S-: PRECLINICAL INVESTIGATION OF A SYNTHETIC CATHINONE AGAINST BEHAVIORAL AND NEUROCHEMICAL EFFECTS OF AND MDPV

A Dissertation Submitted to the Temple University Graduate Board

In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY

By

Helene L. Khalid

May, 2017

Examining Committee Members:

Scott M. Rawls, PhD, Advisory Chair, Department of Pharmacology Barrie Ashby, PhD, Department of Pharmacology Ellen M. Unterwald, PhD, Department of Pharmacology Lynn Kirby, PhD, Department of Anatomy and Cell Biology Allen Reitz, PhD, External Member, Fox Chase Chemical Diversity Center

© Copyright 2017

by Helene L. Khalid All Rights Reserved

ii ABSTRACT

Synthetic cathinones are an emerging class of novel psychoactive substances whose rising rates of abuse have made them a significant public health issue. Recently, synthetic cathinones have emerged as popular of abuse in the United States and

Europe. Illicit dealers have synthesized stable analogues of cathinones and marketed them via the Internet as “legal high” alternatives to commonly abused psychostimulants.

Some adverse effects of synthetic cathinone intoxication include depression, anxiety, myocardial infarction, cardiac dysrhythmias, pulmonary edema, renal failure, stroke and death. Two synthetic cathinones that have gained popularity over the past decade are 4- methylmethcathinone (mephedrone, MEPH) and 3,4-methylenedioxypyrovalerone

(MDPV).

Analogous to other and cathinones, MEPH is a chiral molecule with two enantiomers, R-MEPH and S-MEPH. Pharmacological differences can exist between enantiomers. Given the enantiomeric specificity that exists between the enantiomers of MEPH, the overall goal of this dissertation was to investigate potential therapeutic effects of S-MEPH and to determine the reinforcing effects and potential abuse liability of each enantiomer of MEPH. To date, there are no approved for psychostimulant abuse despite high rates of relapse even with treatment. The lack of pharmacotherapies suggests that new approaches for psychostimulant abuse remain to be identified. Further exploration into the stereochemistry of MEPH will characterize a new approach to manage psychostimulant abuse during acute withdrawal that causes anxiety and relapse.

iii The first experimental chapter of this thesis (Chapter 2) evaluated the effects of S-

MEPH on cocaine and MDPV withdrawal-induced anxiety- and depression-like behavior in the elevated plus maze (EPM) and forced swim test (FST), respectively. EPM and FST were used to test the hypothesis that S-MEPH reduces anxiety-like and depression-like behaviors in rats withdrawn from a 10-day chronic binge cocaine paradigm (10 mg/kg,

3x/day in 1-hr intervals) or binge MDPV paradigm (1 mg/kg, 3x/day in 1-hr intervals).

Control animals received saline injections. Both cocaine and MDPV induced heightened anxiety-like behavior on the EPM compared to saline controls. Rats withdrawn from chronic cocaine exposure received treatment with 10 mg/kg S-MEPH (COC SM 10). S-

MEPH increased the percentage of time spent on the open arms compared to treatment with saline (COC SAL). Similar efficacy was observed for 10 mg/kg of S-MEPH where rats withdrawn from chronic MDPV exposure and treated with S-MEPH during the drug- free period (MDPV SM 10) increased the percentage of time spent on the open arms compared to treatment with saline (MDPV SAL). Treatment with a higher dose of S-

MEPH (30 mg/kg) also produced an increase in time spent on the open arms for rats withdrawn from either cocaine (COC SM 30) or MDPV (MDPV SM 30). However, treatment with 30 mg/kg S-MEPH by itself (SAL S-MEPH) caused a slight, but significant, decrease in time spent on the open arms compared to saline controls (SAL

