessing, 2001).

NE release is at the center of the peripheral thermoregulatory response to MDMA through the induction of heat production and prevention of heat loss. MDMA is an indirect NE 39

agonist (Rothman et al., 2001) and marked increases in NE plasma levels occur in rats and humans following MDMA administration (Sprague et al., 2005; Stuerenburg et al., 2002). Moreover, antagonism of α1- and β3- adrenoceptors reduces MDMA-mediated hyperthermia and rhabdomyolysis in rats (Sprague et al., 2004). NE presumably modulates mitochondrial uncoupling through activation of these receptors during MDMA thermogenesis (Mills et al., 2003). Indeed,

β3- adrenoceptors are present in skeletal muscle, where they regulate the functional expression of UCP3 (Nakamura et al., 2001). α1- Adrenoceptors are also involved in MDMA-mediated vasoconstriction, seemingly preventing dissipation of heat that is produced by UCP3 induction

(Sprague et al., 2003a). Finally, it is speculated that thyroid hormone contributes to UCP3 activation, further enhancing the hyperthermic effects of MDMA, since chronic hyperthyroidism potentiates hyperthermia and hypothyroidism produces intense hypothermia in MDMA-treated rats (Sprague et al., 2007). A detailed summary of the peripheral mechanisms of MDMA-mediated thermogenesis is presented in Figure 1-4. 40 41

Figure 1-4 - Putative Mechanisms of MDMA-Mediated Thermogenesis

Hyperthermia induced by MDMA requires concomitant activation of the hypothalamic-pituitary- adrenal axis and the SNS. MDMA-mediated monoamine release stimulates the hypothalamus and the SNS, causing an increase in plasma NE levels. Subsequently, NE activates α1 and β3 adrenoceptors in SKM cells, elevating intracellular Ca2+ concentrations and increasing the formation of FFA from TG metabolism. Both events induce mitochondrial uncoupling via activation of UCPs (in particular UCP3), which increases heat production. Thyroid hormone is also elevated following MDMA exposure and assists in the thermogenic process through the regulation of UCP gene and protein expression. Additionally, NE prevents heat dissipation by stimulating vasoconstriction through α1 receptor agonism. Together, heat production and loss of heat dissipation cause elevations in body temperature that can lead to hyperthermia. Adapted from (Mills et al., 2004).

Abbreviations: SNS, sympathetic nervous system; SKM, skeletal muscle; PLC, phospholipase C; PIP2, phosphatidylinositol 4, 5-bisphosphate; IP3, inositol trisphosphate; cAMP, cyclic monophosphate; ATP, adenosine trisphosphate; PKA, protein kinase A; FFA, free fatty acids; TG, triglycerides; UCP, uncoupling protein. 42

1.4.2 The 5-HT behavioral syndrome

In addition to hyperthermia, MDMA induces a number of acute, behavioral changes in experimental animals that are referred to as the 5-HT behavioral syndrome. In rodents, the characteristics of this syndrome include hyperactivity, reciprocal forepaw treading, headweaving, hind limb abduction, proptosis, ataxia, penile erection, ejaculation, salivation, defecation, convulsions and death (Spanos and Yamamoto, 1989). The 5-HT syndrome was initially described after concomitant administration of a MAO inhibitor and L-tryptophan to rats, which resulted in the elevation of brain 5-HT levels and all major features described above (Grahame-Smith, 1971).

Subsequent studies have shown similar results upon treatment with other psychostimulants such as p-chloroamphetamine (Green and Kelly, 1976). All these compounds share the ability to cause significant 5-HT release, thus, 5-HT was presumed to be involved in the behavioral changes caused by acute MDMA exposure.

The major feature of the 5-HT behavioral syndrome, and also the most studied, is hyperactivity. MDMA induces significant increases in locomotion in both rats and mice, whereas, nonhuman primates display minimal to no changes in locomotor activity with MDMA (Taffe et al.,

2006; Von Huben et al., 2007). Volunteer subjects report high incidence of increased energy and hyperactivity with recreational MDMA doses (75-150 mg) under controlled laboratory settings

(Baylen and Rosenberg, 2006). Moreover, the 5-HT syndrome triggered by MDMA intoxication manifests differently in humans than in rodents, causing confusion, diaphoresis, cardiovascular complications, increased muscle tone, hyperthermia, tremor and myoclonus (Hall and Henry,

2006). The behavioral response elicited by MDMA differs between species, and also from that of other psychostimulant drugs. For instance, MDMA and amphetamine induce a similar degree of hyperactivity in rodents, whereas, the locomotor and behavioral patterns associated with these 43

two drugs are very distinctive, accentuating differences in their pharmacological mechanism of action (Risbrough et al., 2006).

Amphetamine-mediated hyperlocomotor activity is associated with enhanced DA neurotransmission. The role of DA in locomotion is well established, as evidenced by the increase in locomotor activity with microinjection of DA into the nucleus accumbens of rats (Pijnenburg et al., 1976) and through activation of D1 and D2 receptors with agonist treatment (Salmi et al.,

1998). Moreover, central and systemic administration of pharmacological antagonists of D1 and

D2 receptors are capable of preventing amphetamine-mediated hyperactivity to a similar extent as the selective dopaminergic neurotoxicant, 6-hydroxydopamine (6-OHDA) (Bardo et al., 1999;

Depoortere et al., 2003; Kelly et al., 1975). Much like amphetamine, MDMA induces DA release in the caudate and nucleus accumbens (Yamamoto and Spanos, 1988), areas that compromise the classical “extrapyramidal” motor system. Contrary to amphetamine, this effect is mediated by both nerve impulse and uptake transporter processes (Iravani et al., 2000; Koch and Galloway,

1997). Additionally, MDMA is more potent at releasing 5-HT than DA when compared to amphetamine (Crespi et al., 1997), which suggest that 5-HT is also important to MDMA-induced hyperactivity. Differences in the pharmacology of these drugs are in part responsible for incongruences in their behavioral profile. MDMA stimulates hyperlocomotion in rodents, whilst simultaneously inducing thigmotaxis and reducing exploratory behavior, which manifests as an avoidance of central areas and decreased rearing activity in open-field tests (Gold et al., 1988).

The thigmotaxic properties of MDMA are similar to the behavioral response observed with hallucinogens (Bankson and Cunningham, 2001). Furthermore, the behavioral patterns of MDMA- treated animals exhibit a smoother appearance and include fewer directional changes (Powell et 44

al., 2004). Conversely, amphetamine induces locomotion over the entirety of the field in activity chambers (Gold et al., 1989).

Pharmacological inhibition of SERT showed reductions in MDMA-mediated hyperactivity

(Callaway et al., 1990). 5-HT2A and 5-HT1B receptor antagonists block floor-plane locomotor activity stimulation in MDMA-treated rats; however, suppression of vertical activity (rearing) was unaffected (Kehne et al., 1996; McCreary et al., 1999). The findings indicate that agonism of serotonergic receptors presumably subsequent to increases in 5-HT release is in part responsible for mediating the behavioral effects of MDMA. Similarly, the involvement of dopaminergic pathways in the locomotor effects of MDMA is undeniable. Some studies report that DA efflux into the striatum after systemic MDMA administration is greater or comparable to that of 5-HT

(White et al., 1996). Evidence for the role of DA in the behavioral response to MDMA came from a study by Gold et al. (1989) that demonstrated attenuation of locomotor activity in 6-OHDA- treated rats, which exhibited severe neurotoxicity in the mesolimbic dopaminergic system. In agreement with these results, antagonism of D1 and D2 receptors causes partial inhibition of the hyperlocomotor effects of MDMA, indicating that activation of these receptors follows enhanced

DA release (Bubar et al., 2004). Furthermore, a differential role for these receptors in MDMA- mediated hyperactivity highlighted the involvement of the D1 receptors in linear locomotor patterns, and the importance of D2 receptors to the induction of repetitive circumscribed movements observed with high MDMA doses (30 mg/kg) (Risbrough et al., 2006).

Overall, the data indicate that induction of locomotor activity by MDMA involves a complex interaction between serotonergic and dopaminergic neurotransmission, seemingly orchestrated by the release of 5-HT and DA into the striatum. Work undertaken in this dissertation analyzes the effects of decreased SERT and VMAT2 activity in MDMA-induced hyperactivity. The 45

results from such studies further our understanding of the importance of monoamine transporter-mediated mechanisms in the behavioral response to MDMA.

1.5 MECHANISMS OF LONG-TERM MDMA NEUROTOXICITY

1.5.1 General Comments on Neurotoxicity

Neurotoxicity is classically defined as an adverse reaction that causes both structural and functional damage to the central nervous system (CNS) and/or the peripheral nervous system upon exposure to a specific chemical or physical agent (Tilson, 1990). Much disagreement exists on what constitutes an adverse reaction, however, in general it is considered a direct or indirect change from baseline that threatens an organism’s survival, reproduction and ability to adapt to environmental conditions (Ladefoged et al., 1995; US EPA, 1998). Structural neurotoxic effects manifest as morphological changes in neuroanatomy that occur at any level of CNS organization, whereas functional neurotoxic effects refer to alterations in neurophysiology or behavior, including adverse changes in somatic/autonomic, sensory, motor and cognitive function (WHO,

2001). Interestingly, toxicological and mechanistic analysis of the adverse effects of neurotoxicants have yielded relevant information towards the understanding of neuronal pathways and overall brain function. This knowledge has helped in the identification of potential pharmacological targets for the treatment of neurological disorders.

The capacity of MDMA to inflict serotonergic neuronal damage remains a controversial topic. The observations that MDMA induces axonal degeneration of 5-HT neurons without affecting cell morphology in the dorsal raphe nucleus of rats initiated the first arguments against the potential neurotoxic effects of this psychostimulant drug (O'Hearn et al., 1988; Paris and

Cunningham, 1992). Cell death is considered a hallmark of neurotoxicity (WHO, 2001) but many 46

examples exist of compounds that inflict functional damage to neurons, or other cells in the CNS, in the absence of cell loss, including pesticides and heavy metals such as aluminum (Sanchez-

Santed et al., 2004; Sethi et al., 2009). Standard techniques often employed to show the presence of neurotoxicity include markers of cell death, silver staining and gliosis (Baumgarten and

Lachenmayer, 2004; O'Callaghan and Sriram, 2005; Switzer, 2000). Silver staining is a sensitive marker of cell damage, and dose-dependent increases in silver-positive staining in the parietal cortex, striatum and thalamus have been observed after administration of high MDMA doses (80-

600 mg/kg) (Commins et al., 1987; Jensen et al., 1993). Critics argue that such MDMA doses are

16-100 times higher than recreational human doses, and that the silver staining pattern induced in these studies is largely unspecific, making the findings irrelevant to human exposures (Baumann et al., 2007).

CNS damage is often accompanied by hypertrophy of astrocytes termed “reactive gliosis” and it is distinguished by an increase in expression of glial fibrillary acid protein (GFAP)

(O'Callaghan et al., 1995). High MDMA (75-150 mg/kg) doses lead to significant stimulation of

GFAP levels in relevant brain regions, however, these increases do not correlate with tissue indoleamine deficits or with the degree of GFAP activation elicited by the well-known 5-HT neurotoxicant, 5,7-dihydroxytryptamine (5,7-DHT) (O'Callaghan and Miller, 1993). Comparative studies with MDMA and methamphetamine in SD rats revealed significant reactive gliosis alongside stimulation of microglial markers such as peripheral benzodiazepine receptors and the

OX-6 protein (a marker for class II histocompatibility complex, MHC-2) after methamphetamine treatment (Pubill et al., 2003). Interestingly, enhanced gliosis and microglial activation were absent following MDMA exposure (Pubill et al., 2003). In contrast, icv administration of microglial factor, interleukin 1β (IL-β), exacerbates MDMA-mediated hyperthermia and subsequent 47

serotonergic neurotoxicity (O'Shea et al., 2005b), supporting the involvement of reactive microglia in the neurotoxic response to MDMA.

Another major point of debate in regards to the classification of MDMA as a neurotoxicant is the fact SSRIs produce adverse effects that are similar to the long-lasting neurotoxicity of

MDMA; however, SSRIs are not considered to be neurotoxic. SSRIs are used in the treatment of depression, anxiety, and other personality disorders, and chronic administration of these therapeutic drugs leads to impairment of SERT function, indicated by the inability of the SSRI, fluvoxamine, to modulate SERT-mediated 5-HT clearance from the extracellular space

(Benmansour et al., 1999). It should be noted that the authors reported a lack of tissue 5-HT depletion in SSRI-treated animals, an endpoint marker of toxicity often used to assess the neurotoxic response to MDMA (Benmansour et al., 1999). High doses of SSRIs also induce structural changes to 5-HT nerve terminals, including swelling, fragmentation, and growth abnormalities (Kalia et al., 2000). Unlike morphological damage caused by MDMA, these effects are only transitory, completely dissipating 30 days after treatment. Overall, the aforementioned caveats raise important questions about the categorization of MDMA as a “true neurotoxicant.”

Nevertheless, the amount of evidence in support of the neurotoxic effects of this drug is considerable and the findings are detailed in the next two sections.

1.5.2 MDMA Neurotoxicity in Experimental Animals

The long-term effects of MDMA induce adverse changes in markers of 5-HT nerve terminal integrity in rats (Commins et al., 1987; Schmidt, 1987) and non-human primates (Bowyer et al., 2003; Fischer et al., 1995). One of the hallmarks of MDMA neurotoxicity includes depletion of tissue 5-HT and 5-HIAA levels in serotonergic-rich regions in particular rat neocortex, striatum and hippocampus are vulnerable to these changes. The evaluation of neurochemical markers of 48

MDMA neurotoxicity in rats requires special consideration of strain differences, due to the variability in sensitivities to both the acute and long-term effects of MDMA. For instance, Dark-

Agouti rats require a single dose of MDMA (10 mg/kg) to produce substantial reductions in cerebral 5-HT of 30-50% (O'Shea et al., 1998), whereas, induction of MDMA neurotoxicity in

Wistar and SD rats is often accomplished by multiple drug doses (Aguirre et al., 1998; Shankaran and Gudelsky, 1999).

Tissue indoleamine deficits are thought to be reflective of neurotoxic damage to pre- synaptic nerve endings. In the vast majority of research studies, these neurochemical effects are evaluated 7-14 days post-drug administration and are often used to assess the extent of MDMA neurotoxicity (Capela et al., 2009). Examination of tissue 5-HT levels reveals a biphasic pattern of indoleamine depletion during MDMA neurotoxicity (Schmidt, 1987). Rat cortical 5-HT concentrations significantly decline to 16% of control values between 3 to 6 h post-MDMA administration (10 mg/kg), followed by complete recovery to baseline levels by 24 h. During the second phase, 5-HT levels gradually decline between 1 to 7 days after treatment, to 74% of control values (Schmidt, 1987). Moreover, the second phase of 5-HT depletion constitutes the long-lasting effect of MDMA as demonstrated by De Souza et al. (1990). Drastic decreases in 5-HT content occurred in the frontal cerebral cortex following repeated MDMA exposure (20 mg/kg, twice daily for 4 days), and levels remained below control rats (40-50%) for up to 12 months (De Souza et al.,

1990). The authors concluded that persistent 5-HT depletion after MDMA treatment was indicative of impaired functionality of 5-HT neurons.

MDMA-induced 5-HT deficits are accompanied by the loss of SERT binding sites. SERT is located in the plasma membrane of 5-HT terminals, therefore, its depletion is thought to reflect axonal degeneration of nerve endings mediated by MDMA. SERT reduction is demonstrated by 49

decreases in [3H]paroxetine binding in cortex and hippocampus of rats that persist for up to 32 weeks after MDMA treatment (O'Shea et al., 2006). Similarly, peripheral MDMA administration causes acute and long-term depression of tryptophan hydroxylase (TPH) activity, the rate-limiting enzyme involved in the synthesis of 5-HT (Schmidt and Taylor, 1987). Interestingly, in-vitro TPH activity is unaffected by MDMA (Schmidt and Taylor, 1987), suggesting that in-vivo loss of TPH function is indirectly modulated by MDMA. Furthermore, decreases in TPH activity after a single

MDMA dose are restored by prolonged incubation with dithiothreitol and Fe2+, whereas, restoration of TPH activity under similar conditions, could not be accomplished after a multiple- dose MDMA regimen (Stone et al., 1989). The findings indicate that reversible inhibition of enzymatic function after a single dose of MDMA is likely due to oxidation of sulfhydryl sites within the TPH protein. More importantly, multiple exposure to MDMA induces an irreversible inhibition of TPH activity that is reflective of 5-HT neurotoxicity (Stone et al., 1989).

MDMA-mediated neurodegeneration of 5-HT neurons occurs alongside neurochemical changes. Immunohistochemical analysis utilizing 5-HT, SERT and TPH antibodies confirmed adverse histological changes to neurons in the 5-HT system. Gross depletions in 5-HT immunoreactivity were observed in the brains of MDMA-treated rats, indicative of substantial reductions in 5-HT axonal density (O'Hearn et al., 1988). Regional differences in 5-HT staining revealed that the neocortex, striatum and thamalus sustained the greatest 5-HT loss, with partial sparing of 5-HT axons noted in the hippocampus, lateral hypothalamus and basal forebrain.

Serotonergic nerve terminals were selectively vulnerable to MDMA-mediated neurotoxicity, displaying reduced density of fine, arborized 5-HT axons, and sparing of smooth, straight preterminal fibers, whilst, axons of passage and raphe cell bodies were completely unaffected

(O'Hearn et al., 1988). The phenomenon of MDMA-mediated axotomy of 5-HT terminals, with 50

sparing of cell bodies, is referred to as a “pruning effect” or “retrograde degeneration,” which also occurs during 5,7-DHT serotonergic neurotoxicity (Capela et al., 2008).

Long-lasting functional deficits after MDMA treatment are well documented, further supporting the idea of 5-HT axonal neurodegeneration. Thus, MDMA causes significant decreases in anterograde axonal transport of radiolabeled proline along axonal projections originating in the raphe nuclei 3 weeks after drug-treatment (Callahan et al., 2001). Reduction of anterograde axonal transport is also observed with 5,7-DHT treatment, and parallels MDMA-induced depletion of regional 5-HT and 5-HIAA levels, providing evidence of functional defects after severe axonal injury (Callahan et al., 2001). Furthermore, rats exposed to a high ambient temperature challenge

(30 ºC for 60 min) 4 and 14 weeks after repeated MDMA administration (10mg/kg, daily for 4 days) displayed a prolonged hyperthermic response (Dafters and Lynch, 1998). In contrast, resistance to MDMA-induced hyperthermia and hyperlocomotor activity was observed in rats previously treated with a neurotoxic regimen of MDMA (10 mg/kg, 4x every 2 h) (Shankaran and

Gudelsky, 1999). It can be speculated that persistent impairments in thermoregulation and behavior in response to MDMA correspond to the axotomy of 5-HT neurons in the hypothalamus and striatum, respectively.

Further evaluation of the long-term effects of MDMA revealed reductions in baseline levels of dialysate 5-HIAA, accompanied by attenuation in 5-HT release in response to physiological and pharmacological stimuli (Series et al., 1994). Additionally, memory and learning along with social interaction skills can be impacted long after initial MDMA challenge. Adolescent exposure to MDMA in rats, at postnatal days (PND) 35-60, altered open-field habituation and reduced attention in working memory tests together with the decrease in 5-HT uptake sites in the neocortex (Piper et al., 2005). Similarly, exposure of young Wistar rats (PND 28) to MDMA 51

increased anxiety and reduced social interaction during the abstinence period, and produced a long-lasting reduction in sensitivity to 5-HT2A-mediated behavior (Bull et al., 2004). MDMA treatment also affected cognition in adult rats, measured by the delayed non-match to place performance test, which coincided with 5-HT deficits in cortex, hippocampus and striatum during post-mortem analysis (Marston et al., 1999). Indeed, long-term disruption of sequential and spatial memory learning caused by MDMA is attributed to 5-HT deficits in the hippocampal brain region (Sprague et al., 2003b).

1.5.3 Evidence of Serotonergic Neurotoxicity in Ecstasy Users

Evidence of long-term MDMA neurotoxicity in humans is limited yet convincing. Severe depletion of striatal 5-HT and 5-HIAA levels (50-80%) were found during post-mortem analysis of a frequent ecstasy user (Kish et al., 2000). Although the subject reportedly abused cocaine and heroin months prior to his demise, tissue indoleamine deficits were correlated with chronic

MDMA consumption, given that no other adverse neurochemical effects were detected (Kish et al., 2000). Chronic ecstasy users exhibited lower levels of 5-HIAA in the cerebral spinal fluid (CSF) compared to naïve controls and such reductions were more drastic in female subjects compared to males (McCann et al., 1999; McCann et al., 1994). Of note, CSF 5-HIAA content was negatively correlated to MDMA consumption (Bolla et al., 1998). Furthermore, structural magnetic resonance imaging scans revealed reduced gray matter density in the neocortex, bilateral cerebellum, and midline brainstem of MDMA polydrug users, perhaps reflective of the previously diagnosed psychiatric disorders in these subjects (Cowan et al., 2003). Other neuroimaging studies demonstrated lower density of 5-HT uptake sites in ecstasy users, which again correlated with the intensity of MDMA consumption (McCann et al., 1998; McCann et al., 2005). Many studies have demonstrated long-term functional impairments in memory and learning along with 52

increased incidence of depression, panic disorder, and behavioral alterations with chronic MDMA abuse (Creighton et al., 1991; McGuire, 2000; Rodgers, 2000). Functional deficits in humans often paralleled with markers of 5-HT neurotoxicity (as reviewed by (Capela et al., 2009)).

1.5.4 Molecular Mechanisms of MDMA-Mediated Neurotoxicity

1.5.4.1 Oxidative Stress

The extent of MDMA 5-HT neurotoxicity can be influenced by many factors including metabolism (Monks et al., 2004), enhanced carrier-mediated efflux of neurotransmitters (O'Shea et al., 2005a) and modulation of the acute hyperthermic response (Green et al., 2004). Although the mechanisms underlying the long-lasting neurotoxic effects of MDMA appear to be complex and multifactorial, accumulating evidence suggests a role for oxidative stress in MDMA-induced neuronal injury. Oxidative stress is induced when there is an imbalance in the production of ROS and in the antioxidant defense (Gandhi and Abramov, 2012). Brain tissue is particularly susceptible to oxidative stress-related damage, due to its high oxygen consumption and the abundance of highly peroxidisable substrates (Halliwell, 2006). Indeed, the brain accounts for only

2% of the total body weight, but it consumes 20% of the total oxygen, mostly distributed to neurons and astrocytes (Halliwell, 2006). Not surprisingly, increased production of ROS and subsequent mitochondrial dysfunction have been implicated in the pathogenesis of late onset neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease (Lin and Beal,

2006).

Oxygen possess a high redox potential that allows it to act as an oxidizing agent by accepting electrons from reduced substrates. Despite being highly reactive, oxygen is kinetically stable due to the identical spin states of its two outer orbital electrons (Halliwell, 2006). However 53

under appropriate catalytic conditions, oxygen is partially reduced to oxygen radicals such as

●- ● superoxide (O2 ), and hydroxyl radical (OH ). ROS encompasses both oxygen radicals and non-

1 radicals, including ozone, hydrogen peroxide, and singlet oxygen (O3, H2O2, O2, respectively)

(Halliwell, 2006). Due to the highly reactive nature of these ROS, they can interact with biological macromolecules and alter cellular function, leading to cell death. Excessive ROS results in protein,

DNA and RNA oxidation, and lipid peroxidation, with the subsequent formation of reactive aldehydes such as malondialdehyde and 4-hydroxynonenal, DNA-protein crosslinks, and DNA mutations (Gandhi and Abramov, 2012). Therefore, antioxidant systems are essential for the integrity and survival of neurons, helping them cope with a high oxygen consumption, a long life- span, and the prominent role of nitric oxide in neurotransmission (Droge, 2002). Nitric oxide can form reactive nitrogen species (RNS) in combination with oxygen that nitrate tyrosine residues of proteins, modifying their function and creating oxidative damage (Gandhi and Abramov, 2012).

Figure 1-5 displays the main chemical reactions leading to the production of ROS in the CNS.

The free radical hypothesis of amphetamine-mediated neurotoxicity dates to the 1980s, when Stone et al. (1989) reportedly reversed inactivation of tryptophan hydroxylase activity by

MDMA upon incubation with sulfhydryl-reducing compounds. Hydroxyl radical formation is elevated following systemic MDMA administration, as demonstrated by elevations in extracellular

2,3-dihydroxybenzoic acid (2,3-DHBA) in the striatum and hippocampus of rats (Colado et al.,

1997b; Gudelsky, 1994). Detection of stable hydroxyl adducts of salicylic acid and D- phenylalanine in rat brain is possible after repeated MDMA dosing (10mg/kg, 4x every 2 h)

(Shankaran et al., 1999b). Lipid peroxidation is enhanced in adult female rats in cortical tissue 3 and 6 h subsequent to MDMA exposure. In contrast, fetal rat brain is devoid of such effects, perhaps due to the lower metabolic capabilities of the immature brain (Colado et al., 1997a). 54

These findings suggest a role for metabolism in MDMA-induced ROS production. Furthermore, injection of the spin trap reagent α-phenyl-N-tert-butyl nitrone to Dark Agouti rats prevents

MDMA serotonergic neurotoxicity as evidenced by attenuation of tissue indoleamine deficits and depletion of 5-HT uptake sites in cortex and hippocampus (Colado and Green, 1995).

Neurodegeneration of 5-HT terminals by MDMA was subsequently shown to be reversed with antioxidant treatment, such as sodium ascorbate or L-cysteine (Gudelsky, 1996). Acute hyperthermia has also been implicated in the mechanism of long-term MDMA neurotoxicity

(Broening et al., 1995), seemingly through stimulation of free radical formation and oxidative damage (Colado et al., 1999).

A variety of oxidative stress sources have been identified in the mechanism of MDMA- induced ROS production. Several studies highlight the role of MAO enzymes in the increase of brain hydroxyl radicals following MDMA exposure. Elevation of the cytoplasmic monoamine pool inside 5-HT nerve terminals during the acute pharmacological MDMA response exposes neurotransmitters to MAO-mediated metabolism, perhaps leading to excessive production of

H2O2 and reactive aldehyde intermediates that could be deleterious for neurons (Nagatsu, 2004).

Increases in ROS burden, as a consequence of MAO metabolism, was confirmed upon inhibition of MAOB with L-deprenyl pretreatment (Sprague and Nichols, 1995b). Oxidative stress and membrane lipid peroxidation were attenuated, along with MDMA-mediated tissue 5-HT depletion, and decreases in [3H]paroxetine binding (Sprague and Nichols, 1995b). Similarly, continuous icv infusion of antisense RNA targeted at MAOB resulted in 63% reduction in enzymatic activity and protection against MDMA serotonergic neurotoxicity, without affecting acute hyperthermia 55

Since MAOB resides in the outer membrane of the mitochondria, it is speculated that ROS production derived from MAOB metabolism directly impacts mitochondrial function, further promoting oxidative stress damage to 5-HT nerve endings (Alves et al., 2007). Indeed, binge

MDMA administration (10 mg/kg, 4x, 2 h apart) to male Wistar rats results in long-lasting mitochondrial impairment, including significant elevations in lipid peroxide levels and protein carbonyls. Alterations in mitochondrial DNA encoding for the NDI and NDII subunits of the mitochondrial complex I (NADH dehydrogenase) and COXI of complex IV (cytochrome c oxidase) were also observed (Alves et al., 2007). Moreover, MAOB inhibition by L-deprenyl completely abolished oxidative damage to mitochondrial macromolecules (Alves et al., 2007), suggesting

MAOB’s contribution to mitochondrial injury during long-term MDMA neurotoxicity. A direct correlation between mitochondrial complex I inhibition, accompanied by an increase in superoxide radicals, and MDMA-mediated dopaminergic neurotoxicity in mice confirmed this hypothesis (Puerta et al., 2010).