SAL). Cocaine and MDPV withdrawn rats spent more time in the immobile state, indicative of depression-like behavior in the FST compared to saline controls. Subsequent treatment with 10 mg/kg S-MEPH (COC SM 10), following withdrawal from chronic cocaine exposure, reduced the time spent in the immobile state compared to treatment with saline (COC SAL). Similar efficacy was observed for 10 mg/kg of S-MEPH where

iv rats withdrawn from chronic MDPV exposure and treated with S-MEPH during the drug- free period (MDPV SM 10) decreased the time spent in the immobile state compared to treatment with saline (MDPV SAL). Treatment with a higher dose of S-MEPH (30 mg/kg) also produced a decrease in immobile state for rats withdrawn from either cocaine

(COC SM 30) or MDPV (MDPV SM 30). However, treatment with 30 mg/kg S-MEPH by itself (SAL S-MEPH) caused a reduction in time spent in the immobile compared to saline controls (SAL SAL). The present data suggest S-MEPH has therapeutic potential during early withdrawal from psychostimulant abuse and may be a possible structural and pharmacological template to develop maintenance therapy for psychostimulant abuse.

Although (DA) and (5-HT) release is the primary of S-MEPH, in vitro studies assessed the binding and activity of S-

MEPH to elucidate a possible secondary mechanism that could contribute to the clinical effects of S-MEPH. Indeed, standard (e.g. ) have a dual mechanism of action, and not only increase 5-HT levels, but also block 5-HT2C receptors that help to prevent the initial onset of anxiety reported by patients. Binding and functional assays were performed at the National Institute of Mental Health Psychoactive

Drug Screening Program (PDSP). For radioligand binding, S-MEPH was screened at 10

μM for binding to a battery of receptors including a range of 5-HT, DA, sigma, kappa , , muscarinic, and nicotinic receptor subtypes. It was found that S-

MEPH binds to 5-HT2 receptors (2A, 2B, ) but showed negligible binding for , adrenergic and nicotinic receptors. Functional assays revealed S-MEPH has no activity. These results suggest that S-MEPH may be a possible structural and pharmacological template to develop a maintenance therapy for addicts with acute

v anxiety and depression during early withdrawal by enhancing the release of 5-HT and/or through 5-HT2 receptor interactions.

Chapter 3 investigated whether S-MEPH can reinstate drug-seeking behavior in rats with a history of cocaine exposure. Using the drug intravenous self-administration

(IVSA) model, rats were trained to self-administer cocaine (0.375 mg/kg/infusion) under

FR-1 schedule of reinforcement during daily 2-hour sessions for 12 days. Following acquisition of cocaine self-administration, rats were subject to daily 2-hour extinction sessions. After meeting extinction criteria (less than 15 active lever presses), rats underwent drug-primed reinstatement, where non-contingent injections of R-MEPH (10 mg/kg), S-MEPH (10, 20, 30 mg/kg), or cocaine (10 mg/kg) were administered prior to the reinstatement session. S-MEPH (10, 20, 30 mg/kg) prior to the reinstatement session was not efficacious in producing drug-seeking behavior. R-MEPH-prime injection reinstated cocaine-seeking behavior. Rats reinstated responding after cocaine-prime injection.

After observing differences in priming injections with R-MEPH and S-MEPH during reinstatement to cocaine-seeking (Chapter 3), we further examined the reinforcing effects and potential for abuse of S-MEPH in Chapter 4. Fixed-ratio 1 (FR-1)

(acquisition) and progressive-ratio () schedules of reinforcement were used to determine the rewarding properties and motivational incentive for R-MEPH, S-MEPH as well as racemic MEPH. Rats were trained to self-administer MEPH (0.5 mg/kg/infusion) under a fixed ratio (FR-1) schedule in daily 2-hour sessions for 14 days. After acquisition, rats underwent dose substitution to examine effects of R-MEPH (0.5 mg/kg/infusion) and S-MEPH (0.25, 0.5, 2. mg/kg/ infusion). Dose-substitution studies

vi revealed the lowest (0.25 mg/kg/ infusion) and middle (0.5 mg/kg/ infusion) dose of S-

MEPH significantly increased the number of reinforcers earned, while the highest (2.0 mg/kg/ infusion) dose decreased the number of reinforcers earned during the two hour sessions compared to acquisition of MEPH. Following the observations that rats readily acquire S-MEPH when a history of MEPH administration is already established, in a separate cohort of rats, acquisition of S-MEPH self-administration was studied using three doses of S-MEPH (0.25, 0.5, 2.0 mg/kg/infusion) and R-MEPH (0.5 mg/kg/infusion) on a fixed-ratio 1 (FR-1) and progressive-ratio schedule of reinforcement using drug naïve rats. In this cohort, 50-kHz ultrasonic vocalizations (USVs) were recorded during FR-1 sessions to assess potential stereospecific differences in drug- associated positive affect. Rats were trained to self-administer R-MEPH (0.5 mg/kg/ infusion) or S-MEPH (0.25, 0.5, 2.0 mg/kg/ infusion) for 10 days followed by progressive-ratio for 10 days to determine motivation for drug taking. Rats readily self- administer both enantiomers of MEPH under a FR-1 schedule. Equivalent doses of R-

MEPH and S-MEPH did not significantly differ in number of infusions and active lever presses. Self-administration of R-MEPH elicited greater rates of 50-kHz USVs compared to S-MEPH. Progressive-ratio studies determined that R-MEPH- trained rats had significantly higher breakpoints than that of S-MEPH- trained rats, indicating an increase in motivation to work for reinforcer. These data suggest that escalation of S-MEPH intake occurred when a previous history of MEPH was established; rats exposed to

MEPH are compensating with escalating their intake of S-MEPH to reach the previous euphoric state during acquisition of MEPH. These findings indicate that rats readily acquire both enantiomers of MEPH, which signify an abuse liability for these drugs.

vii However, these data suggest that R-MEPH, rather than the S-MEPH, is responsible for the rewarding and reinforcing effects seen with the racemic form of MEPH, due to the lack of motivation to work for the S-enantiomer.

In conclusion, during early withdrawal from chronic cocaine or MDPV, rats demonstrated heightened anxiety-like behavior on the EPM and increased depression-like behavior in FST. Binding and functional assays indicate affinity for the 5-HT2 receptors

(2A, 2B, 2C) and no agonist activity of S-MEPH. Results show R-MEPH reinstates cocaine-seeking behavior and S-MEPH does not reinstate cocaine-seeking. Furthermore,

R-MEPH and S-MEPH both have reinforcing effects, with R-MEPH having significantly greater reinforcing strength compared to S-MEPH. These studies identified unique differences in the behavioral profile of MEPH enantiomers. This data not only expands the body of literature on the stereospecific effects of MEPH, but also developing the pharmacological profile of MDPV in the context of dependence and addiction. Continued research is needed profiling MEPH and MDPV to develop efficacious pharmacotherapeutics for treating psychostimulant abuse to reduce relapse rates.

viii DEDICATION

To my mother, you have loved me unconditionally and have always been my #1 supporter. You have always pushed me to my fullest potential. You molded me into a hard worker, taught me to never quit and always fight for what I wanted. Through it all, you have been my rock, my support, and shoulder to lean on. I know I can always count on you to put your problems aside to be there when I need you the most. Since my childhood you have sacrificed and worked countless hours to make sure I could afford all of the opportunities that came my way. I strive daily to make you proud so, I dedicate this dissertation to you Mom.

In memory of my beloved brother Kenny Sr, gone too soon. You were my first best friend. You taught me hard work and dedication will get me far in life. You were always there for me when I needed you. Our bond was like no other. You always told me that I was the #1 sis and would brag about me to all your friends. You showed me how I should me loved, taught me self-worth and loved me unconditionally. I can still feel you near and hear your words of encouragement when I feel low. You will always be in my heart and I will always cherish the countless memories we made, forever.