MAO-related metabolism and mitochondrial dysfunction are considered essential to the

MDMA-induced oxidative stress response. However, other prominent sources of ROS have been identified following systemic MDMA administration, including prostaglandin H synthase (PSH)- mediated bioactivation of free radical intermediates. In vitro incubation of PSH-1 and MDMA leads to the formation of MDMA adducts and oxidative DNA damage, evidenced by an increase in 8-oxo-2’-deoxyguanosine levels (Jeng and Wells, 2010). These effects were readily reversed by

PSH-1 inhibition. Moreover, MDMA-treated PSH-1 KO mice displayed reduced DNA oxidation in the CNS and a lack of dopaminergic nerve terminal degeneration (Jeng and Wells, 2010). Excessive neuroinflammation during long-term MDMA neurotoxicity is also suspected to aid in the increase of ROS production and oxidative damage. Thus, acute MDMA exposure is capable of causing 56

aberrant microglial activation and subsequent release of IL-1β (Orio et al., 2004). Anti- inflammatory treatment with minocycline prior to MDMA dosing, prevents the increase in IL-1β release and microglial activation in the frontal cortex of Dark Agouti rats, whilst, protecting against depletion of 5-HT uptake sites.

Other relevant sources of ROS production involved in MDMA neurotoxicity include the metabolism of MDMA to reactive quinone intermediates. These quinone metabolites can undergo redox cycling, leading to the formation of semiquinone radicals, consequently increasing

ROS burden (Macedo et al., 2007). Oxidation of DA within 5-HT nerve terminals has also been suggested to play a role in the generation of free radicals and oxidative stress-related injury

(Bindoli et al., 1992; Spencer et al., 1998). These two mechanisms of ROS production are further discussed in the next sections. Undoubtedly, a variety of factors contribute to the elevation of

ROS observed with acute MDMA treatment, resulting in mitochondrial insult and oxidation of macromolecules, therefore, inducing persistent nerve terminal damage to 5-HT neurons. 57 58

Figure 1-5 - Primary Sources of ROS in the CNS

Molecular oxygen (O2) is predominantly converted to two molecules of water (H2O) during mitochondrial respiration in a four-electron reduction (1). The two-electron product generates hydrogen peroxide (H2O2). Electrons passing through the mitochondrial transport chain can leak ●- ●- and form superoxide radical anions (O2 ) upon reacting with O2. Both H2O2 and O2 can act as oxidizing and reducing reagents in different in vivo systems. A significant source of hydroxyl radicals (OH●) in the brain is the Fenton reaction. According to the Haber-Weiss equation, ferric 3+ ●- 2+ 2+ 3+ iron (Fe ) is reduced by O2 to ferrous iron (Fe ) (2), followed by the oxidation of Fe to Fe with - ● H2O2, yielding hydroxide (OH ) and OH (3). Excess intracellular calcium can induce the production of RNS, such as nitric oxide radical (NO●), by activating nitric oxide synthase (NOS), assisting in the conversion of L-arginine to L-citrulline (4). NO● can diffuse through membranes and rapidly react ●- - - with O2 , which generates peroxynitrite (ONOO ) (5). ONOO is protonated to peroxynitrous acid (ONOOH) at physiological pH, a product capable undergoing hemolytic fission in order to form ● ● nitrogen dioxide (NO2 ) and OH (5). Free radicals are highly reactive and proficient at inducing ●- oxidative damage. Formation of H2O2 can occur through spontaneous dismutation of O2 or, ●- alternatively, from the enzymatic-mediated reduction of O2 by superoxide dismutase (SOD) (6) (Forman and Azzi, 1997). Another important enzymatic reaction that is capable of elevating ROS ●- burden involves an electron transfer from NADPH to molecular oxygen, generating O2 , which is facilitated by NADPH oxidase (NOX) found abundantly in both neuronal and glial cells (Bedard and Krause, 2007). Oxidative deamination of neurotransmitters, including DA, 5-HT and NE by monoamine oxidases (MAO) are relevant sources of H2O2 generation (8). This reaction also produces reactive aldehydes in conjunction with aldehyde dehydrogenase (ALDH) (Tipton et al., 2004). 59

1.5.4.2 DA Contributions

Enhanced DA release has been long implicated in the mechanism of MDMA neurotoxicity.

MDMA induces excess DA efflux in rats by reversing DAT function and stimulating post-synaptic

5-HT2A receptors, which increases both DA synthesis and neurotransmission (Koch and Galloway,

1997; Yamamoto et al., 1995). Direct evidence of the role of DA in MDMA neurotoxicity came from studies showing that selective destruction of DA neurons in the substantia nigra, upon exposure to 6-OHDA, partially restored MDMA-mediated 5-HT deficits (Schmidt et al., 1990c;

Stone et al., 1988). Similar results were obtained by antagonizing DA synthesis with α-methyl-p- tyrosine and a decarboxylase inhibitor (Stone et al., 1988). In contrast, pretreatment with L-DOPA, which elevates DA levels, had an opposing effect, potentiating tissue 5-HT depletion following

MDMA treatment (Schmidt et al., 1990c). Indeed, obstruction of DA neurotransmission via treatment with haloperidol (DA receptor inhibitor) profoundly affected the long-lasting neurotoxic response to MDMA (Schmidt et al., 1990c).

The readily oxidizing nature of DA highlights the importance of this catecholamine in the mechanism of neurodegeneration in Parkinson’s disease (Spencer et al., 1998). DA is more prone to oxidation than most other amines, under a variety of conditions, including enzyme catalyzed,

●- metal catalysis, and by ROS such as O2 . The oxidation products of DA and other catecholamines are H2O2 and semiquinone intermediates. The latter can interact with oxygen or GSH to redox cycle and lead to the production of free radicals that can induce oxidative damage to macromolecules, compromising cell viability. Fornai et al. (2002) investigated the potential of DA release to cause striatal toxicity at post-synaptic sites in male C57 black mice exposed to MDMA.

The authors reported the occurrence of persistent ultrastructural alterations in post-synaptic

GABAergic neurons in the striatum of these animals, which manifested as whorls of concentric 60

membranes that showed increased ubiquitin immunoreactivity (Fornai et al., 2002). Ubiquitin- positive inclusions are common in diseases such as myositis (Gayathri et al., 2000) and cystic fibrosis (Johnston et al., 1998), indicative of the involvement of ubiquitin-proteasome pathways in the neurotoxicity of MDMA, presumably due to an increase in ROS burden and oxidative damage (Fornai et al., 2002). Moreover, the presence of these morphological inclusions was abolished upon treatment with drugs that reduce DA availability (Fornai et al., 2003).

An integrated hypothesis in support of the pivotal role of DA in MDMA neurotoxicity suggests that DA is transported into 5-HT nerve terminals upon MDMA-mediated monoamine release (Sprague et al., 1998). Cytosolic DA is susceptible to oxidation, leading to excess accumulation of ROS that induces a cytotoxic response. Although this hypothesis seems very plausible, it fails to address the fact that 5-HT deficits derived from MDMA treatment also occur in brain regions with minimal DA innervation, such as the hippocampus and frontal cortex

(Sprague et al., 1998). Consequently, DA alone is unlikely to account for the destruction of 5-HT axons and nerve endings at these CNS sites, indicating that other mechanisms are involved or that different mechanisms mediate the long-term neurotoxic effects of MDMA at the various target sites (Capela et al., 2009).

1.5.4.3 The Role of Quinones in MDMA Neurotoxicity

MDMA metabolism is essential to the manifestation of long-term 5-HT neurotoxicity, since although direct injection of MDMA centrally causes induction of monoamine release, it fails to reproduce the marked 5-HT deficits observed with peripheral drug administration (Esteban et al., 2001). Moreover, CYP-derived metabolites of MDMA play a direct role in the neurotoxic response to MDMA. Gollamudi et al. (1989) showed that inhibition of CYP450 activity with SKF-

525A pretreatment could attenuate MDMA-mediated 5-HT depletion in rat cortex, whereas, 61

CYP450 induction with phenobarbital enhanced such neurotoxic response. CYP-mediated demethylenation of MDA and MDMA leads to the formation of reactive catecholamine metabolites (α-MeDA and N-Me-α-MeDA, respectively) that undergo oxidation to their corresponding ortho-quinones (Patel et al., 1991). A number of conjugated quinone products of

α-MeDA and N-Me-α-MeDA have been identified in rat CNS (Jones et al., 2005; Miller et al., 1995) and human urine (Perfetti et al., 2009). In support of this view, many studies have confirmed the neurotoxic potential of quinone metabolites of MDMA when administered by icv or intrastriatal injection to rats (Jones et al., 2005; Miller et al., 1997), in addition to, their ability to induce ROS production and oxidative damage, compromising in vitro cell viability (Capela et al., 2007; Capela et al., 2006; Carvalho et al., 2002). Formation of quinone derivatives of N-Me-α-MeDA and associated ROS production is illustrated in Figure 1-6.

Quinones can act as both oxidants and electrophiles, which gives them the capacity to redox cycle and generate ROS. The electrophilic nature of quinones allows them to form covalent adducts with tissue macromolecules, causing structural damage or functional inactivation (Monks and Lau, 1997). Thus, quinones are considered extremely reactive and highly toxic to cells. The one-electron reduction of quinones yields semiquinone species that react with oxygen to form

●- ●- O2 . O2 has the potential to reduce metal ions, that in turn react with H2O2 to generate hydroxyl radicals, which are suspected to be the reactive species involved in quinone-mediated oxidative damage (Monks and Lau, 1997). Reactive quinone intermediates induce cellular damage through alkylation of proteins and DNA (Miyazaki and Asanuma, 2009). Quinone adduction involves covalent interactions with GSH and other thiol-containing compounds (Hiramatsu et al., 1990), which can lead to detoxication and subsequent elimination from the body; alternatively, preservation or enhancement of biologic (re)activity can occur during conjugation of quinone 62

electrophiles, maintaining the ability to induce oxidative stress and to arylate tissue macromolecules (Monks and Lau, 1997). For example, GSH conjugates are known to interact with enzymes, such as GSH reductase and GSH-S-transferase, at GSH binding sites or critical cysteine residues, causing irreversible inactivation of enzymatic activity (Remiao et al., 2002).

Bacterial plate assays coupled with electrochemical methods confirmed the redox cycle potential of catechol-thioether metabolites of MDMA and their ability to induce substantial ROS

-● production (Felim et al., 2007), presumably through the generation of H2O2 and O2 (Bolton et al.,

2000). N-Me-α-MeDA and its corresponding GSH and NAC conjugates conferred significant ROS- mediated toxicity to cells devoid of endogenous antioxidant defenses (OxyR-), whereas DA and its related catechol-thioether adducts were far less efficient at inducing ROS-mediated cytotoxicity

(Felim et al., 2007). Furthermore, thioether metabolites of MDMA were remarkably inefficient at inducing quinone-mediated cytotoxicity that is promoted through covalent interactions between reactive quinones and endogenous nucleophiles, suggesting that the primary mechanism of

MDMA metabolite-mediated toxicity involves ROS induction through quinone redox cycling (Felim et al., 2007).

The cytotoxicity of reactive quinones arises from their proficiency at increasing ROS.

However, mechanistic studies of DA quinone toxicity have detailed specific pathways that lead to cell death, including mitochondrial dysfunction, inflammation and proteasome impairment

(Miyazaki and Asanuma, 2009). DA oxidation generates prominent ROS production and quinone formation, capable of modifying sulfhydryl-containing compounds. Moreover, DA quinone induction causes changes in mitochondrial permeability as evidenced by an increase in swelling of brain and liver mitochondria (Berman and Hastings, 1999). Formation of DA quinones can be coupled to prostaglandin production via PSH activity, thus, DA quinone reactivity is thought to be 63

closely related to neuroinflammation (Hastings, 1995). Microarray analysis revealed increased expression of genes involved in the pro-inflammatory response in DA quinone-exposed microglia, whereas, several microglial genes with a neuroprotective role, such as purinergic receptor P2X and sestrin 1 were significantly downregulated (Kuhn et al., 2006). DA oxidation is also known to cause proteasome inactivation in neuronal PC12 cells, followed by apoptosis, both of which are prevented with GSH antioxidant treatment and by MAO and DAT antagonisms (Keller et al., 2000).

Thus, DA quinone reactivity is capable of inducing apoptotic cell death through proteasome inhibition in part by increasing ROS burden.

In summary, quinones have the capability to induce substantial ROS production and oxidative damage, leading to cell death through a variety of mechanisms. MDMA metabolism can result in the generation of several quinone species that have been shown to be cytotoxic.

Similarly, excess release of DA following MDMA administration may promote DA quinone formation and reactivity. As such, it is likely that both DA- and MDMA metabolite-derived quinones contribute to the systematic degeneration of serotonergic axons and nerve terminals during MDMA neurotoxicity. Evidence suggests that SERT is responsible for the uptake of MDMA,

MDMA metabolites and DA into 5-HT terminals. Studies in Appendix A are aimed at defining potential interactions of thioether conjugates of N-Me-α-Me-DA with SERT, providing insights into the mechanism of MDMA metabolite-induced neurotoxicity. 64 65

Figure 1-6 - N-Me-α-MeDA-Derived Quinone Formation and ROS Generation

As previously detailed, MDMA is metabolized by CYP enzymes to the catechol intermediate, N- Me-α-MeDA (1), which is readily conjugated by a number of phase II reactions. Alternatively, this catechol intermediate can oxidize to a semi-quinone specie (2) and enter redox cycling in the presence of transition metals, leading to the formation of ortho-quinones (3) and enhanced ROS production. Upon cyclization, ortho-quinones can form aminochromes (4) and other related compounds, ultimately auto-oxidizing and polymerizing into melanin-type polymers. On the other hand, ortho-quinone MDMA derivatives are susceptible to nucleophilic attack by glutathione (GSH), giving rise to 5-(GSH)-N-Me-α-MeDA (5). This thioether metabolite is readily oxidized to yield a GSH-conjugated ortho-quinone specie (7) that also undergoes redox cycle through its corresponding semi-quinone radical (6). Furthermore, the GSH-conjugated ortho-quinone is capable of accepting a second GSH molecule, forming 2,5-bis-(GSH)-N-Me-α-MeDA (8). GSH conjugates of MDMA can be metabolized via the mercapturic acid pathway, producing NAC conjugates with a similar capability to redox cycle and generate ROS (not illustrated in this figure). Adapted from (Capela et al., 2006; Gomez-Toribio et al., 2009). 66

1.5.4.4 The Involvement of Serotonin Transporters

The serotonin reuptake transporter (SERT) belongs to a large family of integral plasma membrane proteins that facilitate the uptake of endogenous biogenic amines from the extracellular space following synaptic neurotransmission (Rudnick, 1997). Comparison of transporter peptides reveals a common transmembrane topology, consisting of 12 membrane spanning segments (Schloss et al., 1992; Worrall and Williams, 1994). The extended N- and C- termini, localized in the cytosol, display putative phosphorylation sites that regulate transport activity. A long extracellular loop containing N-glycosylation sites is important for transport assembly (Blakely et al., 1994). In particular, SERT is responsible for the transport of 5-HT into neurons and peripheral cells including platelets, driven by the transmembrane exchange of NaCl and K+ (Rudnick, 1997). SERT possesses a single extracellular binding site that is simultaneously bound by Na+, Cl- and 5-HT, causing a conformational change that prevents access to the binding pocket from the exterior environment and allows access to the cytosol. After the dissociation of

5-HT and NaCl, K+ enters the same binding site, opening the access from the exterior environment

(Mitchell, 1990).

Given its role in the rapid recycling of effluxed 5-HT, SERT function is implicated in signal amplitude and duration of serotonergic synapses that modulate a variety of 5-HT-related behaviors and pathologies (Anguelova et al., 2003; Comings et al., 1999; Eddahibi et al., 1999;

Hahn and Blakely, 2002; Mynett-Johnson et al., 2000). Furthermore, SERT is a major target site for SSRIs used in the treatment of depression and other affective disorders (Schloss and Williams,

1998), and many drugs of abuse such as MDMA and cocaine (Rothman and Baumann, 2003). The stimulant properties of MDMA and other substituted amphetamines rely upon reversal of 67

monoamine transporter function for the carrier-mediated release of endogenous biogenic amines into the synaptic space (Rudnick, 1997; Rudnick and Clark, 1993).

The involvement of SERT in the long-term serotoninergic neurotoxicity of MDMA is evident by the loss of 5-HT uptake sites measured by [3H] paroxetine binding and the prominent reduction in SERT staining (Battaglia et al., 1987; Schmidt, 1987). However, a few studies have reported that MDMA causes deficits in 5-HT levels and 5-HT uptake sites without affecting SERT protein levels (Wang et al., 2005; Wang et al., 2004). Concluding remarks from these findings suggest that serotonergic depletion following MDMA exposure reflects neuroadaptive processes in 5-HT metabolism driven by SERT protein sequestration as oppose to neurotoxicity.

Nevertheless, recent western blot analysis revealed that the 70KDa band previously thought to correspond to SERT is present in SERT-KO mice and unaffected by treatment with the well- recognized serotonergic neurotoxicant, 5,7-DHT (Xie et al., 2006). Notably, these authors identified a diffuse 63-68KDa band that displays the expected regional brain distribution of SERT that is absent in SERT deficient animals and sensitive to 5,7-DHT and amphetamine treatment.

The authors also demonstrated depletion of 5-HT axonal density with immunohistochemical SERT analysis in parallel with the loss of brain SERT protein after MDMA administration. Thus, the results provide validation that MDMA-induced deficits in 5-HT and SERT levels are likely the consequence of damage inflicted to 5-HT nerve terminals.

Prevention of MDMA-mediated 5-HT depletion with pharmacological SERT inhibition provides further evidence in support of a role for SERT in MDMA neurotoxicity. For example, fluoxetine (10 mg/kg, ip, x2, 60 min apart) treatment protected rats against long-lasting reductions in 5-HT levels and 5-HT uptake sites when administered concomitantly, or 2, 4 or even

7 days prior to MDMA dosing (Sanchez et al., 2001). The protective effects of fluoxetine 68

pretreatment were associated with the presence of fluoxetine and its active metabolite, norfluoxetine, which remained detectable in rat cortex for up 7 days. Both compounds exhibit similar affinity for SERT, acting primarily as 5-HT uptake inhibitors (Bolden-Watson and Richelson,

1993; Wong et al., 1993). In the same manner, fluoxetine administered hours before and after

MDMA treatment afforded protection against tissue indoleamine depletion and the formation of hydroxyl radicals induced by MDMA (Shankaran et al., 1999a). This latter study implicated SERT in the MDMA-mediated oxidative stress response.

Attenuation of 5-HT deficits with selective 5-HT uptake blockers highlights the importance of SERT to MDMA neurotoxicity. Nevertheless, the protective effects of fluoxetine may be a consequence of a pharmacokinetic interaction between this compound and MDMA, as opposed to a direct result of inhibiting SERT function. Much like MDMA, SSRIs are extensively metabolized in the liver by CYPs (Hemeryck and Belpaire, 2002). MDMA is primarily O-demethylenated by

CYP2D1 in rats, and fluoxetine is a potent inhibitor of this isoenzyme (Hemeryck and Belpaire,

2002). Not surprisingly, fluoxetine pretreatment impacted MDMA kinetics by elevating central and peripheral MDMA concentrations, whilst increasing the half-life of MDMA excretion (Upreti and Eddington, 2008). It is possible that fluoxetine treatment abolishes neurotoxicity by interfering with CYP-mediated metabolism of MDMA, preventing the formation of redox active metabolites with enhanced cytotoxic properties (Capela et al., 2009). Although a number of studies implicate SERT in MDMA neurotoxicity, conclusive evidence of the participation of this transporter in the neurotoxic response to MDMA is currently lacking. Experiments in Chapter 3 examine the putative attenuation of MDMA-induced 5-HT deficits in rats lacking functional SERT, as to resolve current caveats surrounding the role of SERT in MDMA neurotoxicity. 69

1.5.4.5 Contributions of Vesicular Transport to MDMA Neurotoxicity

The vesicular monoamine transporter (VMAT) is part of the major facilitator superfamily of transporters (Thiriot and Ruoho, 2001), and consists of 12 putative transmembrane domains

(Peter et al., 1996). The N- and C- termini reside within the cytosol, and a hydrophobic N- glycosylated loop extends to the vesicle lumen (Teng et al., 1998). Two different isoforms of VMAT exist: VMAT1 and VMAT2 (Erickson and Eiden, 1993). VMAT isoforms exhibit varying distribution patterns, with VMAT2 predominantly located in monoaminergic neurons in the CNS, and VMAT1 primarily expressed in neuroendocrine cells in the adrenal medulla and the intestine (Erickson et al., 1996). VMAT2 is responsible for the accumulation and storage of catecholamine and indoleamine neurotransmitters into intra-neuronal storage vesicles. This transport process is accomplished through the ubiquitous H+-ATPase, which utilizes cytosolic ATP to translocate protons into the vesicle, establishing a proton electrochemical gradient. As such, one monoamine molecule is exchanged for two intravesicular protons (Parsons, 2000).

Sequestration of biogenic amines into storage vesicles modulates the availability of monoamines for exocytosis (Fon et al., 1997). VMAT2 function is integral to monoamine activity and maintenance of neuronal health, given that aberrant VMAT2 expression profoundly compromises the viability of cells and organisms. Complete ablation of the VMAT2 protein is lethal to mice, which die only a few days after birth due to an inability to continue growing (Fon et al.,

1997; Takahashi et al., 1997; Wang et al., 1997). In contrast, VMAT2+/- mice are physiologically similar to their wildtype counterparts (Takahashi et al., 1997). Marked decreases in monoamine brain levels, vesicular uptake sites and depolarization-induced DA release occur in VMAT2 heterozygous mice (Fon et al., 1997; Takahashi et al., 1997). 70

VMAT2 deficiency studies in mice revealed susceptibility to exogenous toxicants and psychostimulant drugs perceived to be the consequence of a diminished capacity for vesicular monoamine storage (Taylor et al., 2011). As an example, VMAT2 expression affects the long-term response to methamphetamine. Reduced VMAT2 activity enhanced methamphetamine-induced neurodegeneration and astrogliosis in the striatum of VMAT2 low expression mice (VMAT2 LO), presumably through the exacerbation of DA-derived oxidative damage that occurs in the absence of vesicular DA storage (Guillot et al., 2008). VMAT2 is further implicated in the mechanism of action of psychostimulants by alterations in VMAT2 immunoreactivity and function that occur upon acute and chronic exposure to methamphetamine and cocaine (Brown et al., 2000; Brown et al., 2001; Hogan et al., 2000). Contrary to constitutive VMAT2 deficiency in VMAT2 LO mice, antagonism of vesicular transport with lobeline, a major component of tobacco, confers protection against DA depletion and decreases in VMAT2 protein following methamphetamine administration (Eyerman and Yamamoto, 2005). Disruption of monoamine storage and release subsequent to VMAT2 inhibition underlies the protective effects of lobeline against the acute and long-term toxicities of amphetamines (Dwoskin and Crooks, 2002; Miller et al., 2001; Teng et al.,

1997).

Controversy surrounds the contribution of VMAT2 function to the serotonergic neurotoxicity mediated by MDMA. Much like other amphetamines, MDMA relies upon the inhibition of VMAT2 function to elevate the cytosolic monoamine pool for subsequent synaptic release via reversal of transporter uptake activity. Inhibition of VMAT2 with reserpine pretreatment (5 mg/kg, ip) reverses long-lasting reductions in TPH activity produced by MDMA

(20mg/kg, sc) (Stone et al., 1988), indicative of prevention of serotonergic neurotoxicity. In contrast, subsequent studies revealed that persistent depression of 5-HT uptake sites following 71

MDMA (30mg/kg, sc) administration are unaltered in reserpine-treated rats, refuting the contention that vesicular monoamine stores participate in the mechanism of MDMA neurotoxicity (Hekmatpanah et al., 1989). Notably, the brain monoamine depleting properties of reserpine-only treatment consequent to the blockade of VMAT2 are sustained far beyond cessation of the acute MDMA response (Stone et al., 1988), complicating any interpretations of the effects of decreased VMAT2 activity in MDMA-induced neurotoxicity. In conclusion, the conflicting data on the role of VMAT2 in the neurotoxic effects of MDMA, warrants further investigation. Chapter 4 addresses the involvement of vesicular monoamine stores in MDMA neurotoxicity via reversible antagonism of the VMAT2 protein. Such approach is different from the irreversible inhibition of VMAT2 with reserpine, which results in a more profound and long- lasting pharmacological response (Quinn et al., 1959; Sulser et al., 1964).

1.6 DISSERTATION AIMS

The recreational use of MDMA amongst adolescents and young adults is widespread, despite its harmful effects to the peripheral and central nervous systems (Thomasius et al., 2003).

The acute neuropharmacological response to MDMA consists of an induction in central monoamine release, primarily of 5-HT, that gives rise to a wide variety of psychological and physical symptoms (Green et al., 2003; Liechti et al., 2000b; Liechti and Vollenweider, 2000).

MDMA is also an established serotonergic neurotoxicant that produces long-lasting reductions in tissue indoleamine levels, accompanied by depletion of 5-HT uptake sites and structural damage to 5-HT nerve terminals (Battaglia et al., 1987; Commins et al., 1987; O'Hearn et al., 1988). The deleterious effects of MDMA are evident in both humans and experimental animal models, warranting further investigation into the mechanism of MDMA toxicity. Studies undertaken in this dissertation were aimed at deciphering the involvement of monoamine transporter systems 72

in the acute and long-term toxicities of MDMA. An in-depth understanding of the molecular mechanisms of MDMA pharmacology and subsequent neurotoxicity could shed light on strategies for the treatment of psychostimulant abuse and lethal intoxications. Furthermore, the study of

MDMA-induced serotonergic neurotoxicity provides insight on the function of 5-HT neurons, perhaps impacting the treatment of serotonergic-mediated disorders.

MDMA, and related amphetamines, function predominantly as indirect monoamine agonists, inducing excess release of biogenic amines into the synaptic cleft, thus activating post- synaptic monoamine receptors. Amphetamines are considered substrate-type releasers due to their high binding affinity for plasma membrane transporters that facilitates the uptake of these drugs into nerve terminals, concomitantly with the release of monoamines (Partilla et al., 2006).

As such, MDMA interacts with SERT to enter 5-HT neurons, which in turn reverses transporter function and allows the exit of 5-HT molecules via a carrier-mediated exchange mechanism

(Crespi et al., 1997). Moreover, MDMA is thought to increase the cytosolic 5-HT pool that is available for SERT-mediated transport by disrupting vesicular storage as a consequence of VMAT2 inhibition (Rudnick and Clark, 1993). Given the putative role of SERT and VMAT2 in MDMA-evoked

5-HT release, we hypothesize that these proteins greatly contribute to the physiological, behavioral and neurotoxic effects of MDMA.

Chapter 3 examines the effects of SERT deficiency on MDMA toxicity using a SERT-KO rat model. SERT resides in the plasma membrane of 5-HT nerve endings, aiding in the termination of synaptic neurotransmission via reuptake of 5-HT. Consequently, SERT regulates 5-HT homeostasis and the interaction of serotonergic neurons with exogenous compounds. The present data shows abolishment of tissue indoleamine depletion in MDMA-treated SERT-KO rats, confirming previous studies with pharmacological SERT inhibitors and providing unequivocal evidence for the 73

participation of SERT in MDMA neurotoxicity. SERT-deficient rats treated with MDMA also present attenuated hyperthermia and hyperlocomotor activity, conceivably reflective of reductions in 5-

HT release.