But he said to me, “My grace is sufficient for you, for my power is made perfect in weakness.” Therefore I will boast all the more gladly about my weaknesses, so that

Christ’s power may rest on me. That is why, for Christ’s sake, I delight in weaknesses, in insults, in hardships, in persecutions, in difficulties. For when I am weak, then I am strong. 2 Corinthians 12:9-10 NIV

ix ACKNOWLEDGMENTS

I would like to express my sincere gratitude to everyone who has supported me throughout my graduate career, this work would not have been possible without your endless support.

I would like to thank my advisor, Dr. Scott Rawls, for supporting, guiding, and training me throughout my graduate career. His patience allowed me to think independently and follow my scientific goals. I would also like to thank my thesis committee members: Dr. Barrie Ashby, Dr. Lynn Kirby, and Dr. Ellen Unterwald. You were instrumental in helping me plan my experiments efficiently and gave insightful recommendations as my work progressed. Furthermore, Dr. Allen Reitz, for being my external reader and generously synthesizing the ‘bath salts’ to make all my research possible.

I would like to express my appreciation to my supportive, humorous, and crazy big brothers in the lab, Dr. Ryan Gregg and Dr. Jae Kim. We started this journey together, and it's finally over! Thank you guys for being there for me. You were always a call or email away. You guy have kept me sane and made this progress much easier to deal with. To my current lab members: Christopher Tallarida, Sunil Nayak, Chicora

Oliver, and Dr. Callum I would like to thank you all for your friendship, support, laughter and the insiders; there is no lab like the Rawls’ lab! To our unofficial Rawls lab member Linnet, I love you girl. Thank you for listening to me complain all the time and reading my work. I’d also like to thank my mentees and undergraduates (Yohanka Elisa

Caro, Brona Ranieri, Kathryn DiFurio, Shi Su, and Lili Mo) who have scored endless amounts of USV recordings.

x

Special thanks to the Pharmacology Department and Center for Substance Abuse

Research for financially supporting my research. The Center provides an atmosphere for collaborative research to be conducted as well as a family orientated atmosphere to make you excited to come to work. I’d like to thank a group of people in CSAR, that made it an enjoyable environment and has helped me with my dissertation work: Steve Simmons,

Taylor Gentile, Menahem Doura and Mia Watson. Lastly, I’d like to thank Melva Smith, she has always been so kind and generous, and would print my posters last minute, file all my paperwork and make sure I had everything that I needed. Thank you for keeping me sane and laughing throughout the years.

Last and most importantly I’d like to thank my family and friends whose unconditional love and support kept me strong throughout my journey. You all always encouraged me and never complained when I needed to vent. To my sisters, Nelly and

Netsia, thank you for always listening and being there when I need you. No matter the hour you guys will come running just to make sure that I am okay. I am the most blessed little sister in the world. To my friends, you know who you are, who have been there from start to finish and your support means the world to me. This journey wouldn’t have been possible without your support, prayers, laughter, screenshots, and love. Finally, to my husband Ishmael, I am grateful for your love, support, and dedication throughout this process. This journey has been very stressful but you stayed by my side, endured the long days, nights, and weekends in the lab with minimal complaints, lol. You have motivated and made me push harder at times when I wanted to give up. Thank you giving me the

xi best gift in the world, our daughter, who gave me the final push that I needed to complete my dissertation. I love you both with all my heart.

xii TABLE OF CONTENTS

Page

ABSTRACT ...... iii

DEDICATION ...... iv

ACKNOWLEDGMENTS ...... v

LIST OF TABLES ...... xvii

LIST OF FIGURES ...... xviii

LIST OF ABBREVIATIONS ...... xix

CHAPTER

1. GENERAL INTRODUCTION...... 1

Scientific Rationale...... 1

Cocaine...... 3

Cathinones...... 5

Chirality ...... 8

Mephedrone: Mechanism of Action and Behavioral Effects...... 10

MDPV: Mechanism of Action and Behavioral Effects...... 13

Dopamine Neurotransmission…………………...... 16

Dopamine and Addiction……………...... 19

Serotonin Neurotransmission……………………...... 21

Serotonin and Addiction……………...... 24

General Summary of Objectives...... 26

xiii 2. SYNTHETIC CATHINONES AND STEREOCHEMISTRY: S-

ENANTIOMER OF MEPHEDRONE REDUCES ANXIETY- AND DEPRESSANT-

LIKE EFFECTS IN COCAINE- OR MDPV-ABSTINENT RATS…...... 29

Introduction...... 30

Materials and Methods...... 33

Animals and compounds...... 33

Experimental Procedures……………...... 34

Elevated plus maze (EPM) experiments...... 34

Forced swim test (FST) experiments...... 35

Binding and Functional Assays...... 36

Statistical analysis...... 36

Results...... 37

S-MEPH reduces anxiety-like behavior during psychostimulant withdrawal...... 37

S-MEPH reduces depression-like behavior during psychostimulant withdrawal..38

S-MEPH interacts with 5-HT2 receptor...... 39

Discussion...... 40

Supplementary Materials...... 48

3. EFFECTS OF MEHEDRONE ENANTIOMERS ON REINSTATEMENT OF

COCAINE SEEKING IN RATS...... 50

Introduction...... 50

Materials and Methods...... 53

Animals and drugs ...... 53

Drugs ...... 53

xiv

Catheter Implantation...... 54

Cocaine Self-Administration...... 54

Extinction...... 55

Reinstatement Test...... 55

Data Analysis...... 55

Results...... 56

Cocaine Acquisition and Extinction sessions……………...... 56

R- and S-MEPH-primed reinstatement...... 56

Discussion...... 58

4. STEREOSELECTIVE EFFECTS OF MEPHEDRONE UNDER FIXED

RATIO (FR) AND PROGRESSIVE RATIO (PR) SCHEDULES OF

REINFORCEMENT IN RATS...... 63

Introduction...... 63

Materials and Methods...... 65

Animals ...... 65

Drugs ...... 66

Surgery...... 66

MEPH Self-Administration ...... 66

Acquisition...... 66

Progressive-Ratio Schedule...... 67

Ultrasonic Vocalization (USV) Recording and Analysis...... 68

Data Analysis...... 68

xv Results...... 69

Acquisition of racemic MEPH & Dose-response of enantiomers...... 69

R and S-MEPH Dose-substitution...... 70

Dose-response on fixed-ratio & progressive-ratio schedule of reinforcement...... 71

Fixed-ratio 1...... 71

Progressive-ratio with R and S-MEPH...... 73

Self-administration of R-MEPH elicits greater 50-kHz USVs compared to

S-MEPH...... 75

Discussion...... 76

5. GENERAL DISCUSSION ...... 81

Major Findings...... 82

Implications...... 87

Future Directions and Limitations...... 89

Conclusions...... 94

REFERENCES ...... 96

xvi

LIST OF TABLES

Table Page

2.1 Inhibition (%) of binding of radioligands to receptors by 10 M S-MEPH 48

2.2 Functional assays conducted to test for agonist/antagonist activity of S-MEPH 49

xvii LIST OF FIGURES

Figure Page

1.1 Chemical structures of first and second-generation synthetic cathinones………… 7

1.2 Mephedrone enantiomers structural composition …………………………..…..… 9

1.3 Circuitry of dopaminergic pathways …………………………………………..… 18

1.4 Distribution of the 5-HT2A, 5-HT2B and 5-HT2C receptors in the brain ……… 22

1.5 Functional interactions of neurons …………………………...… 24

2.1 S-MEPH reduces anxiety-like behavior during psychostimulant withdrawal…… 46

2.2 S-MEPH reduces depression-like behavior during psychostimulant withdrawal.... 47

3.1 Cocaine Acquisition and Extinction sessions……………………………….…… 57

3.2 R- and S-MEPH-primed reinstatement …………………………..…………....… 58

4.1 Acquisition of racemic MEPH ………………………………………………...… 70

4.2 R and S-MEPH Dose-substitution …………………………………………….… 71

4.3 Acquisition of R- and S-MEPH...... 73

4.4 Progressive Ratio with R and S-MEPH...... 74

4.5 Characterization of 50-kHz USVs during self-administration of S- and R-MEPH…75

xviii

LIST OF ABBREVIATIONS

MEPH Mephedrone (4-methylmethcathinone)