Chapter 4 considers the effects of VMAT2 inhibition in the acute and long-term toxicities of MDMA. VMAT2 stores monoamine neurotransmitters into intra-neuronal vesicles for rapid exocytotic release during synaptic neurotransmission. This capacity has consequences in determining quantal size, receptor sensitivity and synaptic plasticity (Gasnier, 2000; Pothos et al.,

2000); therefore, VMAT2 is essential for neuronal function and for modulating the mechanism of action of psychostimulant drugs. Treatment with lobeline and its analogs inhibits VMAT2 function and antagonizes methamphetamine-induced self-administration and dopaminergic neurotoxicity in rats, resulting from disturbances in DA storage and release (Beckmann et al., 2010; Eyerman and Yamamoto, 2005). Prominent decreases in [3H]dihydrotetrabenazine (DTBZ) binding, a marker of vesicular uptake sites, occurred concomitant with the loss of SERT immnunoreactivity in non-human primates 2 weeks after exposure to a neurotoxic MDMA regimen. The data suggest that MDMA treatment is capable of inducing enduring alterations in VMAT2 density, perhaps reflective of serotonergic neurodegeneration (Ricaurte et al., 2000). We employed a pharmacological approach, inactivating VMAT2 acutely with Ro4-1284 prior to MDMA challenge.

Ro4-1284 reversibly binds to the VMAT2 protein, displaying a faster and shorter antagonistic effect relative to irreversible inhibitors, such as reserpine (Quinn et al., 1959; Sulser et al., 1964).

Pretreatment with Ro4-1284 demonstrates that compromised VMAT2 function alleviates MDMA- mediated serotonergic neurotoxicity. Similarly, antagonism of the VMAT2 protein diminishes hyperthermia and locomotor induction in rats, perhaps related to the rapid decrease in tissue monoamine levels with Ro4-1284 treatment. Nevertheless, central and peripheral monoamine 74

release following MDMA administration is unaffected by VMAT2 inactivation. Further studies are necessary to elucidate the protective mechanism of Ro4-1284 against the adverse responses to

MDMA. 75

CHAPTER 2: MATERIALS AND METHODS

2.1 MATERIALS

2.1.1 Chemicals and Reagents

3,4-(±)-MDMA hydrochloride was obtained from either Sigma Aldrich

(St. Louis, MO) or from the NIDA Drug Supply Program (Bethesda, MD). 5-HT, 5-HIAA, DA, 3,4- dihydroxyphenylacetic acid (DOPAC), NE, caffeic acid, dihydroxybenzylamine hydrobromide, Ro4-

1284, 3, 3’-diaminobenzidine (DAB), pargyline, ascorbate and HPLC solvents were all purchased from Sigma Aldrich (St. Louis, MO). Antibodies directed against ionized calcium-binding adapter molecule 1 (Iba1) were purchased from Wako Chemicals USA, Inc (Richmond, VA). Antibodies directed against SERT were purchased from Millipore (Billerica, MA). The VECTASTAIN Elite ABC kit used for antibody visualization was obtained from VectorLabs (Burlingame, CA). The MACH4™ antibody detection system was a product of BioCare Medical (Concord, CA). Dulbecco’s Modified

Eagle’s Medium buffered with Hanks Buffered Saline Solution (DMEM/F-12), DMEM: Nutrient

Mixture F-12 (DMEM/F-12), L-glutamine, geneticin, penicillin/streptomycin, N2, B27 and

Lipofectamine 2000 reagent were purchased from Life Technologies (Waltham, MA). All other chemicals were obtained from Sigma Aldrich (St. Louis, MO), Thermo Fisher Scientific (Waltham,

MA), or VWR International (Radnor, PA) unless otherwise stated and were of the highest chemical grade commercially available. N-Me-α-MeDA was synthesized by the Chemical Synthesis Facility at the University of Arizona (Tucson, AZ). 76

2.1.2 Animals and Dose Regimen

2.1.2.1 SERT Deficiency in Rats

Male Wistar wild-type (SERT+/+, WT) rats were obtained from Harlan Laboratories

(Indianapolis, IN) and SERT knockout (SERT-/-, SERT-KO) Wistar rats (both 150-200g) were purchased from GENOWAY S.A. (France). Male rats were exclusively used in this study, based on previous biochemical and functional characterization of the genetic SERT-KO male rat model

(Homberg et al., 2007). Wistar WT and SERT-KO rats received four doses of either MDMA (10 mg/kg, sc) or 0.9% saline 12 h apart. Animals were housed in groups of three per cage and maintained on a 12 h light/dark cycle. Food and water were provided ad libitum. Animals were given a week to acclimate prior to initiating experiments. Procedures were carried out in accordance with the University of Arizona Institutional Animal Care and Use Committee.

2.1.2.2 Pharmacological Inhibition of VMAT2 in Rats

Male Sprague-Dawley (SD) rats weighing ~150g were obtained from Harlan Laboratories

(Indianapolis, IN). Pharmacological inhibition of VMAT2 was achieved by Ro 4-1284 pretreatment.

Ro 4-1284 was dissolved in DMSO and administered to rats (10 mg/kg, ip). Animals dosed with either Ro 4-1284 or DMSO received a single injection of MDMA (20 mg/kg, sc) or 0.9% saline 1 h after pretreatment. Animals were housed in groups of three per cage and maintained on a 12 h light/dark cycle. Food and water were provided ad libitum. Animals were given a week to acclimate prior to initiating experiments. Procedures were carried out in accordance with the

University of Arizona Institutional Animal Care and Use Committee. 77

2.2 METHODS

2.2.1 Confirmation of VMAT2 Inhibition by Ro4-1284

Pharmacological inhibition of VMAT2 is frequently verified by measuring decreases in tissue monoamine levels (German et al., 1981; Staal and Sonsalla, 2000). Male SD rats received

Ro4-1284 (10 mg/kg, ip) and were euthanized via asphyxiation with CO2 followed by decapitation at various timepoints (1 and 12 h). Control animals were euthanized immediately following DMSO administration (0 h timepoint). Brains were removed and striatal tissue was dissected. Tissue samples were processed and analyzed for monoamine content via high performance liquid chromatography coupled to an electrochemical detector (HPLC-ECD) as detailed in sections 2.2.10 and 2.2.11.

2.2.2 Body Temperature Measurements

Mini subcue data-loggers from SubCue (Calgary, Canada) were implanted into the peritoneal cavity of rats. Animals were anesthetized with isoflurane (5% in air) and kept under isoflurane (2% in air) for the duration of the surgery. A small incision (1.5 cm) was made along the top of the right leg, where the subcue datalogger was inserted. Rats were given a 3 to 5 day recovery period. Body temperature was monitored for 24 h prior to any treatment to establish a baseline, and 48 h post-treatment in intervals of 3 min. Ambient temperature was kept at ~22ºC prior to and during the entire treatment period.

2.2.3 Locomotor Activity Assessment

Animals were placed inside open-field activity cages (Coulbourn Instruments) consisting of a clear Plexiglas square box (3.25 x 10 x 7 in) crisscrossed by three photobeam sensor rings. 78

Rats were kept in these cages for 30 min prior to any data recording to allow them to acclimate to the new environment. Computer software supplied by the manufacturer recorded locomotor activity in 15 min bins. For SERT deficiency studies, activity was measured for ~3 h only after the first MDMA (10 mg/kg, sc) or 0.9% saline dose, which included a 45 min baseline period and a 2 h post-treatment period. In the case of VMAT2 inhibition studies, locomotion was recorded for ~3.5 h, which included a 30 min baseline period, a 1 h Ro4-1284 (10 mg/kg, ip) pretreatment period and a 2 h MDMA (20 mg/kg, sc) post-treatment period. Two locomotor activity parameters were analyzed. Horizontal activity was measured as floor plane movement velocity (cm/min) and vertical activity is reported as the number of interruptions of rearing photobeams (entries).

2.2.4 Plasma Norepinephrine Extraction

SD rats were pretreated with Ro4-1284 (10 mg/kg, ip)/DMSO 1 h prior to MDMA (20 mg/kg, sc) or 0.9% saline dosing. Animals were anesthetized with isoflurane (5% induction, 3% maintenance) 1 h following drug treatment and blood was collected from the ventricle of the heart using a syringe containing 100 µL of 100 USP heparin. Blood was centrifuged at 3,000xg for

20 min at 4°C. The plasma supernatant was collected and stored at -80°C until further analysis by

HPLC-ECD.

Catecholamine extraction was accomplished by adding plasma (200 µL) to an Eppendorf tube containing 30 mg of aluminum oxide (Wako Chemical USA; Richmond, VA), and 20 ng of an internal standard, dihydroxybenzylamine hydrobromide, in 1.1 mL of 1.36 M Tris buffer containing

9 mM EDTA (pH 8.6).Tubes were manually shaken for 10 min prior to centrifugation at 1,000xg for 1 min. Supernatant was removed and the alumina pellet was washed with 1 mL of water followed by centrifugation at 1,000xg for 1 min to pellet alumina. This washing process was repeated twice more. Alumina pellets were transferred to a filter cup (UFC30GV00; Millipore; 79

Billerica, MA) containing 500 μL of water and centrifuged at 5,000xg for 10 min at 4°C. Water was discarded and alumina was incubated in 200 μL of 2% (v/v) acetic acid in 100 μM EDTA for 10 min.

Filtrate was recovered by centrifugation at 5,000xg for 10 min at 4°C and immediately analyzed by HPLC-ECD.

2.2.5 HPLC-ECD Analysis of Plasma Norepinephrine

Plasma NE concentrations were determined using a Shimadzu 10ADvp system (Shimadzu

Scientific Instruments; Columbia, MD) with an Eicom P3-CAPC precolumn in-line before an Eicom

CA-5ODS, 2.1x150 mm separation column (Eicom; San Diego, CA). The HPLC was coupled to an

Eicom ECD-700 electrochemical detector (Eicom). Mobile phase consisted of 75.9 mM NaH2PO4,

12.1 mM Na2HPO4, 2.8 mM sodium-1-octanesulfonic acid, 134 μM EDTA, and 12% (v/v) methanol, pH 6.0. Flow rate was 0.18 mL/min. The working electrode was set at +450 mV. Plasma preparations (50 μL) were injected and the NE peak areas were compared against a standard curve to estimate NE levels.

2.2.6 Stereotaxic Surgery for Cannula Placement

Adult male SD rats were anesthetized prior to surgery by intramuscular injection of the following anesthetic cocktail: 33.3 mg/mL ketamine, 10.7 mg/mL xylazine, and 1.3 mg/mL acepromazine- 1 mL/kg, im. Heads were fixed in place on a stereotaxic apparatus and a guide cannula (CXG-4; Eicom) was secured to the skull using dental cement. Cannula coordinates from bregma: anteroposterior, 0.2 mm; mediolateral, ± 3.0 mm; dorsoventral, 7.0 mm (Paxinos and

Watson, 1997). A dummy cannula (CXD-4; Eicom) was inserted inside the guide cannula to prevent tissue debris obstruction. Animals were given a 7-day recovery period before initiation of microdialysis experiments in which they were housed individually. 80

2.2.7 Microdialysis Probe Implantation

Microdialysis probes (CX-I-4-03; Eicom) were lowered through the guide cannula and artificial cerebral spinal fluid (aCSF: 147 mM NaCl, 28 mM KCl, 12 mM CaCl2, and 12 mM MgCl2) was infused via a syringe pump (Razel; Stamford, CT) into the striatum at 1 µL/min. Sample collection began 1 h after initiation of aCSF infusion to allow for equilibration with the surrounding

CNS environment. Dialysate fluid was collected at 30 min intervals for 6 h into 0.5 mL tubes containing 1X antioxidant solution (6.0mM L-cysteine, 2.0mM oxalic acid, 1.3% glacial acetic acid).

Two baseline samples were collected prior to Ro4-1284 (10 mg/kg, ip)/DMSO injection followed by MDMA (20 mg/kg, sc) or 0.9% saline treatment after 1 h. Dialysate samples were assayed via

HLPC-ECD for the detection of monoamine neurotransmitters.

2.2.8 HPLC-ECD Analysis of Dialysate Monoamine Levels

Dialysate monoamine concentrations were measured using a Shimadzu 10ADvp system with an Eicom AC-ODS, 3.0X4.0mm precolumn (Eicom) in-line before an Eicom SC-30DS, 3.0x100 mm separation column (Eicom). The HPLC was coupled to an Eicom ECD-700 electrochemical detector. The mobile phase consisted of 80% 0.1 M citrate-acetate, 20% methanol, 170 mg/L sodium octane sulfonate and 5 mg/L EDTA, pH 3.5. Flow rate was 0.345 mL/min. The working electrode was set at +750 mV. Dialysate samples (~30 μL) were injected and peak areas were compared to a standard curve of DA, DOPAC, 5-HT, and 5-HIAA to achieve quantification.

2.2.9 Immunohistochemistry

WT and SERT-KO Wistar rats were administered a lethal dose of pentobarbital (100 mg/kg, ip) 7 days after MDMA (10 mg/kg, sc, 4x, 12 h apart) or 0.9% saline treatment and transcardially perfused with 4% paraformaldehyde (w/v percentage) in 0.1 M phosphate buffered 81

saline (PBS) (pH = 7.4). Brains were removed and post-fixed in 4% paraformaldehyde overnight.

Brains were stored in 70% ethanol and submitted to the University of Arizona Histology Core for paraffin embedding and slide preparation. Coronal sections (10 µm) were deparaffinized in xylene and rehydrated in ethanol. Sections were incubated in citrate buffer (10 mM citric acid, 0.05% v/v

Tween 20, pH 6.0) for 6 min at 100°C for antigen retrieval. Endogenous peroxidase activity was blocked by immersing slides in 0.3% v/v H2O2 in methanol for 20 min. Microglial detection was achieved by incubation with primary iba1 antibodies (1:1,000 dilution) for 2 h at room temperature and then overnight at 4°C. Antibody binding was detected using the MACH4™ detection system (BioCare Medical; Concord, CA) following instructions provided by the manufacturer. Tissue staining was attained by immersion in DAB and 0.03% hydrogen peroxide for 15 min. Slides were imaged with a Leica DM4000B microscope equipped with a DFC450 camera (Leica Microsystems; Buffalo Grove, IL).

SD rats were given a lethal dose of pentobarbital (200 mg/kg, ip) and transcardially perfused with 4% paraformaldehyde (w/v percentage) in 0.1 M PBS (pH = 7.4) 7 days following

MDMA (20 mg/kg, sc) administration. Brains were extracted and post-fixed overnight at 4ºC in the same solution, and subsequently cryoprotected in 30% sucrose (w/v percentage) in 0.1 M PBS.

Coronal sections of 40 µm were cut using a freezing microtome and stored in cryoprotectant solution at -20ºC until further analysis. Tissue sections were washed with 0.1 M PBS, followed by antigen retrieval with citrate buffer (pH = 6.0) containing 0.03% Triton X-100 for 20 min at 80ºC.

Endogenous peroxide activity was blocked by 30 min incubation with 0.03% hydrogen peroxide in

0.1 M PBS with 0.03% Triton X-100 (PBS-T). Tissue sections were incubated in 5% normal goat serum (NGS) for 90 min. Primary antibody incubation with a polyclonal anti-SERT antibody

(AB9726 1:40000; Millipore; Billerica, MA) in antiserum diluent (3% NGS in PBST) was performed for 48 h at 4ºC. Following rinses with 0.1 M PBS, sections were exposed to biotinylated secondary 82

antibody (goat anti-rabbit) diluted 1:300 in antiserum diluent. Tissue was visualized using avidin– biotin-HRP conjugate solution (VECTASTAIN Elite ABC kit; VectorLabs; Burlingame, CA). Staining was enhanced by incubation with DAB and 0.03% hydrogen peroxide for 10 min. Sections were mounted on collagen-coated slides, dehydrated with increasing concentrations of ethanol and xylenes. Sections were coverslipped and analyzed under a light microscope.

2.2.10 Neurotransmitter Isolation from Brain Tissue

Animals were euthanized 7 days after MDMA dosing via CO2 asphyxiation followed by decapitation. Brains were excised and micro-dissected, collecting the frontal cortex and striatum for neurotransmitter concentration analysis. Tissue was weighed and suspended in 10 volumes of ice-cold 0.1M perchloric acid (134μM EDTA and 263μM octane-sulfonic acid sodium salt) to minimize monoamine oxidation. The tissue was then sonicated for 15 s followed by centrifugation at 16,000 g (4°C) for 20 min. Supernatant was filtered at 0.45 μm and used for monoamine detection via HPLC-ECD.

2.2.11 HPLC-ECD Analysis of Brain Monoamines

Tissue monoamine content was assayed using a Shimadzu 10ADvp system equipped with an ESA C-18, 3 μm, 4.6x80 mm column (Dionex, MA) coupled to an ESA Model 5600A CoulArray system (Dionex, MA). The mobile phase consisted of 35 mM citric acid, 54 mM sodium acetate,

324 μM octane-sulfonic acid sodium salt, 171 μM EDTA, 3% (v/v) methanol, 3% (v/v) acetonitrile, pH 4.0. Flow rate was 0.8 mL/min. Potentials were set at +50, +150, +300, and +450 mV. 50 μL of tissue preparation were used per injection and peak areas were compared to a standard curve of DA, DOPAC, 5-HT, and 5-HIAA to achieve quantitation. 83

2.2.12 In Vitro Cell Cultures

JAR cells were obtained from American Type Culture Collection (ATCC; Manassas, VA).

These cells are derived from human placental tissue, and are often used as a serotonergic cell model due to their capacity to uptake 5-HT (Ramamoorthy et al., 1998; Simantov and Tauber,

1997). JAR cells were cultured in Dulbecco’s Modified Eagle’s Medium buffered with Hanks

Buffered Saline Solution (DMEM/HBSS) supplemented with 10% Fetal Bovine Serum (FBS;

Hyclone; Novato, CA) and 1% penicillin/streptomyocin. Cells were grown to confluency at 37°C,

5% CO2.

RN46A cells were kindly provided by Dr. Whittemore’s research laboratory from the

Kentucky Spinal Cord Injury Research Center at the University of Louisville (Louisville, KY). RN46A cells are an embryonic serotonergic cell line derived from rat raphe neurons that display endogenous SERT expression (Koldzic-Zivanovic et al., 2004; White et al., 1994). Undifferentiated

RN46A cells were cultured at 37ºC, 5% CO2 in DMEM: Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% FBS, 250 µg/mL geneticin, 1% penicillin/streptomycin and 1% L- glutamine. Neuronal differentiation was achieved by incubating cells at 39ºC, 5% CO2 in DMEM/F-

12 supplemented with 100X N-2, 50X B27 and 0.1% bovine serum albumin.

Human Embryonic Kidney (HEK) cells and neuronal SK-N-MC cells were also purchased from ATCC (Manassas, VA). These cell lines were cultured in Eagles Minimal Essential Medium

(EMEM; ATCC) supplemented with 10% FBS. Cells were grown to confluency at 37°C, 5% CO2.

2.2.13 Transient Transfection of hSERT

pCDNA3 containing the cDNA for the human SERT gene (hSERT) was purchased from

Addgene (Cambridge, MA) and used for transient expression of the SERT protein. Transfection 84

was achieved using the lipofectamine 2000 reagent (Invitrogen; Carlsbad, CA) following the protocol provided by the manufacturer. Transfection of the GFP gene was used as a control. In summary, HEK and SK-N-MC cells were seeded in 24-well plates coated with poly-L-Lysine (~3 x

105 cells/well) overnight. cDNA was combined with the lipofectaime 2000 reagent in Opti-MEM

Reduced Serum Medium (Invitrogen) at room temperature for 30 min. Cells were washed with

Opti-MEM and the cDNA-liprofectamine solution was added to each well. Cells were placed at

37ºC, 5% CO2 for 6 h to allow for the uptake of cDNA complexes. Subsequently, transfection medium was removed and replaced with growth medium supplemented with 10% FBS.

Transfected cells were incubated for 24 h prior to any experiments to allow for maximal hSERT expression.

2.2.14 Cellular Uptake Studies

Cellular uptake of [3H]-5-HT into hSERT transfected HEK and SK-N-MC cells was measured

24 h post transfection. JAR cells (2 x 105 cells/well), differentiated RN46A cells (4 x 105 cells/well), and undifferentiated RN46A cells (4 x 105 cells/well) were seeded in 24-well plates 24 h prior to investigation of 5-HT uptake activity. A number of different uptake assays were performed and details for each experiment can be found in the corresponding figure legends. To summarize, cells were washed with warm (~37ºC) uptake buffer (NaCl, 125 mM; KCl, 5 mM; Hepes, 25 mM, glucose, 6 mM; NaHCO3, 5 mM; MgSO4*7H2O, 1.2 mM; KH2PO4, 1.2 mM; CaCl2*2H2O, 2.4 mM; pH, 7.4). Cells were then incubated in warm uptake buffer containing 100 μM pargyline and 100

μM ascorbate for 30 min at room temperature. Cellular uptake was initiated by adding 1 µCi/mL

[3H]5-HT. Nonspecific uptake was determined by incubating cells with 1 µCi/mL [3H]5-HT in the presence of 1 mM, untritiated 5-HT. Addition of cold uptake buffer terminated cellular uptake. 85

Cells were lysed with 0.5 N NaOH and neutralized with 1 N HCl to assess intracellular accumulation of [3H]5-HT by liquid scintillation spectroscopy (Beckman LS 5580; Urbana, IL).

A time-course for 5-HT uptake into hSERT-expressing HEK cells was achieved by incubation with 1 μCi/mL of [3H]5-HT at various time points, either alone or in the presence of excess, untritiated 5-HT (1 mM). Kinetics of [3H]5-HT uptake were performed by saturation analysis, treating HEK-SERT cells with 1 μCi/mL of [3H]5-HT and increasing concentrations of 1 mM

5-HT for 10 min. For inhibition studies, HEK-SERT cells were co-incubated with 1 μCi/mL of [3H]5-

HT and increasing concentrations of MDMA, N-Me-α-MeDA, 5-(GSH)-N-Me-α-MeDA and 5-

(NAC)-N-Me-α-MeDA. Principles of Michaelis-Menten kinetics were applied to determine substrate Km and inhibitor Ki values by non-linear least squares curve fit (GraphPad Prism 5

Software; La Jolla, CA).

2.2.15 Synthesis and Purification of 5-(GSH)-N-Me-α-MeDA and 5-(NAC)-N-Me-α-MeDA

5-(GSH)-N-Me-α-MeDA and 5-(NAC)-N-Me-α-MeDA were synthesized by electrochemical oxidation based on a protocol previously established in our laboratory with minor modifications

(Erives et al., 2008; Jones et al., 2005; Miller et al., 1996). N-Me-α-MeDA (80 mg) was stirred at room temperature for 30 min in 50 mL of HCl (50 mM). Oxidation was induced by applying voltage

(1V) through a BASi Bulk Electrolysis Cell (BASi Inc; West Lafayette, IN) for 30 min. Subsequently,

GSH or NAC were added to the oxidized solution at twice the molar concentration of N-Me-α-

MeDA and stirred until the mixture turned colorless. The solutions were then frozen at -80°C, and lyophilized to dryness. The lyophilized product was dissolved in 1% formic acid and purified by

HPLC (Shimadzu, SCL-10A), using a Beckman Ultrasphere ODS-5 µm particle size 10mm x 250 mm semi-preparative column. Fractions of interest were eluted using methanol and 1% acetic acid

(15:85 v:v) at a flow rate of 2 mL/min. GSH conjugates were monitored at 280nm, whilst NAC 86

conjugates were monitored at 225nm. The recovered eluents were analyzed by tandem mass spectrometry (LC-MS/MS) in full scan mode via ABI/SCIEX 4000 QTRAP hybrid triple-quadrupole linear ion trap mass spectrometer (Applied Biosystems; Foster City, California) using Analyst 1.5.1

Software.

2.2.16 Statistics

Results are presented as absolute values and expressed as the mean ± SE (n = 4-10). For

SERT deficiency studies, effects of repeated MDMA dosing on body temperature were analyzed by summarizing individual temperature responses after each MDMA dose as peak body temperature and area under the curve (∑ºC*h) in order to avoid discrepancies in the timing response of individual animals. ∑ºC*h following MDMA-related temperature spikes was estimated using GraphPad Prism 5. Effects of MDMA on peak temperature and ∑ºC*h after each

MDMA dose were analyzed by one-way ANOVA and subsequent “protected” Fisher-Hayter comparisons between groups. To ensure the nominal 5% significance level, we applied a

Bonferroni p-value correction to the tests associated with overall model significance for each dose period (that is, significance level for individual tests was 0.05/4 = 0.0125 for each dose period). Once this standard was met, we proceeded hierarchically toward more specific comparisons. The effects of body temperature, locomotor activity and dialysate monoamine concentrations following a single dose of MDMA were examined by two-way ANOVA (treatment group x time interval) with time as a repeated measure, followed by post-hoc Bonferroni multiple comparisons. In order to examine the effects of various treatments on dialysate 5-HT levels by two-way ANOVA, undetectable baseline levels of 5-HT were represented as zero. All other results were analyzed by one-way ANOVA followed by post-hoc Fisher-Hayter tests. All analyzes were performed using Stata 11.0 software (College Station, TX) and GraphPad Prism 5 software. 87

CHAPTER 3: EFFECTS OF SEROTONIN REUPTAKE TRANSPOTER DEFICIENCY ON THE ACUTE AND

LONG-TERM TOXICITIES OF 3,4-(±)-METHYLENEDIOXYMETHAMPHETAMINE

3.1 INTRODUCTION AND RATIONALE

The serotonergic system participates in a variety of behaviors, including mood, appetite, sleep and cognition, and plays a role in regulating the gastrointestinal and endocrine systems

(Schloss and Williams, 1998). Disturbed 5-HT homeostasis has been implicated in many psychiatric disorders, such as schizophrenia, eating disorders, obsessive compulsive disorders and drug addiction (Murphy et al., 2004). SERT is responsible for the rapid recycling of 5-HT following its synaptic release during neurotransmission. As such, 5-HT homeostasis is primarily regulated by the action of SERT, which determines the magnitude, duration and spatial distribution of signals that extend to post-synaptic 5-HT receptors (Murphy et al., 1998; Murphy et al., 2004). SERT is capable of transporting other endogenous biogenic amines, including DA (Jones et al., 2004), and exogenous compounds such as 1-methyl-4-(2'-aminophenyl)-1,2,3,6-tetrahydropyridine (2’-NH2-

MPTP), a potent 5-HT neurotoxicant. 2'-NH2-MPTP enters 5-HT neurons via SERT, and induces marked reductions in 5-HT levels and structural damage to 5-HT nerve terminals in mouse brain that simulate MDMA neurotoxicity (Andrews and Murphy, 1993). Similar to other monoamine uptake transporters, SERT is the site of action for many antidepressants, such as fluoxetine

(Prozac) and sertraline (Zoloft) (Schloss and Williams, 1998), and drugs of abuse including MDMA

(Rothman and Baumann, 2003).

SERT has long been implicated in the mechanism of action of a number of 5-HT depleting agents including p-chloroamphetamine, , MDA, and MDMA. These compounds share the ability to cause major in vitro and in vivo 5-HT release (Berger et al., 1992; Crespi et al., 1997;

Johnson et al., 1986; Kankaanpaa et al., 1998), with sustained depletions in 5-HT and 5-HIAA 88

(Clineschmidt et al., 1976; Ricaurte et al., 1985). SERT inhibitors prevent amphetamine- stimulated synaptosomal release (Berger et al., 1992; Rudnick and Wall, 1992). Co-administration of MDMA with benzylpiperazine derivatives, which are weak 5-HT inhibitors, attenuates 5-HT deficiency in vivo (Hashimoto et al., 1992), suggesting that MDMA interacts with SERT at the 5-HT transport site. Pretreatment with the potent SERT blocker, fluoxetine, 2 or 4 days prior to MDMA completely protects against 5-HT deficits and reductions in 5-HT uptake sites (Sanchez et al.,

2001). The acute psychological effects of MDMA in healthy volunteers are also reduced with citalopram, another 5-HT reuptake inhibitor (Liechti et al., 2000a). Collectively, these studies indicate the participation of SERT in the acute and long-term effects of MDMA on the serotonergic system. It is further postulated that SERT serves as carrier for the entry of MDMA, and other amphetamines, into serotonergic neurons, and mediates the rapid efflux of 5-HT into the synaptic cleft (Fuller, 1980), followed by a sustained depletion of this biogenic amine. Morphological damage to serotonergic nerve terminals in various regions of the CNS can also be a consequence of exposure to these amphetamines (Callahan et al., 2001; O'Hearn et al., 1988; Ricaurte et al.,

1985).

The human SERT gene carries a polymorphism (5-HTTLPR) in its upstream regulatory region that results in a 44-base pair deletion/insertion of a repetitive sequence. The dominant short allelic variant decreases the transcriptional efficiency of SERT (Lesch et al., 1996), causing reductions in brain SERT mRNA (Little et al., 1998), SERT protein expression and 5-HT uptake into human blood platelets (Greenberg et al., 1999). Moreover, this common polymorphism has been associated with a number of neuropsychiatric disorders, including anxiety, affective disorders and drug dependence (Caspi et al., 2003; Gerra et al., 2004). Individual variations in 5-HT levels in the

CNS can influence personality traits, thus, they appear to be constitutive and genetically 89

predetermined (Higley and Linnoila, 1997). To investigate the effects of SERT function in the acute and long-term toxicities of MDMA, we used a SERT-KO rat model previously generated by N-ethyl-

N nitrosurea (ENU)-driven targeted selected mutagenesis (Smits et al., 2006). Biochemical analysis revealed that mutant SERT mRNA is targeted for nonsense-mediated decay, and [3H]citalopram binding to brain slices is absent in SERT-KO rats, indicating the abolishment of SERT expression and function (Homberg et al., 2007). Genetic SERT ablation results in disturbances in 5-HT homeostasis, manifested as reductions in maximal rate of 5-HT uptake into hippocampal synaptosomes of 72%, 9-fold increases in basal extracellular 5-HT levels, and decreases in tissue

5-HT content and depolarization-induced 5-HT efflux in SERT-KO rats. In contrast, no changes are observed in 5-HT synthesis and degradation or major adaptations in other monoamine neurotransmitter systems (Homberg et al., 2007).

The restriction of neurobiological changes in SERT-KO animals to the serotonergic system provides a unique research tool to examine the effect of constitutive SERT deficiency in MDMA toxicity, furthering the understanding of this transporter in the mechanism of action of MDMA.

In the present study, we determined the effects of MDMA (10 mg/kg, 4x, 12 h apart) administration in WT and SERT-KO rats. Principles of interspecies scaling predict a dose of

1.28mg/kg (96mg/75kg) in humans to be equivalent to a neurotoxic dose of MDMA of 20mg/kg in rats. Multiple neurotoxic doses of MDMA were used to simulate party raves and similar weekend-long gatherings, where users ingest multiple drug doses.

Acute monoamine release and subsequent activation of post-synaptic monoamine receptors following MDMA treatment is accompanied by marked increases in body temperature that can lead to hyperthermia (Shioda et al., 2008), and enhanced locomotion (Ball et al., 2003;

Ball and Rebec, 2005) in rats. Therefore, we compared the effects of MDMA on body temperature 90

and locomotor activity in WT and SERT-KO animals. Long-term MDMA neurotoxicity in SERT- deficient rats was determined by measuring indoleamine (5-HT and 5-HIAA) concentrations in serotonergic-rich brain regions, 7 days after MDMA treatment, since, neurochemical changes appear as early as 1 week after MDMA exposure (Schmidt, 1987) but can persist for months and even up to 1 year (De Souza et al., 1990). Lastly, selective immunohistochemical staining of microglia was performed using iba1 antigen in order to access differences in microgial occupancy and morphology in WT and SERT-KO rats following MDMA exposure. Reactive gliosis is considered a hallmark of neurotoxicity (O'Callaghan and Sriram, 2005) and MDMA-induced acute microgliosis occurs in rats (O'Shea et al., 2005b; Orio et al., 2004), therefore, aberrant microglial activation has been proposed as a marker of long-term MDMA neurotoxicity. 91

3.2 RESULTS

3.2.1 SERT Deficiency Modulates the Thermoregulatory Response to MDMA after Multiple

Drug Dosing

Saline control animals showed normal fluctuations in body temperature over the 48 h course of treatment, in synchrony with the rodent circadian cycle. During the dark phase, core body temperature slightly rises as a consequence of the increase in physiological and behavioral activity of these nocturnal animals (Figure 3-1B and D), whilst modestly decreasing during the light, and less active phase (Figure 3-1A and C). MDMA (10mg/kg, 4x, 12 h apart sc) induced increases in core body temperature in both WT and SERT-KO rats upon exposure to multiple doses

(Figure 3-1). MDMA-mediated hyperthermia was most prominently observed after the first and third MDMA doses, reaching peak body temperature 2-3 h following drug administration. The thermoregulatory data was analyzed as peak body temperature to estimate the intensity of

MDMA-induced hyperthermia. One-way ANOVA of peak body temperature calculated after each

MDMA dose revealed significant differences between treatment groups after the first and third diurnal MDMA doses [Dose1 F (3, 27) = 27.3, p < 0.0001; Dose3 F (3, 27) = 7.3, p < 0.001] (Figure

3-2A and C). No prominent differences in peak body temperature between the saline and MDMA groups were observed for either the WT or SERT-KO animals after the doses administered during the dark phase lifecycle [Dose2 F (3, 27) = 2.2, p = 0.12; Dose4 F (3, 27) = 2.9, p = 0.057] (Figure 3-

2B and D). Fisher-Hayter post hoc tests revealed that peak body temperatures following the first

(39.5 ± 0.1ºC, p < 0.05) and third (38.7 ± 0.1ºC, p < 0.05) MDMA injections were higher in MDMA- treated WT animals compared to their saline counterparts (38.3 ± 0.07, 37.9 ± 0.1ºC, respectively).

Similarly, SERT-KO MDMA rats reached higher peak body temperatures after the first (39.4 ±

0.2ºC, p < 0.05) and third (38.6 ± 0.2ºC, p < 0.05) MDMA injections in comparison to SERT-KO 92

saline controls (38.3 ± 0.09, 38.0 ± 0.1ºC, respectively). Interestingly, elevations in peak body temperature in WT rats were comparable to those of SERT-deficient animals following multiple

MDMA treatment. Finally, no perceivable differences in peak body temperature were noted between WT and SERT-KO saline rats at any of the dose intervals.

Area under the curve (∑ºC x h) over a 12 h treatment period was estimated following each

MDMA-induced temperature spike as a measurement of the duration of the hyperthermic response (Figure 3-3). One-way ANOVA showed significant overall differences in ∑ºC x h between the treatment groups after the first and third MDMA doses [Dose1 F (3, 27) = 8.9, p = 0.0003;

Dose3 F (3, 27) = 4.1, p < 0.01] (Figure 3-3A and C). Conversely, nocturnal MDMA doses displayed no relevant changes in ∑ºC x h among the treatment groups [Dose2 F (3, 27) = 2.9, p = 0.056;

Dose4 F (3, 26) = 0.56, p = 0.65] (Figure 3-3B and D). Post-hoc analysis revealed that WT MDMA rats exhibited higher ∑ºC x h after the first (437.4 ± 0.8 ∑ºC x h, p < 0.05) and third (434.6 ± 0.8

∑ºC x h, p < 0.05) MDMA-mediated temperature elevations than their saline controls (433.7 ± 0.2,

430.0 ± 1.8 ∑ºC x h, respectively). In contrast, SERT-KO animals exhibited marked increases in ∑ºC x h following the first (434.3 ± 0.8 ∑ºC x h, p < 0.05) MDMA temperature boost only compared to saline counterparts (430.9 ± 1.5 ∑ºC x h). However, when compared to MDMA-treated WT rats,

∑ºC x h at this MDMA dose was significantly lower in the SERT-KO rats (p < 0.05). Again, no significant differences in ∑ºC x h were detected between WT and SERT-KO saline animals throughout the 48 h treatment course. In summary, both WT and SERT-KO rats displayed a similar thermoregulatory response to multiple MDMA dosing in relation to peak body temperature, showing a greater MDMA-related effect after the two diurnal doses. MDMA-treated WT rats had similar increases in ∑ºC x h after these MDMA doses; however, ∑ºC x h in SERT-KO MDMA animals was overall attenuated. 93

3.2.2 Attenuation of MDMA-Induced Hyperactivity in a Genetic Model of SERT Deficiency

MDMA-mediated hyperactivity was assessed using activity chambers that recorded horizontal and vertical locomotor activity after the first MDMA dose (Figure 3-4). Horizontal locomotor activity was sharply stimulated in WT Wistar rats within 15 min of MDMA (10mg.kg, sc) administration, reaching maximal horizontal velocity (289 ± 63 cm/min) 90 min post-treatment and gradually declining thereafter (Figure 3-4B). SERT-KO animals also displayed elevations in horizontal activity upon MDMA dosing. Nonetheless, horizontal velocity increased more gradually in MDMA-treated SERT-KO animals compared to WT counterparts, achieving maximal velocity

(160 ± 19 cm/min) 105 min after drug administration. Two-way repeated measures ANOVA of horizontal velocity revealed significant main effects of treatment group [F (3, 280) = 24.7, p <

0.0001], significant main effects of time interval [F (10, 280) = 10.4, p < 0.0001], and a significant treatment group x time interval interaction [F (30, 280) = 8.7, p < 0.0001]. Further post hoc

Bonferroni comparisons confirmed significant increases in horizontal velocity in MDMA-treated

WT rats compared to saline counterparts throughout the post-treatment period (15-120 min, p <

0.001). Stimulation in horizontal velocity in response to MDMA was delayed in SERT-KO rats, displaying marked increases only between 75-120 min (p < 0.05) after drug administration.

Correspondingly, MDMA-mediated induction in horizontal velocity was distinctly lower in SERT-

KO rats in comparison to WT counterparts between 15-90 min post-treatment (p < 0.05). No significant differences in horizontal velocity were observed between WT and SERT-KO saline rats during the pre- and post- treatment periods. Similarly, one-way ANOVA revealed significant differences in total horizontal velocity averaged over the entire measurement period between the treatment groups [F (3, 28) = 24.4, p < 0.0001]. Post hoc Fisher-Hayter tests indicated that

MDMA induced total horizontal velocity in both WT (186 ± 24 cm/min, p < 0.05) and SERT-KO (94 94

± 11 cm/min, p < 0.05) rats compared to their saline controls (34 ± 4 and 17 ± 3 cm/min, respectively) (Figure 3-4A insert). However, the effects of MDMA on total horizontal velocity were significantly higher in WT rats in contrast to SERT-KO counterparts (p < 0.05). Overall, the results showed that MDMA-mediated induction in horizontal velocity is delayed and attenuated in SERT-

KO animals as a consequence of SERT ablation.

Figure 3-5B illustrates vertical (rearing) activity during the pre- and post- treatment periods in WT and SERT-KO rats to assess changes in exploratory behavior with MDMA (10mg/kg, sc). Two-way repeated measures ANOVA of vertical activity revealed significant time interval effects [F (10, 280) = 2.2, p = 0.018] and a treatment group x time interval interaction [F (30, 280)

= 1.8, p = 0.0076] but no treatment group effects [F (3, 280) = 1.2, p = 0.32]. Post-hoc analysis showed significant decreases in vertical activity in MDMA-treated WT rats in comparison to saline counterparts 15 min (p < 0.05) after drug administration, consistent with previous reports of suppression in early-phase rearing behavior immediately following MDMA exposure (Gold et al.,

1988). No changes in vertical activity were detected in SERT-KO MDMA animals compared to their saline controls or WT counterparts. Furthermore, one-way ANOVA analysis revealed no significant differences in total vertical activity among the treatment groups [F (3, 28) = 1.2, p = 0.32] (Figure

3-5A insert). In summary, MDMA induced a transient decrease in vertical activity in WT rats and no changes in rearing behavior in SERT-KO rats.

3.2.3 Abolishment of Serotonergic Neurotoxicity Mediated by MDMA in SERT Deficient Rats

In order to evaluate the effect of SERT deficiency on long-term MDMA neurotoxicity, neurotransmitter concentrations were determined in brain regions heavily innervated by serotonergic nerve terminals, 7 days after the last dose of MDMA (10 mg/kg, sc). Saline controls showed basal indoleamine concentrations in the cortex (Cx) and striatum (St) of WT rats [5-HTCx 95

= 1.31 ± 0.16, 5-HTSt = 1.095 ± 0.061, 5-HIAACx = 2.41 ± 0.13, 5-HIAASt = 2.30 ± 0.076 pmol/mg of tissue] and SERT-KO rats [5-HTCx = 1.03 ± 0.089, 5-HTSt = 0.82 ± 0.088, 5-HIAACx = 0.88 ±

0.0097, 5-HIAASt =1.16 ± 0.27 pmol/mg of tissue] (Figure 3-6). One-way ANOVA analysis revealed significant differences in 5-HT and 5-HIAA levels in both brain regions among treatment groups

[5-HTCx (F (3, 19) = 3.9, p = 0.025), 5-HTSt (F (3, 19) = 7.1, p = 0.0021), 5-HIAACx (F (3, 19) = 29.0, p < 0.0001), 5-HIAASt (F (3, 19) = 6.4, p = 0.0036)]. SERT deficient rats exhibited lower basal levels of 5-HT (Cx = 21%, St = 25%) and 5-HIAA (Cx = 64%, p < 0.05; St = 49%, p < 0.05; post hoc Fisher-

Hayter tests) in comparison to the WT control group. Reduced tissue indoleamine content in SERT-

KO rats is consistent with previous reports (Homberg et al., 2007). Post hoc Fisher-Hayter tests also confirmed significant decreases in cortical and striatal 5-HT concentrations with MDMA treatment of 48% (p < 0.05) and 50% (p < 0.05), respectively, and in cortical and striatal 5-HIAA concentrations of 62% (p < 0.05) and 56% (p < 0.05) in WT rats. Most revealing, MDMA-treated

SERT-KO rats exhibited higher 5-HT (Cx = 25%, St = 26%) and 5-HIAA (Cx = 29%, St = 37%) concentrations in these brain regions compared to saline counterparts, however, these changes were not significant by post-hoc analysis. Moreover, tissue indoleamine levels were higher in

SERT-KO MDMA rats in both cortex (5-HT = 87%, p < 0.05; 5-HIAA = 23%) and striatum (5-HT =

91%, p < 0.05; 5-HIAA = 58%) in comparison to WT counterparts. Overall, the findings demonstrated that diminished SERT activity affords protection against MDMA-mediated indoleamine depletion.

Long-term neurochemical changes in the dopaminergic system of WT and SERT-KO male rats were determined by measuring striatal DA and DOPAC concentrations 7 days after the last

MDMA dose (Figure 3-7). Long-term MDMA neurotoxicity is selective towards the serotonergic neurotransmitter system in rats (Callahan et al., 2001), therefore, evaluation of tissue catecholamine levels is used as a control. Saline animals show baseline levels of DA and DOPAC in 96

the striatum of WT [DA = 55.50 ± 2.91, DOPAC = 15.39 ± 0.59 pmol/mg of tissue] and SERT-KO rats

[DA= 61.78 ± 2.92, DOPAC = 17.84 ± 4.49 pmol/mg of tissue]. One-way ANOVA analysis revealed no significant differences in striatal catecholamine content among the different treatment groups

[DA F (3, 19) = 1.6, p = 0.21, DOPAC F (3, 19) = 1.9, p = 0.17]. In summary, MDMA had no effect on tissue DA and DOPAC concentrations in both WT and SERT-KO rats.

3.2.4 MDMA Treatment Is Insufficient to Induce Microglial Activation in WT and SERT-KO

rats.

Microglial cells are activated in response to injury, undergoing a number of molecular, morphological and functional changes that coincide with the severity of the neuronal damage

(Kreutzberg, 1996). Upon activation, microglial morphology becomes less ramified and adopts an ameboid-like phenotype that allows for phagocytic activity (Streit et al., 1999; Szabo and Gulya,

2013). In the present study, we evaluated differences in microglial appearance and occupancy, 7 days after the last dose of MDMA (10mg/kg, sc) administered as plausible markers of microglial activation and long-term MDMA neurotoxicity. An antibody against the intracellular Ca2+ binding protein expressed in CNS macrophages, iba1, was utilized for the immunohistochemical evaluation of microglial cells in the frontal cortex, since previous studies show detection of resting and activated microglia with iba1 staining (Ito et al., 1998). High magnification images (400x) revealed no apparent differences in microglial size and morphology between saline and MDMA- treated animals in both WT and SERT-KO rats (Figure 3-8). Furthermore, lower magnification images (100x) were taken in order to determine whether MDMA treatment had any effect on microglial numbers in the frontal cortex. A lack of visual changes in microglial occupancy in WT and SERT-KO animals upon MDMA challenge is illustrated in Figure 3-9. No differences in basal microglial occupancy among WT and SERT-KO saline animals were also confirmed. To summarize, 97

the data suggests that the neurotoxic MDMA regimen utilized herein lacks the capability to induce a discernible microglial response in either WT or SERT-KO rats. 98 99

Figure 3-1 - MDMA-Induced Hyperthermia Occurs in Both WT and SERT-KO Rats after Repeated Drug Administration

Animals received four consecutive doses of MDMA (10mg/kg, sc) at 12 h intervals. Core body temperature was recorded using subcue dataloggers implanted into the peritoneal cavity of experimental animals as previously described under Materials and Methods. Data points display the recorded body temperatures averaged over 30 min intervals for the first (A), second (B), third (C) and fourth (D) MDMA doses. Black arrows indicate the times of drug administration, zero denoting the start of the MDMA treatment. White and black bars at the top of the graphs indicate the duration of the light and dark phases of the 12 h light/dark rodent cycle. Results are expressed as mean ± S.E. (n = 7-8). 100

Figure 3-2 - MDMA Increases Peak Body Temperature in Wistar Rats Regardless of Genotype

Data points show individual peak body temperatures and horizontal lines represent the mean peak body temperature (n = 7-8) for the different treatment groups following the first (A), second (B), third (C) and fourth (D) sc MDMA doses. Values are significantly different from WT saline at (*) p < 0.05, and from SERT-KO saline at (#) p < 0.05. 101

Figure 3-3 - SERT-KO Rats Display Attenuated ∑ºC x h after Multiple MDMA Doses

Data shows ∑ºC x h for each individual animal and the average (n = 7-8) ∑ºC x h for each treatment group over the 12 h treatment period following the first (A), second (B), third (C) and fourth (D) MDMA-mediated body temperature spikes. Values are significantly different from WT saline values at (*) p < 0.05, from SERT-KO saline values at (#) p < 0.05, and from WT MDMA values at (ξ) p < 0.05. 102

Figure 3-4 - SERT-KO Rats Display Delayed and Attenuated Horizontal Velocity after a Single Dose of MDMA

Locomotor activity was measured by placing animals into open-field activity chambers. Baseline levels of locomotion were measured for 45 min, followed by a 120 min post-treatment period in which MDMA (10mg/kg, sc) was administered. Figure shows the mean (SE ± 6-10) horizontal velocity (B) at each time interval. The scatter plot displays total horizontal velocity (A) over the entire 165 min treatment course. Values are significantly different from WT saline values at (*) p < 0.05, from SERT-KO saline values at (#) p < 0.05, and from WT MDMA values at (ξ) p < 0.05. 103

Figure 3-5 - MDMA Causes an Acute Decrease in Vertical Activity in WT Rats and No Changes in SERT-KO Animals

Locomotor activity was measured by placing animals into open-field activity chambers. Baseline levels of locomotion were measured for 45 min, followed by a 120 min post-treatment period in which MDMA (10mg/kg, sc) was administered. Graph shows the average (SE ± 6-10) vertical activity (B) at each time interval and the scatterplot (A) displays the total vertical activity over the entire 165 min treatment course. Values are significantly different from WT saline values at (*) p < 0.05. 104

Figure 3-6 - MDMA-Mediated Indoleamine Depletion is Abolished in SERT-KO Rats Following Multiple Drug Administrations

5-HT and 5-HIAA levels were measured from cortex and striatum 7 days after the last of four MDMA (10mg/kg, sc) injections by HPLC-ECD. Results show individual concentrations of these indoleamines in each treatment group (n = 4-7). Values are significantly different from WT saline values at (*) p < 0.05, from SERT-KO saline values at (#) p < 0.05, and from WT MDMA values at (ξ) p < 0.05. 105

Figure 3-7 - MDMA Does Not Affect Striatal Catecholamine Content in WT and SERT-KO Rats

Striatal DA and DOPAC concentrations were measured by HPLC-ECD, 7 days after the last of four MDMA (10mg/kg, sc) injections. Results show individual concentrations of these catecholamines in each treatment group (n = 4-7). 106

Figure 3-8 - MDMA Has No Effects on Microglial Size or Morphology in Wistar Rats Irrespective of SERT Genotype

Panels display representative images of the frontal cortex captured by light microscopy in WT saline (A), WT MDMA (B), SERT-KO saline (C) and SERT-KO MDMA (D) rats. Microglial cells were stained with iba1, 7 days after MDMA (10mg/kg, 4x, 12 h apart) treatment. Image magnification is 400x and scale bars at the lower right corner of each panel denote 25 µm. 107

Figure 3-9 – MDMA Fails to Cause Any Changes in Cortical Microglial Occupancy in WT or SERT- KO Rats

Representative images of the frontal cortex were captured by light microscopy in WT saline (A), WT MDMA (B), SERT-KO saline (C) and SERT-KO MDMA (D) rats. Microglial cells were stained with iba1, 7 days after MDMA (10mg/kg, 4x, 12 h apart) treatment. Image magnification is 100x and scale bars at the lower right corner of each panel denote 75 µm. 108

3.3 DISCUSSION

The primary objective of this study was to determine the influence of SERT deficiency and of disturbances in 5-HT homeostasis on the physiological, behavioral and neurochemical effects of MDMA. Our data reveal that SERT gene deletion modulates the acute effects of MDMA, including thermoregulation (Figures 3-1) and hyperactivity (Figure 3-4 and 3-5), whilst completely obliterating enduring serotonergic deficits (Figure 3-6). The results indicate that SERT is necessary for the acute and long-term toxicities of MDMA.

MDMA inflicts profound changes in thermoregulation in rats, and multiple doses of

MDMA increase body temperature in both WT and SERT-KO rats (Figure 3-1); however, this thermogenic response is most pronounced after the first and third MDMA doses, as measured by peak body temperature and ∑ºC x h. Differences in body temperature response after multiple

MDMA doses, correlate with thermogenic fluctuations of the circadian cycle. Rodents function under biological rhythms that display physiological and behavioral oscillations, with a duration ranging from 20 to 28 h (Benstaali et al., 2001). Although, circadian rhythmicity is endogenous, it can be influenced by external environmental cues such as daylight. In laboratory rats maintained in a 12 h light/dark cycle, circadian changes in body temperature and locomotor activity occur in synchrony, and their acrophase (time period in the cycle of peak activity) occurs during nighttime

(Benstaali et al., 2001). It is possible that the thermogenic effect of nocturnal MDMA doses was diminished in both WT and SERT-KO rats compared to diurnal MDMA doses due to parallel increases in body temperature related to circadian rhythmicity. Alternatively, attenuation of

MDMA-mediated hyperthermia during nocturnal drug doses could be a consequence of acute depletions in intracellular 5-HT stores following excess 5-HT release during diurnal MDMA doses.

However, this is unlikely given that reductions in tissue 5-HT levels in MDMA-treated rats are 109

recovered by 12 h post-treatment, exhibiting indoleamine concentrations comparable to those of saline controls (Schmidt, 1987).

Overall, SERT-KO animals display a different thermoregulatory effect than their WT counterparts in response to repeated MDMA dosing (4x 10mg/kg, sc). Indeed, similar increases in peak body temperature occurred following the first and third diurnal MDMA doses (Figure 3-2); however, ∑ºC x h was attenuated in MDMA-treated SERT-KO rats (Figure 3-3). Although reductions in ∑ºC x h in KO rats were modest, the findings could have implications in the protective effects of SERT deficiency against MDMA neurotoxicity. The results suggests that SERT-KO rats are exposed to reduced periods of body temperature elevation, which could render them less susceptible to the long-term neurotoxic effects of this amphetamine. Despite a lack of consensus in the literature on the precise role of hyperthermia in MDMA neurotoxicity, some studies report a positive correlation between the magnitude of the thermogenic response and the degree of neurotoxicity. In particular, Malberg and Seiden (1998) demonstrated that low ambient temperatures (20-24ºC) attenuate AUC and prevent tissue 5HT depletions, whereas, elevated ambient temperatures (28-30ºC) potentiate both AUC and the neurotoxic response to MDMA.

Moreover, MDMA metabolite-induced injury to neuronal cortical cultures is aggravated under hyperthermic conditions (40ºC) (Capela et al., 2006). Clomethiazole prevents MDMA-mediated hyperthermia with a concomitant attenuation in hippocampal 2,3-DHBA induction, which is restored by elevating body temperature with a homeothermic blanket (Colado et al., 1999). Thus, acute hyperthermia is thought to contribute to MDMA neurotoxicity through elevations in free radical formation in the brain (Colado et al., 1999). It is conceivable that decreased exposure to elevated body temperatures mediated by MDMA helped alleviate damage to serotonergic neurons in SERT-KO animals, seemingly by reducing ROS production. Nevertheless, hyperthermia 110

is not essential for the manifestation of MDMA neurotoxicity (Broening et al., 1995; O'Shea et al.,

1998), therefore, other SERT-dependent mechanisms are likely to play a more predominant role in determining the neurotoxic response to MDMA upon ablation of SERT function.

SERT-KO rats are insensitive to d-fenfluramine-induced hypothermia (Homberg et al.,

2007), a process driven by the interaction of d-fenfluramine with SERT. In a similar manner, the thermogenic effects of MDMA are in part facilitated by its interaction with the SERT at presynaptic serotonergic terminals, and the subsequent release of 5-HT into the synaptic cleft. Although

MDMA is mainly an indirect 5-HT agonist, it also acts on DA and NE reuptake transporters, causing

DA and NE efflux (Fitzgerald and Reid, 1993). Indeed, pretreatment with D1 receptor antagonists prevents MDMA-mediated hyperthermia in rats, highlighting the importance of DA in MDMA thermogenesis (Shioda et al., 2008). MDMA-evoked 5-HT release may indirectly induce DA efflux via interaction with 5-HT2 receptors, and MDMA-mediated hyperthermia is inhibited with 5-HT2A receptor antagonists, risperidone and ketanserin (Shioda et al., 2008). Nevertheless, critics argue that the hyperthermic effects of MDMA are minimally impacted by central 5-HT release, emphasizing that risperidone is also a weak inhibitor of dopaminergic receptors, and ketanserin can effectively block α1 adrenoceptors, involved in the induction of vasoconstriction during the thermogenic response to MDMA (McCall and Schuette, 1984). This argument is further substantiated by the inability of 5-HT reuptake inhibitors, such as fluoxetine, to prevent hyperthermia produced by MDMA (Gudelsky and Nash, 1996; Sanchez et al., 2001). Nevertheless, prevention of MDMA-induced hyperthermia by risperidone is accompanied by attenuation in 5-

HT and DA release (Shioda et al., 2008), indicating that both neurotransmitters are involved. In agreement with this view, our findings provide considerable evidence of the importance of SERT to MDMA thermoregulation. 111

It is possible that attenuations in ∑ºC x h in SERT-KO rats following the diurnal MDMA- induced temperature spikes results from disturbances in carrier-mediated 5-HT release in the absence of functional SERT. Similar increases in peak body temperature in KO and WT animals after the diurnal MDMA doses likely reflects the maintenance of MDMA-evoked release of other monoamines in SERT-deficient animals. Consistent with this hypothesis, studies in SERT-KO mice revealed minimal to no changes in extracellular 5-HT concentrations in the prefrontal cortex after

MDMA treatment, with similar increases in extracellular DA levels occurring in the striatum of WT and SERT-KO mice (Trigo et al., 2007). In vivo microdialysis experiments are necessary to confirm presumed disruption of MDMA-mediated 5-HT efflux as a consequence of the absence of SERT.

Excess monoamine release following MDMA treatment also stimulates dose-dependent behavioral changes that include enhanced locomotion, and all major features of the 5-HT syndrome. The increased locomotor activity observed with MDMA is primarily the result of striatal

DA release. Studies in freely moving rats have shown that DA neurotransmission and locomotor activity stimulation are blocked by treatment with D2 receptor antagonists prior to MDMA exposure (Ball et al., 2003). The role of 5-HT release in MDMA-induced locomotion is thought to occur via indirect modulation of the DA system. Pharmacological blockade of 5-HT2A receptors decreases MDMA-mediated striatal excitation in parallel with a suppression in locomotor activity

(Ball and Rebec, 2005). Our results reveal attenuation in MDMA-induced horizontal velocity in

SERT-deficient rats (Figure 3-4). The delayed stimulation in MDMA-mediated hyperactivity observed in SERT-KO rats is consistent with elevations in late-phase locomotor activity with high- dose MDMA treatment (30mg/kg) in mice deficient in 5-HT1B receptor. Correspondingly, D1 and

D2 KO mice display reductions in late-phase hyperlocomotion after MDMA treatment, confirming that dopaminergic activity contributes to the delayed behavioral response of this psychostimulant drug (Risbrough et al., 2006). We hypothesize that direct DA efflux mediated by MDMA is most 112

likely unaffected in SERT-KO rats, whereas perturbations in SERT-mediated 5-HT release affects striatal DA neurotransmission, which is manifested as reductions in MDMA-induced horizontal hyperactivity. Altogether, the data confirm previous findings of a critical role for 5-HT pathways in the behavioral effects of MDMA, highlighting the involvement of SERT in the initiation and maintenance of enhanced locomotor activity.

In contrast, a single dose of MDMA had no effect on total vertical activity in both WT and

SERT-KO male rats (Figure 3-5), which coincides with studies that demonstrate male rats are less sensitive to the locomotor effects of MDMA, in particular to the stimulation of rearing behavior

(Palenicek et al., 2005; Walker et al., 2007), although, MDMA-treated WT rats display an acute decrease in vertical locomotion, a phenomenon that has been previously reported (Gold et al.,

1988; Timar et al., 2003). Finally, in agreement with a study analyzing cocaine supersensitivity in

SERT-KO rats, our results show that SERT-deficiency does not impact basal locomotor activity levels, despite induction of a depressive phenotype (Olivier et al., 2008).

Abolishment of SERT activity has profound effects on the long-term neurotoxicity of

MDMA. MDMA selectively targets the serotonergic system in both humans (Bolla et al., 1998) and rats (Commins et al., 1987; Schmidt et al., 1987). Consistent with this view, MDMA had no effects on tissue catecholamine levels in the striatum of both WT and SERT-deficient rats (Figure 3-7).

Importantly, MDMA-mediated indoleamine depletion was abolished in SERT-KO rats in both cortex and striatum (Figure 3-6). In fact, 5-HT and 5-HIAA levels were slightly higher in MDMA- treated KO animals compared to saline controls, although this trend was not statistically significant. Alleviation of MDMA neurotoxicity can be primarily correlated to the absence of functional SERT, since KO rats displayed marked reductions in brain indoleamine levels indicative of disturbed 5-HT homeostasis (Figure 3-6). Noteworthy, tissue catecholamine content appeared undisturbed in these animals, suggesting no major adaptations in the dopaminergic system, and 113

most likely in other non-serotonergic neurotransmitter systems, as previously demonstrated

(Homberg et al., 2007). The present findings thereby support a critical role for SERT in MDMA neurotoxicity.

Although many hypotheses on the molecular mechanism of MDMA neurotoxicity have been put forward, the involvement of SERT in this process is indisputable. The fact that reductions in presynaptic SERT protein are one of the hallmarks of MDMA-mediated neurotoxicity is perhaps most revealing. The selectivity of MDMA towards the serotonergic system can in part be attributed to its higher affinity for SERT compared to the DA and NE transporters in rat synaptosomes (Rothman et al., 2001). In vitro studies have demonstrated MDMA inhibits SERT to a greater extent than to DAT, reversing its function, and causing major neurotransmitter release via calcium dependent (Crespi et al., 1997) and independent (Johnson et al., 1986) mechanisms.

Furthermore, 5-HT uptake inhibitors can block in vivo MDMA-induced 5-HT and DA release

(Gudelsky and Nash, 1996) and neurotoxicity in rat brain (Sanchez et al., 2001) but DAT uptake inhibitors, such as GRB 12909, fail to prevent MDMA-mediated monoamine release (Mechan et al., 2002).

The protective mechanisms of SERT deficiency in MDMA neurotoxicity are likely the consequence of prevention of cellular oxidative stress. Indeed, pharmacological inhibition of SERT attenuates hydroxyl radical generation (Shankaran et al., 1999b), thus averting oxidative damage that can be detrimental to 5-HT neurons. Interestingly, MDMA-induced ROS production has been regarded as a cause of SERT inhibition (Sprague and Nichols, 1995a; Sprague and Nichols, 1995b).

However, further evidence suggests that MDMA increases ROS burden by interfering with SERT function (Shankaran et al., 2001; Shankaran et al., 1999a). SERT proteins are capable of transporting DA (Jones et al., 2004), therefore, excess accumulation of DA in the striatum may permit the uptake of this catecholamine into depleted 5-HT nerve terminals (Sprague et al., 1998). 114

MAOB-mediated deamination of DA generates substantial ROS (Sprague and Nichols, 1995b).

Production of DA-derived ROS is implicated in membrane lipid peroxidation and alterations to tissue macromolecules that ultimately cause cell death (Berman et al., 1996). Additionally, it is speculated that thioether MDMA metabolites can enter 5-HT neurons through SERT-mediated transport. These metabolites exhibit lower oxidation states than the parent catechols (Zhang et al., 2000), facilitating the formation of ROS and reactive ortho-quinones through redox cycling.

Quinone-derived thioether metabolites are highly electrophilic and often interact with DNA and proteins, by forming covalent bonds or through arylation (Miyazaki and Asanuma, 2009).

Adduction of cellular macromolecules can result in their inactivation or malfunction, thus inducing cytotoxicity. For example, thioether metabolites of 1,4-benzoquinones (BQ), known to cause nephrotoxicity in rats, selectively bind lysine and glutamic acid residues within cytochrome c, which affects domains involved in protein-protein interactions (Fisher et al., 2007). Indeed, oxidatively modified adducts of salicylic acid and protein carbonyls are detected upon MDMA exposure (Alves et al., 2007; Shankaran et al., 1999b). Given the oxidative reactivity and electrophilic properties of quinone thioether intermediates, it is reasonable to conclude that cathecholamine metabolites of MDMA contribute to MDMA-induced serotonergic axotomy by elevating ROS and oxidative damage. Hence, the absence of functional SERT in KO rats may alleviate 5-HT deficits by hindering the entry of DA and neurotoxic metabolites of MDMA into serotonergic terminals.

Finally, MDMA (10 mg/kg, 4x, 12 h apart) failed to induce persistent changes in microglial morphology (Figure 3-8) and occupancy (Figure 3-9) in both WT and SERT-KO rats. Herdon et al.

(2014) showed a biphasic increase in microglial stained area (µm2) in rat cortex in parallel with an increase in phagocytic activity , which peaked at 12 and 72 h after MDMA (20mg/kg, sc) exposure; however, 7-days post-MDMA treatment, the elevation in microglial occupancy had subsided, 115

which coincides with the present data. Although excessive gliosis and microglial activation are suspected to persist long after MDMA treatment, serving as a reliable marker in the assessment of MDMA neurotoxicity (O'Callaghan et al., 1995; O'Callaghan and Miller, 1993), our results contradict with such a contention. Even though brain monoamine deficits are considered reliable endpoint measurements of amphetamine-induced toxicity, they are not direct markers of neuronal terminal injury. Hence, further analysis is required to corroborate prevention of MDMA- mediated serotonergic degeneration in Wistar rats devoid of any SERT activity. 116

CHAPTER 4: EFFECTS OF VESICULAR MONOAMINE TRANSPORTER 2 INHIBITION IN THE IN VIVO

TOXICITIES OF 3,4-(±)-METHYLENEDIOXYMETHAMPHETAMINE

4.1 INTRODUCTION AND RATIONALE

The ability of MDMA and other substituted amphetamines to stimulate synaptic release is attributed to the interference of transport mechanisms at the plasma membrane and storage vesicles (Schuldiner et al., 1993). Conceivably, MDMA binds monoamine uptake transporters located in the plasma membrane, inducing the release of biogenic amines via a carrier-mediated exchange mechanism (Crespi et al., 1997). Disruption of VMAT2 function is speculated to assist in

MDMA-induced release of 5-HT, DA and NE by elevating the concentration of these biogenic amines in the cytosol, allowing for their rapid efflux into the synaptic space (Rudnick and Clark,

1993). Schuldiner et al. (1993) demonstrated that MDMA competes with the well-known vesicular inhibitor, reserpine, for the substrate binding site in the VMAT2 protein, suggesting the direct interaction of MDMA with this transporter. Additionally, MDMA appears to interfere with VMAT2 function by dissipation of the ATP-driven transmembrane pH gradient involved in vesicular transport of biogenic amines (Schuldiner et al., 1993). Most revealing, MDMA-evoked outflow of

[3H] monoamines from rat brain slices is reduced in the presence of reserpine, in a dose- dependent manner, with 5-HT release displaying particular susceptibility to the effects of reserpine treatment (Fitzgerald and Reid, 1993). Likewise, efflux of 5-HT was markedly attenuated in reserpine-treated raphe neurons (Gu and Azmitia, 1993). MDMA and other amphetamines are well-known substrates of VMAT2. They exhibit low affinity for [3H]DTBZ binding but high potency at inhibiting vesicular [3H]DA uptake and releasing preloaded [3H]DA from rat vesicular fractions

(Partilla et al., 2006). MDMA likely enters intra-neuronal vesicles and depletes monoamine storage by reversing VMAT2 function (Partilla et al., 2006). 117

VMAT2 stores DA, 5-HT, NE, epinephrine and histamine into synaptic vesicles for exocytotic release. VMAT2 pumps biogenic amines against a concentration gradient by exchanging two intra-vesicular protons for one substrate molecule (Parsons, 2000). Modulation of vesicular loading via VMAT2 regulation has a profound influence on neurotransmission and neuronal health (Pothos et al., 2000). For example, disruption of VMAT2 function can lead to DA mishandling, by increasing DA levels in the cytosol available for oxidation, thereby compromising

DA neurons and modulating their interactions with exogenous compounds (Takahashi et al.,

1997). Consequently, VMAT2 has been implicated in the mechanism of psychostimulant abuse

(Dwoskin and Crooks, 2002). For instance, lobeline, a natural alkaloid found in “Indian tobacco,” perturbs monoamine storage and release by interfering with VMAT2 (Teng et al., 1998).

Interestingly, acute inhibition of VMAT2 by a lobeline analog, UKCP 110, prevents in vitro methamphetamine-mediated DA release and self-administration in rats, without development of tolerance (Beckmann et al., 2010). Thus, lobeline analogs with an enhanced selectivity for VMAT2 inhibition have been proposed for the treatment of psychostimulant abuse (Crooks et al., 2011).

Contributions of VMAT2 to the in vivo mechanisms of MDMA toxicity have been studied through irreversible inhibition of this transporter via reserpine pretreatment. However, the findings are inconclusive, with some demonstrating that reserpine impacts MDMA-mediated neurotoxicity (Schmidt et al., 1990c; Stone et al., 1988), whilst others show it has no effects

(Hekmatpanah et al., 1989; Schmidt and Taylor, 1987). The results are further confounded by the pharmacological response that reserpine treatment alone appears to have, complicating the interpretation of the data. In the present study, we implemented a different approach, antagonizing VMAT2 function with Ro4-1284, a benzoquinolazine derivative that inhibits VMAT2 reversibly. Unlike MDMA, Ro4-1284 is not a substrate for VMAT2 but rather an uptake inhibitor, 118

displaying similar potency at inhibiting [3H]DTBZ binding and vesicular [3H]DA uptake. Moreover, uptake inhibitors of VMAT2 exert partial release of [3H]DA from synaptic vesicles (Nickell et al.,

2011; Partilla et al., 2006). Ro4-1284 exhibits monoamine-depleting activity similar to reserpine

(German et al., 1981), however, these effects are transient, usually dissipating within a few hours after treatment. More specifically, the dose of Ro4-1284 (10mg/kg, i.p.) utilized in this analysis induces maximal reduction of striatal DA content in rats at 1 h post-treatment. DA levels remain significantly depleted in these animals for up to 6 h (Staal and Sonsalla, 2000). Experiments detailed in this chapter include a comprehensive examination of the effects of VMAT2 dysfunction on the thermoregulatory, behavioral and neurotoxic response to MDMA, studies which are currently lacking. We hypothesize that pharmacological antagonism of VMAT2 would profoundly impact the acute and long-term toxicities of MDMA in our rat model due to the importance of this transporter to the mechanism of action of MDMA and other amphetamine drugs. 119

4.2 RESULTS

4.2.1 Pharmacological Inhibition of VMAT2 Decreases Brain Monoamine Levels

Pharmacological antagonism of VMAT2 is known to cause prominent depletion of brain monoamines (German et al., 1981; Staal and Sonsalla, 2000). Thus, indoleamine and catecholamine content in the striatum of SD rats was assessed at 1 h and 12 h following Ro4-1284

(10mg/kg, ip) administration. One-way ANOVA revealed significant differences in tissue concentrations of 5-HT [F (2, 15) = 16.5, p < 0.0002)], DA [F (2, 15) = 53.9, p < 0.0001] and their respective metabolites, 5-HIAA [F (2, 15) = 68.7, p < 0.0001) and DOPAC [F (2, 15) = 67.6, p <

0.0001] (Figure 4-1). Post-hoc analysis revealed significant depletions in DA content of 69% (p <

0.05) 1 h after Ro4-1284 exposure. To a lesser extent, decreases in striatal 5-HT (35%, p < 0.05) were also noted in Ro4-1284-treated animals compared to DMSO controls. On the contrary, tissue concentrations of monoamine metabolites 5-HIAA and DOPAC were elevated (50% and 67%, respectively; p < 0.05) from controls 1 h post-Ro4-1284 treatment. Importantly, changes in striatal monoamine content were absent in SD rats at 12 h after Ro4-1284 dosing. Thus, Ro4-1284 induced rapid reductions in tissue DA and 5-HT concentrations that completely dissipated 12 h post-treatment.

4.2.2 Inhibition of VMAT2 Protects Rats against MDMA-Mediated Hyperthermia

As expected, a single dose of MDMA (20 mg/kg, sc) induced a thermogenic response in rats, causing elevations in core body temperature that reached a maximum of 40.7 ± 0.1ºC, 2.5 h after drug administration; body temperatures remained above those of saline controls for up to

12 h (Figure 4-2). Pretreatment with the VMAT2 inhibitor, Ro4-1284 (10mg/kg, ip), attenuated

MDMA-mediated hyperthermia in rats. Following MDMA administration, the Ro4-1284/MDMA 120

treatment group displayed a transient drop in body temperature to 36.7 ± 0.6ºC, returning to baseline levels 3 h post-treatment. Two-way repeated measures ANOVA of core body temperature revealed significant effects of treatment group (F (3, 675) = 25.7, p < 0.0001), significant effects of time interval (F (27, 675) = 5.6, p < 0.0001), and a significant treatment x time interaction (F (81, 675) = 9.1, p < 0.0001). Bonferroni post hoc tests confirmed a significant hyperthermic effect in the MDMA-treated rats as compared to their saline counterparts between

1.5 - 5.5 h post-treatment (p < 0.001). Notably, Ro4-1284 treatment alone had no profound effects on body temperature. Body temperatures recorded in the Ro4-1284/MDMA treatment group were significantly different from both the Ro4-1284/saline group at the 2 h time interval (p <

0.01), and the MDMA group between 1.5 -5 h post-treatment (p < 0.001). In summary, Ro4-1284 pretreatment abolished hyperthermia in MDMA-exposed rats, instead inducing an acute decrease in body temperature.

4.2.3 Elevation of Peripheral NE Levels with MDMA Treatment Is Unaffected by Inactivation

of VMAT2 Function

The thermoregulatory response to MDMA is facilitated by the parallel induction of central and peripheral components. NE is at the center of the peripheral response, coordinating the increase in heat production from skeletal muscle tissue and loss of heat dissipation through induction of vasoconstriction (Mills et al., 2004). In order to investigate any potential peripheral effects that Ro4-1284 might have on MDMA-induced hyperthermia, plasma NE concentrations were measured 30 min prior to the first significant elevation in body temperature observed following MDMA dosing (1 h post-treatment) (Figure 4-3). One-way ANOVA revealed significant differences in plasma NE levels among the various treatment groups (F (3, 20) = 9.5, p < 0.0004)

(Figure 4-3). Post hoc Fisher-Hayter tests showed significant increases in plasma NE levels in the 121

MDMA-only group (~123%) and Ro4-1284/MDMA group (~149%) when compared to saline controls (Figure 4-3). Ro4-1284 treatment alone had no effects on NE levels. Moreover, peripheral

NE concentrations in the MDMA-only group were similar to the Ro4-1284/MDMA group. Thus,

Ro4-1284 pretreatment failed to modulate the acute stimulation in NE levels in the periphery after MDMA exposure.

4.2.4 Inhibition of VMAT2 Attenuates Induction of Locomotor Activity in MDMA-Treated

Rats

The behavioral response to MDMA was measured using activity chambers that recorded various aspects of locomotion. Two specific parameters were selected for this analysis: horizontal velocity (hyperactivity) and vertical activity (rearing; exploratory behavior). MDMA (20 mg/kg, sc) stimulated horizontal locomotor activity in SD rats. Animals attained maximal activity (547 ± 46 cm/min), 60 min post-MDMA treatment (Figure 4-4B). In combination with Ro4-1284, MDMA also induced horizontal locomotor activity. Horizontal velocity in the Ro4-1284/MDMA treatment group reached maximal activity (170 ± 35 cm), 75 min following MDMA dosing. Two-way repeated measures ANOVA of horizontal hyperactivity revealed significant main effects of treatment group

(F (3, 299) = 57.8, p<0.0001), significant main effects of time interval (F (13, 299) = 26.1, p<0.0001) and a significant treatment x time interaction (F (39, 299) = 16.9, p<0.0001). Further Bonferroni post-hoc analysis confirmed significant increases in horizontal velocity 15 - 120 min after MDMA administration compared to saline controls (p < 0.001). Horizontal activity was unaffected by Ro4-

1284 treatment alone. Moreover, post hoc tests revealed significant inductions in MDMA- mediated horizontal locomotion in the presence of Ro4-1284, displaying marked elevations in movement velocity over controls 75 - 120 min following drug exposure (p < 0.01). However, when 122

compared to MDMA-only treatment, increased horizontal velocity was significantly reduced in the Ro4-1284/MDMA group throughout the post-treatment period (p < 0.001).

MDMA represses exploratory behaviors immediately following drug dosing, however, high-dose treatment augments late-phase rearing activity above that of control rats (Gold et al.,

1988). Consistent with these observations, MDMA-mediated increases in vertical activity occurred more gradually compared to horizontal locomotion, reaching maximal activity (14.2 ±

10.2) 90 min post-treatment (Figure 4-5B). In contrast, induction of vertical activity with MDMA was absent in animals pretreated with Ro4-1284. Rearing activity displayed significant treatment group effects (F (3, 299) = 4.5, p = 0.013) and a significant time x treatment interaction (F (39, 299)

= 1.63, p = 0.013) but no significant time interval effects (F (13, 299) = 1.4, p = 0.15). Enhanced vertical activity was noted in the MDMA-treated group between 75 - 105 min post-treatment (p

< 0.01). Stimulation of rearing behavior mediated by MDMA was completely suppressed with Ro4-

1284 pretreatment between 75 - 105 min after drug administration (p < 0.01). At last, Ro4-1284 treatment alone had no effects on rearing activity.

Locomotor activity results were also analyzed as total % Δ from baseline (Figure 4-4A and

4-5A). One-way ANOVA showed significant differences in % Δ from baseline for total horizontal velocity (F (3, 23) = 6.1, p < 0.0032) and total vertical activity (F (3, 22) = 4.9, p < 0.0092). Fisher-

Hayter pairwise comparisons confirmed significant increases in the % Δ from baseline of horizontal and vertical activity with MDMA treatment (p < 0.05). Although % Δ from baseline for horizontal velocity was elevated in Ro4-1284/MDMA-treated animals, this effect was not significantly different from their saline counterparts. In contrast, % Δ from baseline for total vertical activity was lower in the Ro4-1284/MDMA group compared to MDMA-only treatment (p

< 0.05). In summary, Ro4-1284 pretreatment delayed and attenuated MDMA-induced horizontal locomotion, whilst completely abolishing stimulation in rearing behavior. 123

4.2.5 Pharmacological Inactivation of VMAT2 Has Minimal Effects on MDMA-Induced

Monoamine Release in Rat Striatum

Microdialysis probes were implanted into the striatum via stereotaxic surgery, 5-7 days prior to dialysate collection (Figure 4-6). Recovered samples were assayed for indoleamine and catecholamine analysis via HPLC-ECD. Baseline concentrations of these neurotransmitters in the striatum were as follows: [5-HT, below detection levels; 5-HIAA, 246.6 ± 14.7; DA, 3.6 ± 0.3;

DOPAC, 1112.4 ± 61.1 pg/10 µL of dialysate]. MDMA (20mg/kg, sc) treatment had a profound effect on extracellular monoamine concentrations in this brain region, elevating 5-HT and DA levels, meanwhile reducing levels of corresponding metabolites, 5-HIAA and DOPAC (Figures 4-7 and 4-8). Two-way repeated measurements ANOVA of dialysate neurotransmitter levels revealed significant treatment effects [5-HT (F (3, 296) = 15.97, p < 0.0001); DA (F (3, 275) = 13.8, p <

0.0001); 5-HIAA (F (3, 297) = 3.7, p = 0.02); DOPAC (F (3, 275) = 28.2, p < 0.0001)], significant time effects [5-HT (F (11, 286) = 29.58, p < 0.0001); DA (F (11, 275) = 23.2, p < 0.0001); 5-HIAA (F (11,

297) = 12.6, p < 0.0001); DOPAC ( F(11, 279) = 69.8, p < 0.0001)] and a significant treatment x time interaction [ 5-HT (F (33, 286) = 10.13, p < 0.0001); DA (F (33, 275) = 8.4, p < 0.0001); 5-HIAA (F

(33, 297) = 4.1, p < 0.0001; DOPAC (F (33, 275) = 18.6, p < 0.0001)]. Furthermore, post hoc

Bonferroni comparisons confirmed prominent increases in 5-HT levels in rat striatum with MDMA throughout the post-treatment period (p < 0.0001), which appeared to be unaffected by Ro4-

1284 pretreatment (Figure 4-7). Similarly, striatal DA concentrations were stimulated in all

MDMA-treated rats compared to controls, 60-210 min post-treatment (p < 0.001) (Figure 4-8).

Although DA concentrations appeared to be slightly higher with Ro4-1284/MDMA treatment compared to MDMA alone, the trend did not reach statistical significance. Conversely, MDMA caused a marked reduction in DOPAC levels 60-240 min post-treatment (p < 0.001). Ro4-1284 124

alone significantly elevated DOPAC levels in the striatum immediately after dosing, an effect that persisted for 210 min in the Ro4-1284/saline group (p < 0.05). Upon MDMA injection, DOPAC concentrations decreased in the Ro4-1284/MDMA group and remained depleted 90-240 min post-treatment in respect to saline controls (p < 0.001). Importantly, elevated DOPAC levels resulting from Ro4-1284 pretreatment were sustained 30-90 min following MDMA exposure (p <

0.001). In contrast, striatal 5-HIAA concentrations decreased more gradually with MDMA treatment, reaching statistical significance over the saline control group between 210-240 min post-drug administration (p < 0.05) (Figure 4-7). Extracellular 5-HIAA content significantly increased 30 min after Ro4-1284 treatment and remained elevated for 60 min (p < 0.05).

Following MDMA treatment, 5-HIAA levels in the Ro4-1284/MDMA group were slightly lower, however, the change is not statically significant in comparison to saline controls. Overall, Ro4-

1284 pretreatment had minimal effects on MDMA-induced release of 5-HT and DA in rat striatum.

However, Ro4-1284 elevated striatal concentrations of corresponding metabolites, 5-HIAA and

DOPAC, indicative of increased monoamine turnover. Additionally, the diminishing effects of

MDMA on extracellular monoamine metabolites is delayed with Ro4-1284 challenge.

4.2.6 Prevention of MDMA-Mediated Indoleamine Depletion with Acute Antagonism of

VMAT2 Function

In order to determine the effect of VMAT2 inhibition on MDMA neurotoxicity, tissue neurotransmitter concentrations were assessed in the cortex (Cx) and striatum (St), 7 days after

MDMA (20 mg/kg, sc) exposure. Basal indoleamine levels in saline-treated animals were as follows: [5-HTCx = 1.64 ± 0.22, 5-HTSt = 1.52 ± 0.16, 5-HIAACx = 1.64 ± 0.081, 5-HIAASt = 1.92 ±

0.095 pmol/mg of tissue) (Figure 4-9). One-way ANOVA revealed significant differences in neurotransmitter levels among the treatment groups [5-HTCx (F (3, 21) = 6.98, p < 0.0019), 5-HTSt 125

(F (3, 20) = 10.92, p < 0.0002), 5-HIAACx (F (3, 21) = 20.23, p < 0.0001), 5-HIAASt (F (3, 21) = 34.92, p < 0.0001)]. Fisher-Hayter pairwise comparisons exhibited significant reductions in 5-HT concentrations in both cortex (~50%, p < 0.05) and striatum (~60%, p < 0.05) of MDMA-treated rats when compared to saline controls. Similarly, decreases in cortical (62%, p < 0.05) and striatal

(65%, p < 0.05) 5-HIAA levels were apparent after MDMA administration. Tissue indoleamine content was unaffected by Ro4-1284 treatment, as evidenced by similar neurotransmitter concentrations observed in the Ro4-1284/saline and saline-only treatment groups. Furthermore,

5-HT and 5-HIAA levels in Ro4-1284/MDMA-exposed rats were comparable to those of saline controls, but significantly higher from those of the MDMA treatment group in both brain regions analyzed (p < 0.05). Thus, pretreatment with Ro4-1284 prevented MDMA-induced 5-HT deficits.

The selectivity of MDMA towards the serotonergic system was confirmed by evaluating changes in tissue catecholamine levels in SD rats 7 days following peripheral MDMA administration (20 mg/kg, sc). Saline-treated animals displayed basal DA and DOPAC levels in rat striatum: [DA = 56.88 ± 1.92, DOPAC= 16.41 ± 0.91 pmol/mg of tissue] (Figure 4-10). One-way

ANOVA indicated no significant differences in catecholamine content among the treatment groups [DA (F (3, 21) = 1.46, p = 0.25; DOPAC (F (3, 21) = 1.84, p = 0.17]. As expected, MDMA failed to induce damage to the dopaminergic system in rats. More importantly, the data asserts that

Ro4-1284 treatment alone or in combination with MDMA has no enduring effects on brain catecholamine content.

4.2.7 Inhibition of VMAT2 Reduces Persistent Serotonergic Nerve Terminal Damage in Rats

Exposed to MDMA

Immunohistochemical staining of SERT is considered a sensitive marker of 5-HT nerve terminal damage in MDMA neurotoxicity (Xie et al., 2006). Low magnification images illustrate 126

widespread changes in SERT staining pattern upon MDMA treatment in rat striatum (Figure 4-11).

Saline treated animals displayed basal levels of SERT protein in this brain region. Higher magnification images revealed staining of 5-HT nerve endings (fibrous appearance) and terminal buttons (granular pattern) (Figure 4-12). MDMA induced severe reductions in SERT protein expression, with decreased axonal and terminal button staining 7 days post-treatment (Figure 4-

12B). Furthermore, the loss in SERT protein reflects a decrease in striatal 5-HT nerve terminal density. In contrast, treatment with Ro4-1284 alone had no discernable effects on SERT staining.

Similarly, Ro4-1284/MDMA-treated animals displayed minimal to no changes in 5-HT nerve terminal marker expression from saline controls (Figure 4-12D). Individual variability in SERT staining patterns, in response to the different treatments, paralleled the results obtained in the neurochemical assay. Overall, these findings suggest that Ro4-1284 pretreatment attenuated long-term reductions in serotonergic nerve terminal density in rat striatum associated with

MDMA neurotoxicity. 127

Figure 4-1 - Acute Effects of Ro4-1284 on Striatal Indoleamine and Catecholamine Content

Concentrations of 5-HT, DA and corresponding metabolites, 5HIAA and DOPAC were assessed in striatum at 1 h and at 12 h following Ro4-1284 (10mg/kg, ip) treatment by HPLC-ECD as described under Materials and Methods. Results show individual concentrations of these monoamines in each treatment group. Values are different from the DMSO control group at (*) p<0.05 and from the Ro4-1284 (12 h) group at (#) p<0.05. 128 129

Figure 4-2 - Pretreatment with Ro4-1284 Prevents MDMA-Mediated Hyperthermia in Rats

Temperature probes implanted into the peritoneal cavity of SD rats recorded core body temperature in 3 min intervals. Animals were given Ro4-1284 (10mg/kg, ip) 1 h prior to MDMA (20mg/kg, sc) or saline (control) treatment. The black arrow represents the time of Ro4-1284 pretreatment, and zero indicates the time of MDMA treatment. Results are expressed as means ± S.E. (n=5-8 in each group) and represent temperature measurements collected from the four different treatment groups. Values are different from the saline group at (*) p < 0.05, from Ro4- 1284/saline group at (#) p < 0.05 and from MDMA group at (ξ) p < 0.05. 130

Figure 4-3 - Ro4-1284 Pretreatment Has No Effect on Acute Induction of Plasma NE Levels after MDMA Administration

SD Rats were given Ro4-1284 (10mg/kg, ip) 1 h prior to a single dose of MDMA (20mg/kg, sc). Blood was collected 1 h following MDMA treatment and plasma NE levels were assayed by HLPC- ECD. Scatter plots display plasma NE levels from individual experimental subjects and the mean (SE ± 6) for each treatment group. Values are different from the saline group at (*) p < 0.05 and from Ro4-1284/saline group at (#) p < 0.05. 131 132

Figure 4-4 - Effects VMAT2 Inhibition on MDMA-Induced Horizontal Locomotor Activity

Locomotor activity was monitored by placing animals into open-field activity chambers. Baseline levels of locomotor activity were measured for 30 min, followed by a 60 min pretreatment period with either DMSO or Ro4-1284 and a 120 min post-treatment period after MDMA (20mg/kg, sc) or saline administration. The graph shows the mean (SE± 6-7) horizontal velocity (B) at each time interval and the scatter plot displays the %Δ of total horizontal velocity from baseline (A). The black arrow represents the time of Ro4-1284 pretreatment and zero indicates the time of MDMA treatment. Values are different from the saline group at (*) p < 0.05, from Ro4-1284/saline group at (#) p < 0.05 and from MDMA group at (ξ) p < 0.05. 133 134

Figure 4-5 - Effects VMAT2 Inhibition on the Stimulation of Vertical Activity with MDMA Treatment

Locomotor activity was monitored by placing animals into open-field activity chambers. Baseline levels of locomotor activity were measured for 30 min, followed by a 60 min pretreatment period with either DMSO or Ro4-1284 and a 120 min post-treatment period after MDMA (20mg/kg, sc) or saline administration. The graph shows the mean (SE± 6-7) vertical activity (B) at each time interval and the scatter plot displays the %Δ of total vertical activity from baseline (A). The black arrow represents the time of Ro4-1284 pretreatment and zero indicates the time of MDMA treatment. Values are different from the saline group at (*) p < 0.05, from Ro4-1284/saline group at (#) p < 0.05 and from MDMA group at (ξ) p < 0.05. 135

Figure 4-6 - Stereotaxic Representation of Microdialysis Probe Implantation into Rat Striatum

Coronal section illustrating the placement of microdialysis probes in the area of the brain corresponding to the striatum, or caudate putamen (anteroposterior, +0.2 mm; mediolateral, ± 3.0 mm; dorsoventral, 7.0 mm). (A) guide cannula, 4.0mm; (B) cellulose membrane, 3.0 mm. Image was adapted from the Paxinos and Watson rat brain atlas (Paxinos and Watson, 1997). 136 137

Figure 4-7 - Dialysate Indoleamine Levels Following Ro4-1284 and MDMA Treatment in Rat Striatum

Animals received Ro4-1284 (10mg/kg, ip), followed by a single injection of MDMA (20mg/kg, sc) 1 h later. 30-min striatal dialysate samples were collected via in vivo microdialysis and assayed for indoleamine content through HLPC-ECD analysis. Results display the mean ± S.E. (n = 6-8 in each group) dialysate concentrations in each treatment group. Basal extracellular 5-HT levels were below level of detection, thus, values are presented as pg/ 10µL of dialysate. Extracellular 5-HIAA levels are expressed as % of baseline. The black arrow represents the time of Ro4-1284 pretreatment and zero indicates the time of MDMA treatment. Values are different from the saline group at (*) p < 0.05, from Ro4-1284/saline group at (#) p < 0.05 and from MDMA group at (ξ) p < 0.05. 138 139

Figure 4-8 - Dialysate Catecholamine Levels Following Ro4-1284 and MDMA Treatment in Rat Striatum

Animals received Ro4-1284 (10mg/kg, ip), followed by a single injection of MDMA (20mg/kg, sc) 1 h later. 30-min striatal dialysate samples were collected via in vivo microdialysis and analyzed for catecholamine content through HLPC-ECD analysis. Results show the mean ± S.E. (n = 6-8 in each group) extracellular DA and DOPAC concentrations expressed as % of baseline. The black arrow represents the time of Ro4-1284 pretreatment and zero indicates the time of MDMA treatment. Values are different from the saline group at (*) p < 0.05, from Ro4-1284/saline group at (#) p < 0.05 and from MDMA group at (ξ) p < 0.05. 140

Figure 4-9 - Ro4-1284 Pretreatment Affords Protection against MDMA-Mediated Serotonin Depletions

5-HT and 5-HIAA levels were measured from cortex and striatum 7 days after MDMA exposure by HPLC-ECD. Ro4-1284 was administered 1 h prior to MDMA treatment (20mg/kg, sc). Results show individual indoleamine concentrations in each treatment group. Values are different from the saline group at (*) p < 0.05, from Ro4-1284/saline group at (#) p < 0.05, from MDMA group at (ξ) p < 0.05. 141

Figure 4-10 - Ro4-1284 Pretreatment Does Not Inflict Long-Lasting Changes in Catecholamine Content

DA and DOPAC levels were measured from striatum 7 days after MDMA exposure by HPLC-ECD. Ro4-1284 was administered 1 h prior to MDMA treatment (20mg/kg, sc). Results show individual catecholamine concentrations in each treatment group. 142 143

Figure 4-11 - VMAT2 Inhibition Prevents Widespread Reductions in Striatal SERT Staining Produced by MDMA

Representative images of the striatum were captured by light microscopy. Coronal sections were stained for SERT in animals treated with saline (A), MDMA (B), Ro4-1284/saline (C) and Ro4- 1284/MDMA (D), 7 days post-drug administration. Image magnification is 100x and scale bars at the lower right corner of each panel denote 75 µm. 144 145

Figure 4-12 - Ro4-1284 Pretreatment Attenuates Loss of Serotonergic Nerve Terminal Density in Rat Striatum Following MDMA Dosing

Representative images of the striatum were captured by light microscopy. Coronal sections were stained for SERT in animals treated with saline (A), MDMA (B), Ro4-1284/saline (C) and Ro4- 1284/MDMA (D), 7 days post-drug administration. Image magnification is 400x and scale bars at the lower right corner of each panel denote 25 µm. 146

4.3 DISCUSSION

VMAT2 is involved in the storage of catecholamine and indoleamine neurotransmitters within neurons, thereby influencing quantal size, receptor sensitivity and synaptic plasticity

(Gasnier, 2000; Pothos et al., 2000; Sulzer and Pothos, 2000). VMAT2 function is also crucial to the pharmacological mechanism of many psychostimulant drugs. Compounds that selectively target VMAT2 disrupt monoamine storage and release, hindering the behavioral and biochemical effects of amphetamine and methamphetamine. The present findings demonstrate that pharmacological inhibition of VMAT2 with Ro4-1284 pretreatment protects rats against the acute and long-term toxicities of MDMA. Furthermore, the data identifies a role for VMAT2 in the in vivo mechanism of action of MDMA.

Ro4-1284 (10mg/kg, ip) caused significant reductions in striatal monoamine content at 1 h post-treatment, confirming disruption of VMAT2 function at the time of MDMA administration

(Figure 4-1). Interestingly, Ro4-1284 was more potent at depleting tissue DA levels compared to

5-HT levels. The findings are consistent with previous reports that indicate brain 5-HT is less sensitive to the effects of the acute VMAT2 inhibitor, tetrabenazine, than DA (Pettibone et al.,

1984). Moreover, increases in corresponding DA and 5-HT metabolites, DOPAC and 5-HIAA, also occurred with Ro4-1284 treatment, reflecting stimulation in monoamine turnover. Indeed, DA and 5-HT deficits resulting from VMAT2 antagonism by tetrabenazine are reversed by inhibition of monoamine oxidases (Pettibone et al., 1984), which likely contributes to the increase in deaminated metabolites observed with Ro4-1284 treatment.

The thermoregulatory response to MDMA is profoundly impacted by Ro4-1284 pretreatment. A single neurotoxic dose of MDMA (20mg/kg, sc) induced prominent hyperthermia in rats that was completely abolished in the presence of Ro4-1284 (Figure 4-2). Irreversible 147

inhibition of VMAT2 with reserpine also attenuates MDMA-mediated hyperthermia (Sabol and

Seiden, 1998). Interestingly, reserpine-only treatment causes significant hypothermia that persists beyond the administration of MDMA (Sabol and Seiden, 1998) and beyond the presence of the drug in the brain and plasma (Brodie et al., 1956). Thus, it is difficult to discern whether the protective effects of reserpine against the thermogenic effects of MDMA are directly correlated to its vesicular depleting activity or the consequence of an induction of hypothermia. In the present study, and despite Ro4-1284-induced reductions in tissue 5-HT and DA levels (Figure 4-

1), body temperature was unaffected by Ro4-1284 treatment alone. Acute and chronic exposure to Ro4-1284 (40mg/kg, ip) can cause intense hypothermia in rats due to its proficiency at inhibiting VMAT2 function and reducing tissue monoamine levels (Yehuda et al., 1999). However, the lower dose of Ro4-1284 administered in this study was insufficient to elicit hypothermia.

Hyperthermia produced by MDMA involves activation of central post-synaptic 5-HT2A and D1 receptors (Mechan et al., 2002; Shioda et al., 2008). Therefore, we speculate that Ro4-1284 prevents MDMA-induced hyperthermia by stimulating monoamine depletion; however, additional studies are needed to investigate this hypothesis. Moreover, MDMA in conjunction with Ro4-1284 decreases body temperature, perhaps by further enhancing the ability of Ro4-1284 to diminish brain DA and 5-HT levels.

MDMA-mediated hyperthermia resembles facultative thermogenesis in warm-blooded animals, requiring activation of thermoregulatory control centers in the brain alongside stimulation of the sympathetic nervous system (Mills et al., 2004). Intravenous infusion of NE increases body temperature in rats (Ribeiro et al., 2000) and plasma NE release plays a critical role in modulating the peripheral thermogenic effects of MDMA through induction of heat generation and prevention of heat dissipation. In the present study, plasma NE concentrations were 148

markedly elevated 60 min after MDMA dosing (Figure 4-3), supporting the contention that enhanced NE levels in the periphery are important for the manifestation of MDMA-mediated hyperthermia. Nonetheless, the 2.2-fold increase in NE levels observed after MDMA treatment is considerably lower than the 35-fold increase previously reported by Sprague at al. (2005). The use of a lower MDMA dose in our studies could perhaps account for such discrepancy along with other differences in the experimental procedure. Furthermore, Ro4-1284 pretreatment had no effect on MDMA-related induction of circulating NE levels. Given that Ro4-1284 completely abolished hyperthermia produced by MDMA, the findings are confounding and suggest that peripheral VMAT2 inhibition contributes minimally to the protective mechanism of Ro4-1284.

Depression of VMAT2 function reduced horizontal velocity in MDMA-treated rats (Figure

4-4). DA neurotransmission is a critical mediator of the extrapyramidal motor system, and suppression of locomotor activity in reserpine-treated mice has been attributed to depletion of brain catecholamines (Smith and Dews, 1962). MDMA-induced elevation of locomotor activity is associated with enhanced striatal neuronal activity and modulation of D1/2 receptors (Ball et al.,

2003). Thus, attenuation in horizontal hyperactivity mediated by MDMA is perhaps related to the robust DA-depleting activity of Ro4-1284 treatment (Figure 4-1). Rats with deficiencies in SERT

(Chapter 3) and mice with 5-HT1B receptor deficiencies (Scearce-Levie et al., 1999) exhibit delayed onset of horizontal hyperactivity following peripheral MDMA administration. In light of the fact that 5-HT pathways are seemingly important to the early-phase locomotor effects of MDMA

(Scearce-Levie et al., 1999), decreases in brain 5-HT following Ro4-1284 administration may be responsible for the late onset of MDMA-induced horizontal activity. As such, the data supports a role for VMAT2 in the initiation and maintenance of horizontal hyperlocomotion upon MDMA dosing. In contrast, abolishment of rearing behavior (Figure 5) is most likely the consequence of 149

prominent striatal DA depression with Ro4-1284 treatment, given that antagonism of D1/2 receptors prevents induction of vertical activity following MDMA administration in rats (Bubar et al., 2004). Indeed, genetic ablation of the 5-HT1B receptor, and 5-HT uptake inhibition by fluoxetine, block stimulation of floor-plane locomotion without affecting vertical activity, which suggests that exploratory behaviors, such as rearing, are minimally impacted by 5-HT pathways stimulated by MDMA (Callaway et al., 1990; Scearce-Levie et al., 1999).

Overall, reversible antagonism of VMAT2 with Ro4-1284 attenuated MDMA-induced hyperthermia and hyperactivity. Compelling evidence exists for the involvement of enhanced monoamine release and neurotransmission in the acute responses to MDMA. Acute VMAT2 inhibition caused alterations in brain monoamine concentrations evident by decreases in striatal

5-HT and DA with Ro4-1284 treatment. Similarly, VMAT2 deficiency in mice elicits reductions in brain DA, 5-HT, and NE levels (Wang et al., 1997), and the appearance of depressive-like behaviors, including locomotor retardation (Fukui et al., 2007), and enhanced sensitivity to the locomotor effects of psychostimulants, such as amphetamine and cocaine (Wang et al., 1997).

Moreover, selective inhibition of VMAT2 with N-1,2-dihydropropyl lobeline analogs reduce methamphetamine-induced DA release from brain slices and affords protection against the behavioral effects of this drug (Beckmann et al., 2010). Collectively, these findings suggest that modulation of vesicular monoamine storage remarkably influences the pharmacology of amphetamines; therefore, it should be anticipated that VMAT2 blockade by Ro4-1284 modulates acute MDMA toxicity seemingly through disruption of monoamine storage and release.

In vivo brain microdialysis was employed to examine the effects of acute VMAT2 inhibition on MDMA-mediated monoamine release. The potent monoamine-depleting properties of Ro4-1284 have been extensively demonstrated (German et al., 1981; Staal and Sonsalla, 2000); 150

however, studies often evaluate changes in tissue monoamine levels, most likely reflective of intracellular concentrations of these neurotransmitters. Evaluation of the effect of Ro4-1284 on monoamine levels in the extracellular space is currently lacking. According to our data, Ro4-1284 treatment alone does not influence dialysate 5-HT and DA concentrations in the striatum, whereas, extracellular concentrations of corresponding metabolites, 5-HIAA and DOPAC, were noticeably augmented, confirming stimulation in monoamine turnover (4-6 and 4-7). The data coincides with the elevation in tissue 5-HIAA and DOPAC levels observed with Ro4-1284 (Figure

4-1) and reflects increases in monoamine oxidation within the cytosol consequential to the loss of VMAT2 function that lead to accumulation of 5-HIAA and DOPAC in the extracellular space.

Furthermore, stimulation in monoamine metabolite levels with Ro4-1284 was reversed by MDMA treatment, although 5-HIAA concentrations appeared to be more resistant to the depleting activity of MDMA. MDMA is capable of inhibiting MAOs in order to enhance efflux of parent monoamines (Leonardi and Azmitia, 1994), thus, accounting for the reversal of Ro4-1284- mediated increases in dialysate 5-HIAA and DOPAC concentrations upon MDMA exposure.

Noteworthy, Ro4-1284 markedly delayed depletion of monoamine metabolites by MDMA, however, the significance of these effects is unknown. The fact that Ro4-1284 did not impact basal

5-HT and DA levels in the striatum is consistent with other studies using other VMAT2 inhibitors

(Eyerman and Yamamoto, 2005; Meyer et al., 2013) and with our findings that Ro4-1284-only treatment has no effects on thermoregulation and locomotor activity.

Most surprisingly, elevations in extracellular 5-HT and DA content in rat striatum following

MDMA administration were minimally affected by Ro4-1284 pretreatment. Contrasting results have shown that MDMA-evoked 5-HT efflux is partially blocked by VMAT2 inhibition with reserpine (Fitzgerald and Reid, 1993; Gu and Azmitia, 1993; Sabol and Seiden, 1998). 151

Nonetheless, other studies revealed that MDMA and other amphetamines are capable of inducing synaptic monoamine release from reserpine-insensitive pools (Johnson et al., 1991). Altogether, our results do not support the contention that Ro4-1284 modulates the acute MDMA response by reducing excess monoamine release. In fact, MDMA in conjunction with Ro4-1285, appeared to enhance extracellular DA concentrations slightly over MDMA treatment alone. A plausible explanation for such phenomenon could be the putative stimulation of DA synthesis as a compensatory mechanism for the vesicular diminishing activity of Ro4-1284, thereby augmenting

MDMA-mediated DA release. Indeed, depletion of brain monoamines with reserpine has been shown to increase catecholamine synthesis by inducing tyrosine hydroxylase (TH) activity

(Carlsson et al., 1977; Mueller et al., 1969). Although conceivable, no further evidence supports the enhancement of MDMA-mediated monoamine release upon depression of VMAT2 function.

In addition, selective antagonism of VMAT2 with the lobeline derivative, GZ-793A, attenuated methamphetamine-evoked DA release in the nucleus accumbens shell of rats by interfering with vesicular storage and TH activity (Meyer et al., 2013). The disparity between the present results and those of Meyer et al. (2013) perhaps relies on incongruences in the mechanism of action of

MDMA and methamphetamine, or differences in the selectivity of the two compounds used for the inhibition of VMAT2 protein. Indeed, the parent compound, lobeline, possess lower affinity for VMAT2 than GZ-793A (Horton et al., 2011) and much like Ro4-1284 is inefficient at inhibiting stimulant-mediated DA release in vivo (Eyerman and Yamamoto, 2005; Meyer et al., 2013).

Notably, lobeline prevents hyperthermia (Eyerman and Yamamoto, 2005) along with increased stereotypy, locomotor activity and self-administration in methamphetamine-exposed rats

(Harrod et al., 2001; Miller et al., 2001; Neugebauer et al., 2007; Tatsuta et al., 2006). 152

The fact that Ro4-1284 had such antagonizing effects on the thermoregulatory and behavioral response to MDMA, without affecting MDMA-induced monoamine efflux in the striatum is most perplexing. Although central monoamine release, primarily of 5-HT, is mechanistically important to the manifestation of acute MDMA toxicity, studies have failed to show a direct correlation between hyperthermia and the induction of extracellular 5-HT following repeated MDMA dosing (Rodsiri et al., 2008). Likewise, 5-HT reuptake inhibitors prevent excess

5-HT and DA release, as well as, long-term neurotoxicity (Gudelsky and Nash, 1996; Sanchez et al., 2001) but have no effect on MDMA-mediated hyperthermia (Malberg et al., 1996; Schmidt et al., 1990b). In contrast, substantial evidence exist to suggest a role for 5-HT receptor function in

MDMA-induced hyperthermia and hyperlocomotor activity (Docherty and Green, 2010; Gudelsky and Yamamoto, 2008). Remarkably, antagonism of post-synaptic 5-HT2A receptors reduces the increase in monoamine levels centrally, and attenuates the thermal response to MDMA (Shioda et al., 2008). Meanwhile, blockade of 5-HT2A/2C receptors decreases MDMA-induced hyperactivity and striatal neuronal activity (Ball and Rebec, 2005). It is possible that Ro4-1284 has an indirect antagonistic effect at post-synaptic monoaminergic receptors, thus interfering with the neuropharmacological effects of MDMA without altering monoamine release. However, at present, there is no data that supports this hypothesis.

Another explanation for the perceived lack of inhibition of MDMA-evoked monoamine release by Ro4-1284, relies on the fact that inductions in neurotransmitter levels were only measured in the dorsal striatum (caudate nucleus and putamen). Thermoregulatory centers reside within the hypothalamus (Rothwell, 1994) and evidence suggests that MDMA-induced hyperthermia requires activation of hypothalamic pathways (Stephenson et al., 1999).

Correspondingly, induction in dorsal striatal monoamine release and neuronal activity is thought 153

to be involved in the stereotypy (continuous sniffing, head bobbing, circling and mouthing) produced by MDMA, rather than in its locomotor activity effects, which requires input from the nucleus accumbens (Freed and Yamamoto, 1985; Kelly et al., 1975). In summary, increases in striatal 5-HT and DA concentrations examined herein are not reflective of the thermogenic and hyperlocomotor activity response to MDMA, thus requiring further investigation of the effects of

Ro4-1284 pretreatment on MDMA-mediated monoamine release in other brain regions, including the hypothalamus and nucleus accumbens.

Pharmacological inhibition of VMAT2 with Ro4-1284 afforded protection against the long- term neurotoxic response to MDMA. Deficits in tissue 5-HT and 5-HIAA 7 days following MDMA exposure were completely abolished in the presence of Ro4-1284 (Figure 4-9). In agreement with the neurochemical assay findings, Ro4-1284-treated rats showed resistance to decreases in SERT staining mediated by MDMA, displaying minimal to no signs of a decline in serotonergic axonal density (Figure 4-12). Reserpine pretreatment has yielded inconclusive evidence regarding the effect of VMAT2 inhibition on MDMA neurotoxicity. Hekmatpanah et al. (1989) confirmed depletion of brain DA and 5-HT content at the time of reserpine (5m/kg, ip) administration, but noted no changes in MDMA-mediated reduction of 5-HT uptake sites. Conversely, a study by

Schmidt et al. (1990b) reported a protective effect of reserpine (5mg/kg, sc) against MDMA

(20mg/kg, sc) neurotoxicity by measuring decreases in TPH activity, since tissue 5-HT levels remained markedly attenuated 1 week after reserpine-only treatment. The utilization of distinct endpoint markers of neurotoxicity may, in part, account for such discrepancies in the results.

Additionally, the persistent monoamine depleting activity of reserpine could be a confounding factor when evaluating the neurotoxic effects of MDMA. Serotonin deficits develop gradually 1 to

7 days after MDMA exposure (Schmidt, 1987), a time window seemingly important for the 154

manifestation of lasting biochemical and structural damage to 5-HT terminals caused by the analogous serotonergic neurotoxicant, 5,7-DHT (Baumgarten and Bjorklund, 1976; Capela et al.,

2008). Unlike reserpine, the monoamine depleting properties of Ro4-1284 were short-acting, as evident by the full recovery of DA and 5-HT concentrations at 12 h post-treatment (Figure 4-1), and by the lack of alterations in baseline tissue monoamine content (Figure 4-9 and 4-10) and

SERT staining (Figure 4-12) 7 days after Ro4-1284 challenge. Altogether, our findings provide convincing evidence of the involvement of VMAT2 in MDMA neurotoxicity.

The etiology of MDMA neurotoxicity remains largely unknown. Furthermore, little is known about the role of VMAT2 in the long-lasting effects of MDMA, making it difficult to speculate on the mechanism driving Ro4-1284 neuroprotection against MDMA. Evidence suggests that VMAT2 is reduced upon psychostimulant treatment. Declines in VMAT2 immunoreactivity occur immediately after exposure to a neurotoxic regimen of methamphetamine (Eyerman and

Yamamoto, 2005; Riddle et al., 2002), which validates the prevailing view that depression of

VMAT2 function precedes the development of long-term neurotoxicity mediated by amphetamines (Brown et al., 2000; Riddle et al., 2002). Similarly, enduring loss of VMAT2 ligand binding has been reported after repeated high-dose psychosimulant treatment (Frey et al., 1997;

Hogan et al., 2000; Ricaurte et al., 2000) and VMAT2 protein expression is attenuated in the striatum of old mice (10 week-olds) 7 days following MDMA administration (Reveron et al., 2005).

One possible explanation for the protective mechanism of Ro4-1284 against long-term

MDMA neurotoxicity could lie in the concomitant attenuation of acute hyperthermia. Speculation exists about the role of hyperthermia in MDMA neurotoxicity through the exacerbation of free radical formation (Green et al., 2003). Free radical production is enhanced in MDMA-treated animals (Colado et al., 1997b; Gudelsky, 1994), which presumably assists in the 155

neurodegenerative process of serotonergic terminals. Ro4-1284 pretreatment completely obliterated MDMA-induced hyperthermia in SD rats, perhaps rendering them less susceptible to

MDMA neurotoxicity. However, it is unlikely that attenuation of MDMA thermogenesis is entirely responsible for prevention of neurotoxicity. For instance, hyperthermia is not essential to the neurotoxic effects of MDMA, since 5-HT deficits occur in Dark Agouti rats exposed to a low-dose

MDMA regimen (4mg/kg, twice daily for 4 days) in the absence of hyperthermia (O'Shea et al.,

1998). Correspondingly, a number of compounds afford neuroprotection without altering the thermoregulatory response to MDMA, including 5-HT reuptake inhibitors (Malberg et al., 1996;

Sanchez et al., 2001) and anti-oxidants (Colado et al., 1997b; Shankaran et al., 2001; Yeh, 1999).

Furthermore, VMAT2 inhibition with lobeline 2 h after dissipation of methamphetamine-induced hyperthermia attenuated enduring DA depletion, indicating that the neuroprotective effects of lobeline are partly independent of its ability to modulate the temperature response to methamphetamine (Eyerman and Yamamoto, 2005). These findings also highlight the importance of late-phase events in the manifestation of psychostimulant-mediated neurotoxicity, which most likely occur following the initial pharmacological effects (Eyerman and Yamamoto, 2005). Hence,

Ro4-1284 likely acts through a combination of temperature dependent and independent mechanisms to reverse MDMA neurotoxicity. An investigation of the effects of Ro4-1284 in the presence of MDMA thermogenesis is necessary in order to confirm such conjecture.

Alternatively, the neuroprotective response elicited by Ro4-1284 pretreatment is perhaps most closely related to its monoamine depleting activity as a consequence of VMAT2 antagonism.

In particular, the profound depression of brain DA content produced by Ro4-1284 could account for the abolishment of MDMA neurotoxicity. The pivotal role of DA in MDMA-mediated neurotoxicity has been long recognized. In support of this view, attenuation of DA availability 156

prevents serotonergic deficits (Schmidt et al., 1990a; Schmidt et al., 1991; Stone et al., 1988), whereas, stimulation of DA synthesis and neurotransmission aggravate injury to 5-HT neurons upon MDMA treatment (Aguirre et al., 1998; Schmidt et al., 1991). The readily oxidizable nature of DA, resulting in the accumulation of ROS and reactive quinone species, could provide a mechanism by which DA metabolism within 5-HT neurons contributes to MDMA neurotoxicity.

DA quinones covalently modify cysteine residues giving rise to 5-cysteinyl-catechols at the active sites of a number of proteins, including parkin, TH and DAT (Machida et al., 2005; Whitehead et al., 2001; Xu et al., 1998). These covalent modifications interfere with protein function, compromising cellular integrity. Therefore, quinone formation derived from DA auto-oxidation has been implicated in the pathogenesis and progression of Parkinson’s disease (Ogawa et al.,

2000) and the long-term neurotoxicity of methamphetamine (LaVoie and Hastings, 1999).

DA is primarily oxidized in the cytosol by MAOB, which generates H2O2 and reactive aldehyde intermediates (Nagatsu, 2004). ROS production from MAOB-derived metabolism of DA can adversely impact mitochondrial function. A detailed analysis of oxidatively modified mitochondrial proteins in rat liver following MDMA treatment reveals extensive alterations in proteins involved in energy supply, fatty acid metabolism, antioxidant response and chaperone function (Moon et al., 2008). Mitochondrial dehydrogenase 2 (ALD2), 3-ketoacyl-CoA thiolases and ATP synthase are of particular interest, due to their prominent inactivation (Moon et al.,

2008). Inhibition of ALD2 leads to accumulation of toxic lipid aldehydes (Jayanthi et al., 1999), which coupled with insufficient supply of ATP from free fatty acid metabolism, due to inactivation of 3-ketoacyl-CoA thiolases and ATP synthase (Eaton et al., 2000), could eventually result in cell death and tissue organ damage (Easton et al., 2003; Jayanthi et al., 1999). Similarly, DA quinones induce changes in mitochondrial membrane permeability by interacting with the mitochondrial 157

permeability transition pore (PTP) (Berman and Hastings, 1999). PTP opening causes depolarization of transmembrane potential, osmotic swelling and loss of oxidative phosphorylation (Berman and Hastings, 1999). As such, PTP modulation has been associated with a number of mechanisms of neuronal cell injury, including excitotoxicity, ischemia and dopaminergic neurotoxicity in Parkinson' disease (Cassarino et al., 1998; Nieminen et al., 1996;

Uchino et al., 1995; Zamzami et al., 1996). In summary, DA-mediated ROS production and quinone reactivity may stimulate mitochondrial dysfunction, providing a rationale for the contribution of DA to the systematic degeneration of serotonergic nerve terminals during MDMA neurotoxicity.

Contributions from other neurotransmitter systems to MDMA neurotoxicity, 5-HT in particular, cannot be disregarded. In spite of the fact that decreases in 5-HT content by Ro4-1284 treatment were less pronounced compared to DA, 5-HT pathways have been implicated in the neurotoxic response to MDMA (Sanchez et al., 2001). In agreement with this hypothesis, data presented in Chapter 3 revealed alleviations in MDMA-mediated indoleamine depletion with SERT deficiency. Altogether, it is tempting to suggest that inhibition of VMAT2 with Ro4-1284 affords protection against MDMA neurotoxicity primarily by diminishing tissue monoamine levels and consequently interfering with the pharmacological effects of this psychostimulant drug. However, our preliminary results indicate that MDMA-evoked monoamine release is undisturbed in the presence of Ro4-1284. 158

CHAPTER 5: CONCLUDING REMARKS

5.1 SUMMARY

Studies undertaken in this dissertation provide convincing evidence for the involvement of SERT and VMAT2 in the mechanisms of MDMA toxicity. The acute pharmacological response to MDMA and related amphetamines results in the indirect modulation of monoamine neurotransmitter systems, predominantly of 5-HT origin, through excess synaptic release and reuptake inhibition (Johnson et al., 1986; Steele et al., 1987). In contrast, long-lasting neurotoxic effects of MDMA induce adverse biochemical and morphological changes that compromise 5-HT nerve terminal integrity, with sparing of raphe cell bodies, in a process referred to as retrograde degeneration (Capela et al., 2008). Work aimed at deciphering a role for the , SERT, in the acute and long-term effects of MDMA revealed attenuation of hyperthermia and locomotor activity alongside complete alleviation of 5-HT deficits with MDMA dosing in the absence of functional SERT. Furthermore, a pharmacological inhibition model was implemented to investigate potential contributions of the vesicular transporter, VMAT2.

Antagonism of VMAT2 protein prevented hyperthermia and MDMA neurotoxicity, whilst substantially reducing the behavioral locomotor effects of this psychostimulant drug.

SERT is a NaCl sensitive symporter that resides in the plasma membrane of serotonergic terminals, facilitating the rapid recycling of 5-HT from the synaptic cleft following neurotransmission. In this capacity, SERT regulates 5-HT homeostasis and serves as a primary target site for antidepressants and a number of drugs of abuse, including MDMA (Rothman and

Baumann, 2003; Schloss and Williams, 1998). Indeed, MDMA-evoked monoamine efflux in vitro and from rat brain is blocked by fluoxetine, an established SERT inhibitor (Berger et al., 1992;

Gudelsky and Nash, 1996). Additionally, MDMA exhibits greater potency at inhibiting SERT 159

compared to either DAT or NET in rat synaptosomes (Rothman et al., 2001). Indeed, differences in the neurotoxic profile of substituted amphetamines have been attributed, in part, to their differences in monoamine transporter potency (McCann and Ricaurte, 2004; Rudnick and Wall,

1992). Based on these findings, it is tempting to speculate that the interaction of MDMA with

SERT is integral to the pharmacological mechanism of this amphetamine. A prevailing hypothesis proposes that MDMA binds to SERT, acting as a substrate, and thereby inducing excess 5-HT release via a carrier-mediated exchange mechanism (Crespi et al., 1997; Gudelsky and Nash,

1996).

SERT deficiency in rats modestly reduces the time of exposure to elevated body temperatures upon multiple MDMA dosing, which may be a contributing factor in the concomitant prevention of MDMA neurotoxicity. Similarly, stimulation of horizontal velocity produced by MDMA was markedly delayed and attenuated with depression of SERT activity, emphasizing the importance of this transporter to the initiation and maintenance of the locomotor activity response to MDMA. Sufficient evidence supports a role for central monoamine release, and subsequent activation of post-synaptic receptors, in the thermoregulatory and behavioral effects of MDMA. For instance, pharmacological inhibition of 5-HT2A and D1 receptors averts MDMA-mediated hyperthermia (Mechan et al., 2002; Shioda et al., 2008), and risperidone, a non-selective 5-HT2A antagonist, prevents hyperthermia in conjunction with the release of 5-HT and DA in the hypothalamus (Shioda et al., 2008). In a similar manner, the locomotor response to MDMA is orchestrated by agonism of 5-HT2A and 5-HT1B receptors (Kehne et al., 1996;

McCreary et al., 1999; Scearce-Levie et al., 1999), and by activation of D1 and D2 receptors consequent to enhanced DA efflux in the nucleus accumbens (Bubar et al., 2004; Risbrough et al.,

2006). Thus, it can be assumed that SERT deficiency hinders acute MDMA toxicity by preventing 160

transporter-mediated increases in 5-HT levels in the CNS. The fact that marked elevations in peak body temperature and locomotor activity were still observed following MDMA treatment reveals that the release of other biogenic amines is seemingly preserved upon ablation of SERT expression. In particular, increases in delayed locomotor activity in SERT-KO rats reflect the stimulation of dopaminergic pathways presumably involved in the induction of late-phase hyperactivity by MDMA (Risbrough et al., 2006).

The precise mechanism of MDMA neurotoxicity remains elusive. Nevertheless, SERT has been previously recognized as a putative modulator of the long-term neurotoxic response to

MDMA. Selective blockade of the SERT protein by fluoxetine treatment diminished 5-HT deficits, implicating SERT in MDMA neurotoxicity (Sanchez et al., 2001; Shankaran et al., 1999a). However, concerns have been raised about the possible inhibitory effect of fluoxetine on MDMA metabolism (Upreti and Eddington, 2008), which could provide a rationale for the neuroprotection afforded by this compound. Indeed, MDMA metabolism is a requirement for neurotoxicity (Esteban et al., 2001; Gollamudi et al., 1989) and direct injection of MDMA metabolites into the CNS replicates the damage to the serotonergic system inflicted by peripheral administration of the parent drug (Bai et al., 1999; Miller et al., 1997). Results from the present study validate the importance of SERT in MDMA neurotoxicity. SERT deficiency eliminated persistent 5-HT depletions in brain regions vulnerable to the effects of MDMA. Moreover, restrictive impairment in basal tissue 5-HT levels led us to conclude that abolishment of MDMA neurotoxicity is related to diminished SERT activity and discernable disturbances in 5-HT homeostasis.

The protective effects of SERT deficiency may be a consequence of the absence of transport mechanisms that permit the uptake of MDMA and its neurotoxic metabolites into 161

serotonergic nerve terminals. Thioether metabolites of MDMA can undergo redox cycling to their corresponding ortho-quinones (Capela et al., 2006; Gomez-Toribio et al., 2009), catalyzing substantial ROS production and oxidative damage that compromises neuronal cell viability

(Capela et al., 2007; Capela et al., 2006) Undoubtedly, oxidative stress is increased upon MDMA treatment through elevations in membrane lipid peroxidation, MAO-mediated oxidation of monoamines and mitochondrial dysfunction (Colado et al., 1997a; Moon et al., 2008; Sprague and Nichols, 1995b). Most revealing, SERT appears to assist in the stimulation of ROS burden, since attenuation of free radical formation in rat brain is accomplished with fluoxetine treatment

(Shankaran et al., 1999a). In addition, studies emphasize significant contributions of DA to MDMA neurotoxicity, thus proposing that DA is transported into depleted 5-HT neurons, where it causes prominent oxidative damage subsequent to its deamination by MAOB (Sprague et al., 1998). In agreement with this hypothesis, MDMA and α-MeDA-derived metabolites stimulate in vitro DA uptake via SERT (Jones et al., 2004). As such, SERT deficiency may obstruct the transport of excess

DA into serotonergic neurons, influenced by MDMA, thereby preventing nerve terminal degeneration.

Disruption of VMAT2 function is speculated to enhance monoamine release following

MDMA administration by promoting the accumulation of biogenic amines within the cytosol that are available for transport-mediated efflux into the synaptic space (Rudnick and Clark, 1993).

VMAT2 protein controls the storage of catecholamine and indoleamine neurotransmitters into synaptic vesicles, thus influencing neurotransmission and neuronal integrity. Decreased VMAT2 expression causes disturbances in brain DA levels and dopaminergic signaling (Fon et al., 1997;

Takahashi et al., 1997), whilst VMAT2 deficient mice exhibit α-synuclein pathology and behavioral abnormalities that resemble Parkinson’s disease (Taylor et al., 2011). Notably, perturbations in 162

monoamine sequestration affect the interaction of neurons with exogenous compounds, including amphetamines (Gainetdinov et al., 1998; Guillot et al., 2008). Much like other psychostimulants, MDMA is a well-recognized substrate of VMAT2 (Partilla et al., 2006). Not surprisingly, abatement of VMAT2 activity with reserpine decreases MDMA-induced monoamine release from rat brain and cultured neurons (Fitzgerald and Reid, 1993; Gu and Azmitia, 1993;

Sabol and Seiden, 1998).

Through utilization of a pharmacological model of reduced VMAT2 function, we confirmed an essential role for VMAT2 in acute MDMA toxicity. The thermogenic effects of MDMA were obliterated by pretreatment with the reversible VMAT2 inhibitor, Ro4-1284.

Correspondingly, horizontal hyperlocomotion stimulated by MDMA was delayed and attenuated, whereas, enhanced rearing behavior was absent in Ro4-1284-treated rats. The perceivable attenuation in hyperthermia and locomotor activity with VMAT2 inhibition is congruent with the decrease in tissue 5-HT and DA levels and the increase in monoamine turnover produced by Ro4-

1284 at the time of MDMA treatment. Furthermore, it is conceivable that Ro4-1284 reverses

MDMA-induced hyperthermia through disruption of vesicular monoamine storage, both centrally and in the periphery. MDMA-mediated hyperthermia requires activation of thermoregulatory control centers in the brain alongside stimulation of the SNS (Mills et al., 2004). Such a mechanism could explain the complete abolishment of MDMA-mediated hyperthermia by Ro4-1284, with only a reduction in the locomotor activity response, which is controlled primarily through modulation of central DA neurotransmission. Nonetheless, elevations in plasma NE levels after

MDMA dosing were insensitive to Ro4-1284 pretreatment, contradicting the hypothesis that putative peripheral effects of Ro4-1284 participate in the prevention of MDMA thermogenesis.

Moreover, in vivo microdialysis revealed a lack of alterations in MDMA-mediated increases in 163

striatal 5-HT and DA in the presence of Ro4-1284. However, Ro4-1284 treatment delayed the depression of monoamine metabolites, 5-HIAA and DOPAC, after MDMA dosing. Although the nature of such effects remains to be determined, the findings could have repercussions with respect to prevention of acute-phase toxicity of MDMA by Ro4-1284. Thus, despite the inability of Ro4-1284 pretreatment to alter MDMA-induced monoamine release in the striatum, the data emphasizes the critical role of VMAT2 in the thermoregulatory and behavioral response to

MDMA.

Contributions of VMAT2 to MDMA neurotoxicity were further considered in this study.

Investigations into the mechanism of action of amphetamines showed that VMAT2 staining is significantly attenuated upon acute and chronic exposure to these compounds (Brown et al.,

2000; Brown et al., 2001; Hogan et al., 2000). Importantly, MDMA causes an enduring loss in

SERT uptake sites in the striatum that are accompanied by decreases in VMAT2, reflective of compromised serotonergic terminal viability (Ricaurte et al., 2000). Acute inhibition of VMAT2 function antagonized the neurotoxic effects of MDMA, as evidenced by the absence of tissue 5-

HT depletions and perturbations in serotonergic axonal density. The neuroprotection afforded by

Ro4-1274 pretreatment is likely orchestrated by temperature dependent and independent processes. Given that elevated body temperature exacerbates hydroxyl radical formation produced by MDMA (Colado et al., 1997b), it is reasonable to assume that elimination of MDMA- mediated hyperthermia helps alleviate the subsequent neurotoxicity by reducing oxidative stress.

Alternatively, severe decline in brain DA content prior to MDMA exposure could also account for the protective effects of Ro4-1284. The requirement for an intact dopaminergic system for the occurrence of 5-HT deficits after MDMA exposure suggests that DA is essential to MDMA neurotoxicity (Schmidt et al., 1990c; Stone et al., 1988). Moreover, the pro-oxidant nature of DA 164

provides a viable mechanism by which catecholamines can stimulate neurodegeneration of 5-HT terminals with MDMA treatment. MAO-mediated metabolism of DA within 5-HT neurons may stimulate the production of ROS and reactive quinone intermediates (Sprague et al., 1998).

Specifically, DA quinone reactivity has been linked to cytotoxicity via induction of mitochondrial impairments, reactive gliosis, and apoptosis (Miyazaki and Asanuma, 2009). Thus, one could infer that disruption of VMAT2 activity by Ro4-1284 pretreatment impedes DA availability, consequently reducing ROS burden and damage to serotonergic nerve endings. It should be noted, however, that MDMA-evoked DA release was minimally affected by Ro4-1284 treatment, requiring additional experiments to interpret such inconsistencies.

In closing, work performed in this dissertation implicates serotonin and vesicular monoamine transporters in the in vivo acute and long-term mechanisms of MDMA toxicity. In particular, the data emphasizes that diminished activity of these transporters profoundly impacts thermoregulation, behavior and neurotoxicity mediated by MDMA. Nevertheless, studies aimed at understanding the antagonistic effects of Ro4-1284 against MDMA toxicity are inconclusive, warranting further analysis. In this regard, the present results assist in understanding the mechanisms underlying amphetamine-induced alterations in the biochemistry and morphology of neurons, revealing insightful knowledge into the normal and abnormal functioning of the CNS with the anticipation of providing options for the treatment of drug abuse and neurodegenerative pathologies. 165

5.2 FUTURE DIRECTIONS

The data presented in Chapters 3 and 4 delineate important contributions of SERT and

VMAT2 proteins to the immediate and long-lasting effects following exposure to MDMA.

However, the precise mechanisms by which decreased function of these transporters conveys protection against MDMA-mediated hyperthermia, hyperactivity and neurotoxicity remains to be detailed. The acute effects of MDMA are mainly characterized by the prompt release of monoamine neurotransmitters, leading to the activation of post-synaptic receptors that are responsible for MDMA neuropharmacology. The indirect agonist properties of MDMA rely upon the interaction with SERT for the transport-mediated efflux of 5-HT. Hence, we speculate that

SERT deficiency diminishes the thermoregulatory and behavioral responses to MDMA by obstructing excess release of 5-HT from pre-synaptic terminals.

In vivo microdialysis is a sampling technique that has been extensively utilized to evaluate the effects of MDMA on monoaminergic neurotransmission in the CNS. This technique allows for the quantitation of neurotransmitter levels in real-time, thus revealing valuable information on the immediate neurochemical changes that occur upon exposure to MDMA. Indeed, brain microdialysis studies in awake rats displayed dose-dependent increases in extracellular 5-HT and

DA concentrations in the striatum and medial prefrontal cortex after peripheral MDMA administration (Baumann et al., 2005; Gudelsky and Nash, 1996; Yamamoto and Spanos, 1988).

Microdialysis experiments could provide insights into potential alterations in MDMA-evoked monoamine release consequent to the loss of SERT expression and perceivable disturbances in 5-

HT homeostasis in SERT-KO rats. Numerous studies suggest that stimulation of 5-HT activity impacts the release of other biogenic amines as evidenced by blockade of MDMA-induced DA release, and NE-related cardiovascular symptoms via inhibition of 5-HT uptake (Koch and 166

Galloway, 1997; Liechti and Vollenweider, 2000). While SERT deficiency may severely impact accumulation of dialysate 5-HT levels, it is our prediction that the release of other neurotransmitters is for the most part preserved, reflected in the marked elevations of peak body temperature and late-phase locomotor activity observed in MDMA-treated SERT-KO rats. In support of this view, mice deficient in SERT, or in 5-HT1B receptor, showed a lack of stimulation in

5-HT release with MDMA accompanied by prominent increases in DA concentrations in the extracellular space (Scearce-Levie et al., 1999; Trigo et al., 2007).

In addition to analyzing monoamine levels in the extracellular space via microdialysis, the pharmacokinetics of MDMA and MDMA metabolite accumulation in the CNS of SERT-KO rats can be determined. Our laboratory has employed microdialysis to show that thioether metabolites of

MDMA persist in the brain following multiple MDMA dosing (Erives et al., 2008). Notably, cellular uptake of MDMA and DA is accomplished via the SERT (Jones et al., 2004; Verrico et al., 2007), and it has been proposed that MDMA and its catecholamine metabolites gain entry into 5-HT terminals via SERT-dependent mechanisms. Based on this evidence, it is reasonable to suggest that the absence of functional SERT can conceivably enhance accumulation of MDMA and MDMA metabolites in the brain parenchyma. Indeed, SERT-mediated sequestration of MDMA into pre- synaptic 5-HT terminals is speculated to be partly responsible for the high concentration of MDMA in rat brain relative to plasma, implying that SERT activity can impact MDMA pharmacokinetics

(Capela et al., 2009; Chu et al., 1996). Moreover, putative reduction in the transport of thioether

MDMA metabolites into 5-HT neurons could provide a rationale for the abolishment of MDMA neurotoxicity in SERT-deficient rats.

SERT-KO rats were insensitive to MDMA-mediated depletion of tissue indoleamine levels in serotonergic-rich brain regions with known vulnerability to this amphetamine. Thus, it is 167

enticing to conclude that the absence of SERT offers resistance against MDMA neurotoxicity.

Although brain monoamine levels allow for a rapid and reliable assessment of neurodenegerative processes in response to amphetamine-induced toxicity, they are not direct markers of neuronal injury. In fact, the neuroadaptive hypothesis within the MDMA field argues that neurochemical impairments resulting from MDMA exposure are merely a reflection of changes in 5-HT biosynthesis derived from the inhibition of TPH activity, as opposed to an indication of serotonergic axotomy (Baumann et al., 2007). While valid, these observations are contradictory to the substantial evidence in support of MDMA neurotoxicity, which demonstrates that serotonergic deficits occur parallel to morphological and functional abnormalities, and appear to endure far beyond cessation of the initial pharmacological response (De Souza et al., 1990;

Marston et al., 1999; Piper et al., 2005; Sabol et al., 1996; Scanzello et al., 1993). In reality, it is likely that both neuroadaptive and neurotoxic processes coexist, providing an explanation for the recovery of brain 5-HT content in the hypothalamus (Sabol et al., 1996) but the severe 5-HT depletion and denervation of serotonergic nerve terminals still apparent at 52 weeks after MDMA treatment in various cortical regions (Sabol et al., 1996; Scanzello et al., 1993).

A detailed histological evaluation of MDMA-exposed SERT-KO rats is necessary to further establish the neuroprotective effects of SERT deficiency inferred by our studies. Ablation of SERT function complicates the implementation of immunohistochemical techniques, given that decreases in SERT protein and 5-HT uptake sites are the gold standard when assessing structural serotonergic injury derived from MDMA treatment. Nevertheless, alternative sensitive methods exists to examine neurodegeneration, such as silver staining and gliosis. High-dose MDMA treatment leads to the accumulation of silver-positive, argyrophillic deposits containing fragmented dendrites and degenerating nerve terminals (Commins et al., 1987; Jensen et al., 168

1993). Furthermore, neuroinflammation is often associated with CNS injury and amphetamine- derived gliosis is an acceptable marker of neurotoxicity (O'Callaghan and Miller, 1993;

O'Callaghan and Sriram, 2005). Correspondingly, anti-inflammatory treatment with minocycline prior to MDMA dosing, prevents increases in IL-1β release and microglial activation in rat cortex, whilst protecting against depletion of 5-HT uptake sites (Orio et al., 2010). The neuroprotective mechanism of minocycline is attributed to its ability to hinder the release of ROS and RNS from activated microglia via the inhibition of enzymes involved in the production of cellular oxidants, including NOX and PSH2 (Wu et al., 2002). Interestingly, our results display a lack of alterations in microglial occupancy in WT and KO rats, 7 days after repeated MDMA dosing. Notably, the combined 40 mg/kg dose of MDMA utilized in these studies may be insufficient to induce a glial response, given that much larger MDMA doses (75-150 mg/kg) are often required to stimulate reactive gliosis in other experiments (O'Callaghan and Miller, 1993). Additionally, other glial markers can be investigated, including elevations in IL-1β levels and OX-42 staining, both indicators of microglial activation (Orio et al., 2004). It is essential to emphasize that these neurotoxic markers do not directly evaluate damage inflicted to the serotonergic nervous system and should be supplemented with histological staining of 5-HT nerve terminals via antibodies against TPH and 5-HT.

Future studies should also address the role of SERT-dependent oxidative stress in the induction of MDMA neurotoxicity. Reports of an attenuation in MDMA-induced ROS production with decreased SERT activity, both in in vivo and in vitro, implicate this transporter in the cellular oxidant response to MDMA (Jones et al., 2004; Shankaran et al., 1999a). These findings have led us to conclude that SERT-deficient rats most likely lack the capability to uptake readily oxidizable compounds, including DA and thioether MDMA metabolites, thereby decreasing ROS burden and 169

neurodegeneration of 5-HT nerve terminals. A thorough analysis of oxidative stress markers in

SERT-KO rats challenged with MDMA is pending, including evaluation of hydroxyl radical formation, membrane lipid peroxidation, mitochondrial impairment, and oxidative damage to macromolecules. All of the aforementioned mechanisms of oxidative stress-related injury are reportedly stimulated following peripheral MDMA dosing (Alves et al., 2007; Colado et al., 1997a;

Colado et al., 1997b; Gudelsky, 1994; Moon et al., 2008; Sprague and Nichols, 1995b).

Data in Chapter 4 highlights the importance of VMAT2 in MDMA toxicity. Given that the interaction of MDMA with VMAT2 enhances the cytosolic monoamine pool and facilitates efflux of these biogenic amines, it should be expected that inhibition of this transporter with Ro4-1284 pretreatment would antagonize the acute effects of MDMA. Accordingly, Ro4-1284 prevented

MDMA-mediated hyperthermia and hyperactivity. However, MDMA-evoked 5-HT and DA release in rat striatum remained mostly undisturbed, in contrast to reports indicating that VMAT2 contributes to amphetamine-induced toxicity by interfering with monoamine storage and release

(Beckmann et al., 2010; Dwoskin and Crooks, 2002; Meyer et al., 2013). Despite these findings, there are reasons to suspect that the perceived lack of effects of Ro4-1284 on striatal 5-HT and

DA release may not be reflective of the diminished hyperthermic and hyperlocomotor response.

As an example, MDMA-induced hyperthermia is initiated in the hypothalamus, and stimulation of locomotor activity primarily involves activation of dopaminergic pathways in the nucleus accumbens (Freed and Yamamoto, 1985; Stephenson et al., 1999). Likewise, increases in DA levels in the caudate were more prominent and occurred much rapidly compared to the nucleus accumbens following high-dose MDMA treatment (10 mg/kg), which suggests that regulation of

MDMA-mediated DA release is region-specific (Yamamoto and Spanos, 1988). As such, it is tempting to speculate that acute inhibition of VMAT2 could differentially affect stimulation of 170

dialysate monoamine concentrations in the various brain regions susceptible to MDMA.

Therefore, examination of MDMA-mediated monoamine release in other regions of the CNS, including hypothalamus and nucleus accumbens is necessary to validate such an interpretation.

Alternatively, it is interesting that Ro4-1284 pretreatment delayed reductions in monoamine metabolites induced by MDMA. The data trends towards an overall attenuation in

MDMA-induced depletion of 5-HIAA and DOPAC, although the findings were not significant in all cases. These results are perhaps indicative of the antagonistic effect of Ro4-1284 on MDMA- mediated inhibition of monoamine metabolism, which could have implications in the protective mechanism of Ro4-1284 against acute MDMA toxicity. MDMA enhances the release of biogenic amines, in part, by blocking MAO-derived deamination of neurotransmitters (Leonardi and

Azmitia, 1994). Pharmacological studies have revealed a close relationship between monoamine degradation and post-synaptic receptor activity (Chase, 1973). For example, activation of central

DA receptors with apomorphine depresses DA turnover through a feedback mechanism that modulates impulse-driven activity in the pre-synaptic neuron (Anden et al., 1967; Nyback et al.,

1970). The opposite effect may also be possible, in which changes in monoamine metabolism influence receptor activity. In this regard, Ro4-1284 might indirectly inhibit post-synaptic serotonergic and dopaminergic receptor activity by accelerating monoamine turnover in the presence of MDMA, thus attenuating hyperthermia and hyperactivity. In light of this hypothesis, presumed alterations in monoamine metabolism in response to Ro4-1284 should be investigated by determining changes in MAO activity. Pharmacological manipulation of monoamine metabolism can also provide insights into the protective effects of Ro4-1284.

The inability of Ro4-1284 to prevent MDMA-mediated elevations in NE plasma levels suggests that Ro4-1284 has minimal effects on the peripheral MDMA response. VMAT2 is found 171

in neuronal cells of the central, peripheral and enteric nervous system. Moreover, hyperthermia is regulated by neuronal pathways in the hypothalamus and the SNS (Mills et al., 2004). Hence, we predict that Ro4-1284 reverses MDMA-induced hyperthermia through disruption of vesicular monoamine storage and neurotransmission, both centrally and in the periphery, which is supported by the robust inhibitory effects of Ro4-1284 on MDMA-induced hyperthermia. Future studies should analyze additional peripheral markers of MDMA thermogenesis, such as increases in skeletal muscle lysis, serum creatine kinase, plasma thyroid hormone levels, mitochondrial UCP expression and depletion of ATP levels in skeletal muscle tissue (Brown and Yamamoto, 2003;

Cunningham, 1997; Rusyniak et al., 2005; Sprague et al., 2003a; Sprague et al., 2004; Sprague et al., 2007). Alternatively, pharmacological activation of α1 and β3 adrenoceptors might partially restore MDMA-mediated hyperthermia in the presence of Ro4-1284, given the critical role of these receptors in augmenting heat production and vasoconstriction during MDMA thermogenesis (Sprague et al., 2004).

The protective mechanisms of Ro4-1284 against MDMA-mediated serotonergic neurotoxicity should also be addressed. Prevention of hyperthermia with acute VMAT2 inhibition may contribute to the abolishment of serotonergic deficits in MDMA-exposed rats seemingly by decreasing ROS burden. Nonetheless, hyperthermia is not a requirement for neurotoxicity, and temperature dependent effects of Ro4-1284 are likely to play a major role in attenuating the neurotoxic response to MDMA. Two approaches can be considered in order to confirm alleviation of MDMA neurotoxicity by Ro4-1284 pretreatment with the occurrence of hyperthermia. In one instance, rats are exposed to elevated ambient temperatures (~27ºC) to overcome the diminishing effects of Ro4-1284 on MDMA-induced hyperthermia. Warm ambient temperatures

(26-30ºC) potentiate hyperthermia produced by MDMA (Malberg and Seiden, 1998), whereas, 172

hypothermia observed with concomitant MDMA and NMDA receptor antagonist treatment is reversed by maintaining a room temperature of 26.5ºC, instead, causing intense hyperthermia

(Farfel and Seiden, 1995). Alternatively, Ro4-1284 can be administered immediately after cessation of the hyperthermic effects of MDMA (~7 h post-treatment). Consistent with this idea, post-treatment with VMAT2 inhibitor, lobeline, prevents methamphetamine-induced dopaminergic neurotoxicity in the presence of hyperthermia (Eyerman and Yamamoto, 2005).

Finally, the monoamine depleting activity of Ro4-1284, as a consequence of decreased

VMAT2 function, could be partly responsible for the obliteration of MDMA neurotoxicity. In particular, tissue DA levels were susceptible to the effects of Ro4-1284. In light of the fact that DA is a critical component of MDMA neurotoxicity, future mechanistic evaluation of the contribution of reduced dopaminergic activity to the protective effects of Ro4-1284 should be determined.

This would involve the use of pharmacological agents that increase DA synthesis and neurotransmission. Indeed, stimulation of dopaminergic activity exacerbates MDMA neurotoxicity (Aguirre et al., 1998; Schmidt et al., 1991). 173

APPENDIX A: EFFECTS OF 3,4-(±)-METHYLENEDIOXYMETHAMPHETAMINE AND ITS THIOETHER

METABOLITES ON THE IN VITRO FUNCTION OF THE SEROTONIN REUPTAKE

TRANSPORTER

A.1 INTRODUCTION AND RATIONALE

MDMA metabolism is an integral step in the attenuation of its pharmacological activity, ultimately leading to its elimination from the body. Nevertheless, a plethora of evidence supports a pivotal role for metabolism in MDMA neurotoxicity. In humans, CYP-induced O- demethylenation of MDMA to N-Me-α-MeDA is the first step in the metabolic pathway of this amphetamine derivative. Interestingly, pharmacological modulation of phase I and II enzymes involved in MDMA metabolism has profound consequences to the in vitro cytotoxicity and in vivo serotonergic deficits produced by MDMA (Antolino-Lobo et al., 2010; Gollamudi et al., 1989). The fact that direct injection of MDMA into rat CNS fails to replicate the neurotoxic profile observed following peripheral administration reveals that MDMA metabolism is necessary for the manifestation of neurotoxicity (Esteban et al., 2001).

Conjugation of reactive MDMA ortho-quinones with GSH and NAC results from the oxidation of corresponding catecholamine intermediates, leading to the formation of thioether metabolites. Multiple thioether conjugates of N-Me-α-MeDA and α-MeDA have been identified in rat brain (Erives et al., 2008; Jones et al., 2005), where they accumulate after repeated MDMA administration (Erives et al., 2008). Moreover, thioether MDMA metabolites exhibit a high redox potential that facilitates ROS production (Felim et al., 2007), which is a requirement for the occurrence of MDMA-mediated cytotoxicity (Bai et al., 1999; Carvalho et al., 2002; Carvalho et 174

al., 2004; Miller et al., 1997). Thus, it is tempting to suggest that thioether metabolites of MDMA are the ultimate toxicants in MDMA neurotoxicity.

The neurotoxic profile of redox-active MDMA intermediates is well established, and a number of cytotoxicity mechanisms, involving excessive ROS generation, have been suggested.

However, an understanding of how the parent drug and/or metabolites preferentially target the serotonergic system remains elusive. One could further speculate that both the parent drug and metabolites are transported into 5-HT neurons or facilitate the transport of reactive compounds

(such as DA) with the capability of inducing a potent cellular oxidant response. The selectivity of

MDMA and MDMA metabolites towards 5-HT nerve terminals likely requires the participation of

SERT. Loss of SERT protein and 5-HT uptake sites occur during long-term MDMA neurotoxicity

(Callahan et al., 2001; Schmidt, 1987; Xie et al., 2006), suggesting the degeneration of serotonergic axons and nerve terminals. Moreover, the neuropharmacological mechanism of

MDMA is dependent upon stimulation of excess 5-HT efflux via SERT activity. The results presented in Chapter 3 reveal disturbances in the acute and long-term effects of MDMA in SERT- deficient rats, which confirms the importance of this transporter in the mechanism of action of

MDMA.

The role of SERT as a primary site of interaction, and an important mechanism of MDMA- mediated in vitro cytotoxicity, was previously examined in our laboratory. Jones et al. (2004) reported that α-MeDA-derived metabolites of MDMA, 5-(GSH)-α-MeDA and 2,5-bis-(GSH)-α-

MeDA, are more proficient at inhibiting SERT function and inducing ROS generation than the parent compounds, MDMA and MDA. α-MeDA is formed from the O-demethylenation of MDA mediated by CYPs and it constitutes the main route of MDMA metabolism in rats (Chu et al.,

1996). Conversely, N-Me-α-MeDA-related conjugates are the major metabolites identified in 175

human urine (Schwaninger et al., 2011a). Data on the effects of thioether metabolites of MDMA derived from N-Me-α-MeDA on SERT activity is currently lacking. Thus, we sought to investigate the putative interaction of mono-conjugated products of N-Me-α-MeDA, 5-(GSH)-N-Me-α-MeDA and 5-(NAC)-N-Me-α-MeDA, with SERT. Our experimental design consisted of measuring cellular

5-HT uptake in the presence of these MDMA metabolites in an attempt to determine any effects on in vitro SERT function. The findings could have implications in the mechanism of MDMA metabolite-mediated neurotoxicity and the understanding of serotonergic pathologies related to

5-HT reuptake malfunctions. 176

A.2 RESULTS

A.2.1 Synthesis and Identification of Thioether MDMA Metabolites

GSH and NAC conjugates of N-Me-α-MeDA were synthesized by electrochemical oxidation through the use of a potentiostat. Crude reactions were purified by HPLC-UV analysis, yielding individual peaks for the different metabolites (Figure A-1). Fractions of interest were collected and analyzed by tandem mass spectrometry (LC-MS/MS) to confirm the identity of the various N-Me-α-MeDA products. Tandem mass spectrometry has been previous utilized as a reliable method to detect MDMA and other designer drugs, including their respective metabolites

(Clauwaert et al., 2001; Maurer et al., 2000; Nordgren and Beck, 2003). The peak labeled as 1 in the HPLC-UV chromatogram, resulting from the reaction of N-Me-α-MeDA with GSH, corresponded to 5-(GSH)-N-Me-α-MeDA (Figure A-1A). The expected parent ion for 5-(GSH)-N-

Me-α-MeDA (m/z = 486.9) was identified by mass spectrometry and subsequent MS/MS fragmentation revealed patterns consistent with other GSH adducts, illustrating the presence of

N-Me-α-MeDA ions (m/z = 181) upon the loss of GSH (m/z = 307), and the loss of glutamate ions

(m/z = 129), and an ion depicting the cleavage of the S-C bond of the cysteine residue in the GSH molecule (m/z = 274) (Figure A-2) (Grillo and Hua, 2003; Guan et al., 2003; Mutlib et al., 2000).

Peak 2 from the HPLC-UV purification analysis corresponded to 5-(NAC)-N-Me-α-MeDA (Figure A-

1B). The predicted parent ion for 5-(NAC)-N-Me-α-MeDA (m/z = 342.9) was identified by LC-MS.

MS/MS fragmentation revealed the loss of NAC ions (m/z = 162), and ions corresponding to the cleavage of the S-C bond of the cysteine residue in the NAC molecule (m/z = 130) (Figure A-3), a fragmentation pattern characteristic of mercapturic acids (Lang et al., 2000; Urban et al., 2003).

In summary, thioether metabolites of N-Me-α-MeDA were successfully synthesized by electrochemistry as substantiated by tandem mass spectrometry analysis. 177

A.2.2 N-Me-α-MeDA-Derived Metabolites of MDMA Fail to Modulate 5-HT Uptake into

hSERT-Expressing Human Embryonic Kidney Cells

Various in vitro cellular models were initially considered to examine the effects of thioether metabolites of MDMA on SERT function. The human SERT gene was transiently transfected into immortalized human embryonic kidney (HEK) cells and neuroblastoma (SK-N-MC) cells due to their lack of endogenous SERT expression. SK-N-MC cells were selected based on their neuronal origin, meanwhile, HEK cells possess exceptional transfectability, and are widely used to study transport mechanisms in vitro (Ahlin et al., 2008; Sissung et al., 2010). Two additional cell lines were tested with presumed endogenous SERT expression. JAR cells are a human placental serotonergic cell line that have the capability to transport 5-HT (Ramamoorthy et al., 1998;

Simantov and Tauber, 1997). Finally, RN46A cells, derived from embryonic rat raphe neurons, are a temperature sensitive cell line that adopts a neuronal phenotype upon differentiation at 39ºC

(White et al., 1994). Expression of SERT has been noted under undifferentiated (33ºC) and differentiated conditions (Koldzic-Zivanovic et al., 2004; White et al., 1994).

Functional SERT expression in the different cell models was assessed by measuring cellular uptake of [3H]5-HT, either alone or in the presence of excess, untritiated 5-HT (Figure A-

4.). Intracellular accumulation of [3H]5-HT is observed in all cells transiently transfected with hSERT and in endogenously SERT-expressing JAR cells. More importantly, addition of excess 5-HT significantly reduced [3H]5-HT uptake in these three cell models, to levels measured in the GFP- transfected controls, indicating that 5-HT gains intracellular access via transport-mediated mechanisms that likely involve SERT. Interestingly, [3H]5-HT accumulation in differentiated and undifferentiated RN46A cells was insensitive to the addition of untritiated 5-HT, which suggests that these cells do not uptake 5-HT via SERT. Other studies are consistent with our present 178

findings, showing a lack of 5-HT uptake activity in RN46A cells despite endogenous mRNA expression of SERT (Yammamoto et al., 2013).

Potential effects of GSH and NAC MDMA conjugates were examined in hSERT-expressing cells based on preliminary results from the functional SERT expression assay. HEK-hSERT cells displayed the highest capacity to uptake [3H]5-HT in addition to their well-recognized amenability to genetic manipulation. Time-course analysis of [3H]5-HT uptake into HEK-hSERT cells demonstrated that specific 5-HT transport occurs rapidly, showing a linear increase in intracellular

[3H]5-HT accumulation from 1-10 min following incubation, which plateaus after 60 min (Figure

A-5A). Furthermore, saturation studies in hSERT-transfected HEK cells were performed with co- incubation of 1 μCi/mL of [3H]5-HT and varying concentrations of cold 5-HT (0-1000µM). The calculated values for Km and Vmax were as follows: 12.5 ± 2.3 µM and 368 ± 59 pmol/min/mg of protein (Figure A-5B).

Putative modulation of SERT function by MDMA and its metabolites was investigated by measuring uptake of [3H]5-HT in the presence of increasing concentrations of MDMA, N-Me-α-

MeDA, 5-(GSH)-N-Me-α-MeDA and 5-(NAC)-N-Me-α-MeDA (0-1000µM). A lower estimated Ki value for MDMA (17.08 ± 2.2 µM) compared to N-Me-α-MeDA (189.8 ± 21.2 µM), suggests that the parent compound is more potent at inhibiting [3H]5-HT uptake than its CYP-formed metabolite

(Figure A-6.). Conversely, the kinetic profiles of 5-(GSH)-N-Me-α-MeDA and 5-(NAC)-N-Me-α-

MeDA showed no inhibitory effect on [3H]5-HT uptake activity with the increase in metabolite concentration. A summary of the kinetic analysis further substantiates these findings, displaying a dose-dependent inhibition of [3H]5-HT uptake by MDMA and N-Me-α-MeDA (Figure A-7).

MDMA significantly reduces [3H]5-HT transport into HEK-SERT cells to 34% (p < 0.001) of control values at a concentration of 30 µM, whilst almost abolishing uptake at 300 µM (4.2% of control; 179

p < 0.001). Prominent decreases in [3H]5-HT uptake (35% of control; p < 0.001) with N-Me-α-

MeDA treatment were only observed at high substrate concentrations (300 µM), indicating that

N-Me-α-MeDA is less efficient at inhibiting SERT function than MDMA. Finally, no significant changes were noted in [3H]5-HT uptake at the various concentrations of 5-(GSH)-N-Me-α-MeDA and 5-(NAC)-N-Me-α-MeDA examined. Unlike MDMA and N-Me-α-MeDA, thioether MDMA conjugates lack the capability to hinder SERT function in our hSERT-expressing in vitro model. 180

Figure A-1 - Purification of Thioether Metabolites of N-Me-α-MeDA

Thioether conjugates of N-Me-α-MeDA were synthesized by electrochemistry and purified through HPLC-UV analysis. Chromatographs display isolation of GSH (A) and NAC (B) products of N-Me-α-MeDA monitored at 280 and 225 nm, respectively. Labeled peaks correspond to thioether metabolites of MDMA subsequently identified by MS/MS. 181 182

Figure A-2 - MS/MS Spectrum of 5-(GSH)-N-Me-α-MeDA

Peak 1 recovered from HPLC-UV analysis was identified as 5-(GSH)-N-Me-α-MeDA by LC-MS/MS. The calculated molecular weight of 5-(GSH)-N-Me-α-MeDA is 487.54 daltons. A dominant peak of m/z = 486.9 was identified by mass spectrometry. The spectrum displays the MS/MS scan of the dominant peak at a collision energy of 40eV. Illustrated in the figure is the fragmentation pattern characteristic of mercapturic acids, such as the loss of glutamate (-129), GSH (-307) and GSH minus the S residue (-274). The characteristic fragmentation pattern produces ions with a m/z of 357, 213 and 181 daltons. 183 184

Figure A-3 - MS/MS Spectrum of 5-(NAC)-N-Me-α-MeDA

Peak 2 recovered from HPLC-UV analysis was identified as 5-(NAC)-N-Me-α-MeDA by LC-MS/MS. The expected molecular weight of 5-(NAC)-N-Me-α-MeDA is 342.12 daltons. A dominant peak of m/z = 342.9 was identified by mass spectrometry. The spectrum displays the fragmentation pattern of this dominant peak at a collision energy of 30eV. Illustrated in the figure are the two main fragmentation patterns observed in the spectrum. The loss of 162 daltons (ion of m/z = 180.7) corresponds with an expected loss of NAC from the parent compound. The loss of NAC minus the S residue (-130 daltons) produces ions with a m/z of 213 daltons, a fragmentation pattern characteristic of mercapturic acids. 185

Figure A-4 - Cellular Uptake of 5HT into Various In Vitro Serotonergic Cell Models

Total uptake of 1 µCi/mL of [3H]-5HT was measured by liquid scintillation spectroscopy in cell models transiently transfected with hSERT (HEK-SERT and SK-N-MC-SERT) and in cell lines with endogenous SERT expression (JAR and RN46A). GFP transfections served as a control for functional SERT expression. RN46A cells cultured at 33ºC present a fibroblast-like morphology and a differentiated neuronal phenotype when cultured at 39ºC. Inhibition of specific [3H]-5HT transport is accomplished by addition of excess, untritiated 5-HT (1 mM). Data are expressed as the mean (n=3) ± S.E.M. Values are significantly different from [3H]-5HT treatment at (*) p < 0.001. 186 187

Figure A-5 - Kinetics of 5HT Uptake into HEK Cells Expressing hSERT

Time-course of [3H]5-HT uptake (A) was determined by incubating HEK-hSERT cells with 1 μCi/mL of [3H]5-HT at various time points either alone or in the presence of excess, cold 5-HT (1 mM). Specific 5-HT transport was calculated by subtracting uptake in the presence of excess 5-HT from total [3H]5-HT uptake. Kd and Vmax values were determined from saturation analysis (B), treating HEK-hSERT cells with 1 μCi/mL of [3H]5-HT and increasing concentrations of cold 5-HT for 10 min. Data are expressed as the mean (n=3) ± S.E.M. 188 189

Figure A-6 - Kinetics of MDMA, N-Me-α-MeDA, 5-(GSH)-N-Me-α-MeDA and 5-(NAC)-N-Me-α- MeDA-Mediated Modulation of 5-HT Uptake into hSERT-Transfected HEK Cells

Plots display [3H]-5-HT uptake in the presence of MDMA and N-Me-α-MeDA conjugates. Ki values were determined by co-incubation of cells with 1 μ Ci/mL of [3H]5-HT and increasing concentrations of MDMA, N-Me-α-MeDA, 5-(GSH)-N-Me-α-MeDA and 5-(NAC)-N-Me-α-MeDA. Data are expressed as the mean (n= 3) ± S.E.M. 190

Figure A-7 - 5-(GSH)-N-Me-α-MeDA and 5-(NAC)-N-Me-α-MeDA Fail to Inhibit 5-HT Uptake into hSERT-Transfected HEK Cells in a Dose-Dependent Manner

Graphical representation of the kinetics analysis of MDMA and MDMA metabolite-mediated modulation of [3H]5-HT uptake. Data are expressed as the mean (n= 3) ± S.E.M of [3H]5-HT uptake as % of control (0 µM). Values are significantly different from control treatment at (*) p < 0.001. 191

A.3 DISCUSSION

Preliminary results from this study do not support the hypothesis that N-Me-α-MeDA- derived metabolites of MDMA interact with SERT. Quantitation of [3H]5-HT uptake into hSERT- expressing HEK cells in the presence of 5-(GSH)-N-Me-α-MeDA and 5-(NAC)-N-Me-α-MeDA showed no effect on intracellular 5-HT accumulation with increasing concentrations of the two test compounds (Figure A-6), indicative of a lack of modulation of SERT function. Conversely,

MDMA and N-Me-α-MeDA displayed dose-dependent inhibition of [3H]5-HT uptake, with MDMA being a more potent inhibitor of SERT activity than its CYP-formed metabolite (Ki = 17 and 190

µM, respectively).

Inhibition of in vitro SERT function with MDMA treatment is well established. Consistent with previous reports, MDMA appears to be a weaker inhibitor of this transporter relative to other known SERT-specific compounds, such as SSRIs, used in the treatment of depression and anxiety disorders (Barker et al., 1998; Mortensen et al., 1999). For example, kinetic analysis of SERT- transfected HeLa cells reported Ki values of MDMA in the low µmol range similar to our results, whereas, calculated values for fluoxetine and citalopram were in the nmol range (Mortensen et al., 1999). Although MDMA demonstrates less potency at inhibiting 5-HT uptake than SSRIs, interaction of this amphetamine with SERT appears to be involved in the long-term neurochemical and morphological lesions to 5-HT nerve terminals observed during MDMA neurotoxicity

(Malberg et al., 1996; Sanchez et al., 2001) (Chapter 3). In contrast, adverse effects on the serotonergic system caused by high-dose treatment with SSRIs, fluoxetine (4 x 114 mg/kg, po) and sertraline (4 x 286 mg/kg, po), are less severe compared to MDMA (4 x 20mg/kg, sc) and do not appear to persist beyond 18 h post-treatment (Kalia et al., 2000). Collectively, these studies imply 192

that processes other than the possible interaction of MDMA with SERT are important in the development of long-term MDMA neurotoxicity.

One event contributing to MDMA neurotoxicity is the formation of thioether MDMA metabolites that can induce ROS production and cause oxidative damage to 5-HT neurons.

Consistent with this hypothesis, systemic metabolism of MDMA appears to be required for the manifestation of serotonin deficits (Esteban et al., 2001). A number of studies from our laboratory have demonstrated that GSH conjugates of α-MeDA i) are metabolized in rat brain, ii) are readily transported across the blood brain barrier and iii) inflict behavioral and neurotoxic effects comparable to the parent compound, MDMA (Bai et al., 2001; Bai et al., 1999; Miller et al., 1997;

Miller et al., 1996; Miller et al., 1995). Additionally, 5-(GSH)-α-MeDA and 2,5-bis-(GSH)-α-MeDA inhibit [3H]5-HT uptake into hSERT-transfected SK-N-MC cells more efficiently than MDMA.

Hence, it is conceivable that the in vivo interaction of MDMA with SERT can also occur indirectly through metabolite-mediated effects (Jones et al., 2004). Based on the electrophilic properties of

5-(GSH)-α-MeDA and 2,5-bis-(GSH)-α-MeDA, one could speculate that these thiother MDMA metabolites interact with nucleophilic cysteine residues located in the extracellular loops of the transmembrane SERT protein, thereby modulating SERT activity (Chen et al., 1998). Perhaps most revealing is the fact that 5-(GSH)-α-MeDA and 2,5-bis-(GSH)-α-MeDA increase the selectivity of

SERT for DA, stimulating transport of DA into hSERT-expressing SK-N-MC cells to a greater extent than MDMA (Jones et al., 2004). The findings support the long recognized role of DA in MDMA neurotoxicity and provide a means by which DA may enter serotonergic nerve terminals and subsequently underdo oxidation, contributing to ROS production.

Contrary to 5-(GSH)-α-MeDA and 2,5-bis-(GSH)-α-MeDA, our data shows that 5-(GSH)-N-

Me-α-MeDA and 5-(NAC)-N-Me-α-MeDA have no inhibitory activity at SERT compared to the two 193

parent compounds, MDMA and N-Me-α-MeDA, suggesting that conjugation with GSH and NAC may consequently reduce the affinity of N-Me-α-MeDA metabolites for this transporter.

Inconsistencies in our findings with those of Jones et al. (2004) could arise from a number of factors. Structural differences between α-MeDA and N-Me-α-MeDA may ultimately account for disparities in the ability of their respective catecholamine metabolites to inhibit SERT subsequent to conjugation with GSH. α-MeDA is derived from MDA, a neuropharmacologically active metabolite of MDMA with enhanced behavioral and neurotoxic properties compared to the parent drug (Hiramatsu et al., 1989; Moore et al., 1996). Furthermore, rats exposed to a single high-dose (150 nmol) or multiple doses (30 nmol x 4 every 12 hr), of 5-(GSH)-N-Me-α-MeDA and

5-(NAC)-N-Me-α-MeDA via intrastriatal administration displayed no signs of 5-HT depletion

(observations from our laboratory, Herndon et al., 2013), whereas, direct injection of 5-(GSH)-α-

MeDA, 2,5-bis-(GSH)-α-MeDA and 5-(NAC)-α-MeDA into the CNS at similar doses produces long- lasting serotonergic deficits analogous to systemic MDA administration (Bai et al., 1999). Thus, it appears that thioether metabolites of α-MeDA are more potent neurotoxicants than the metabolites derived from N-Me-α-MeDA, perhaps due, in part, to their greater competency at inhibiting SERT function.

Another plausible explanation for the inability of N-Me-α-MeDA metabolites to interact with SERT may be a consequence of the absence of hyperthermic conditions. Much like α-MeDA intermediates, elevated temperature (40ºC) can modulate SERT function, preferentially increasing uptake of DA rather than 5-HT into hSERT-expressing HEK cells (Saldana and Barker,

2004). Indeed, compelling evidence exists for the involvement of hyperthermia in MDMA neurotoxicity (Colado et al., 1998; Farfel and Seiden, 1995; Malberg and Seiden, 1998;

McNamara et al., 2006). Hyperthermia may promote 5-HT terminal damage by inducing free 194

radical formation (Green et al., 2003). Consistent with this hypothesis, exposure of rat cortical cultures to hyperthermic conditions (40ºC) potentiates ROS generation and neuronal cell death mediated by thioether metabolites of α-MeDA and N-Me-α-MeDA (Capela et al., 2007; Capela et al., 2006). Altogether, these findings indicate that hyperthermia, might to some extent, contribute to MDMA neurotoxicity by enhancing the redox potential of thioether MDMA conjugates and perhaps even influence their interaction with SERT. In the present study, kinetic analysis of [3H]5-

HT uptake in the presence of 5-(GSH)-N-Me-α-MeDA, 2-(NAC)-N-Me-α-MeDA was performed at room temperature (~22 ºC), warranting additional studies that investigate the effects of elevated ambient temperature on MDMA metabolite-mediated inhibition of SERT.

The lack of inhibition of [3H]5-HT uptake by thioether metabolites of N-Me-α-MeDA could also be related to SERT expression levels in our in vitro cellular model. SERT-mediated uptake of a number of substrates reportedly varies according to cell type. For instance, hSERT-expressing

HEK cells are more efficient at transporting 5-HT and DA than HeLa cells also transfected with hSERT, seemingly due to differences in surface SERT expression (Saldana and Barker, 2004).

Importantly, both 5-HT transport and 5-HT-induced current are stimulated with concomitant increase of SERT expression levels in xenopus oocytes (Ramsey and DeFelice, 2002). Moreover, high SERT expression levels correlate with a decreased capacity of d-amphetamine, cocaine and paroxetine to inhibit 5-HT uptake (Ramsey and DeFelice, 2002). hSERT-transfected HEK cells displayed the greatest efficiency of [3H]5-HT uptake relative to the other “serotonergic” cells models initially tested (Figure A-4), perhaps reflective of enhanced SERT expression levels, which may to some extent negatively impact the interaction of thioether N-Me-α-MeDA metabolites with SERT. Given that SERT expression profoundly influences the function and pharmacology of 195

serotonin transporters, future analysis should take into consideration cell-specific differences in the surface levels of these transporters.

Overall, considerable evidence substantiates the role of metabolism in the long-term neurotoxic response to MDMA. More specifically, GSH and NAC conjugates of N-Me-α-MeDA accumulate in rat brain after repeated MDMA dosing (Erives et al., 2008) and display potent neurotoxicant properties (Capela et al., 2007; Capela et al., 2006; Jones et al., 2005). Despite the perceivable lack of inhibition of SERT function by 5-(GSH)-N-Me-α-MeDA and 5-(NAC)-N-Me-α-

MeDA, additional experiments are necessary in order to address the aforementioned caveats. A putative interaction of thioether MDMA metabolites with SERT could have implications i) in the selectivity of these compounds for 5-HT nerve terminals ii) in the potential mechanism of transport of MDMA and its metabolites into 5-HT neurons and ii) in the underlying mechanisms of MDMA metabolite-mediated neurotoxicity. 196

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