R-MEPH R-(d/(+))-mephedrone

S-MEPH S-(l/(-))-mephedrone

MDPV Methylenedioxypyrovalerone

COC Cocaine

DA Dopamine

5-HT Serotonin

NE

DAT Dopamine transporter

SERT Serotonin transporter

NET Norepinephrine transporter

CPP Conditioned place preference

ICSS Intracranial self-stimulation

EPM Elevated plus maze

FST Forced swim test

NAcc Nucleus accumbens

xix CHAPTER 1

GENERAL INTRODUCTION

Scientific Rationale

Drug abuse in the United States is a major public health concern that has significant negative consequences on the judicial system (increased crime associated with drug use) and healthcare system (specialized treatment, ED visits, illnesses, and prolonged hospital stays), as well as a profound economic burden (approximately $200 billion). Drug addiction is a frequently relapsing disorder that is regarded as the compulsion to seek and take drugs despite negative consequences. Addiction leads to dysfunction of neurotransmitter systems that leads to biological, psychological, social and/or spiritual characteristic manifestations. In 2013, there were approximately 24.6 million current illicit drug users in the United States aged 12 or older, of which 1.5 million were cocaine users [SAMHSA, 2013]. Cocaine, (METH), and 3,4- methylenedioxymethamphetamine (MDMA, ‘Ecstasy’) are among the traditionally abused illicit psychostimulants. Psychostimulants are highly addictive and eventually lead to tolerance and dependence. The common routes of administration for optimal effects are injection, snorting, or smoking. Users may experience, euphoria, increased alertness, elevated mood, anxiety, elevated heart rate, elevated blood pressure, irregular heart rate, chronic insomnia, uselessness, seizures, aggression, psychosis, heart failure, and/or paranoia, especially when used in high doses or repeatedly. In recent years, an unprecedented rise in use of “legal high” alternatives to scheduled drugs such as cocaine and METH has been observed. “Legal high” alternatives are designed to mimic the intoxicating effects of commonly abused illicit drugs [Meyers et al., 2015]. Among the

1 recreational drugs that are being synthesized are synthetic cathinones, widely known as

“bath salts”, which are beta-ketone derivatives [Winstock et al., 2011;

German et al., 2014; Meyers et al., 2015]. Two synthetic cathinones that have gained popularity in the U.S. and Europe are mephedrone (4-methylmethcathinone, MEPH) and

3,4-methylenedioxypyrovalerone (MDPV). Relapse is common among addicts; most addicts (between 80-90%) will relapse within 12 months following drug abstinence

[Hendershot et al., 2011]. Physical withdrawal symptoms are manageable but the psychological withdrawal effects make it difficult to treat. A combination of therapies may need to be instituted such as cognitive-behavioral therapy, self-help support groups, other behavioral therapies, and/or change in environment. There is currently no approved to treat psychostimulant addiction to cocaine and the designer cathinones

MEPH and MDPV [Sofuoglu et al., 2013]. Given the severe consequences of psychostimulant addiction, new approaches to manage psychostimulant abuse during withdrawal and relapse need to be identified.

To identify stereospecific behavioral effects associated with individual MEPH enantiomers, the reinforcing properties and potential for abuse liability of each MEPH enantiomer will be determined. To further expound on the mechanisms of MEPH enantiomers, the behavioral and neurochemical effects of S-MEPH in animal models of cocaine-induced and MDPV-induced anxiety, depression, and relapse will be characterized. We hypothesize that S-MEPH will reduce both cocaine- and MDPV- induced anxiety and depression and prevent relapse during withdrawal through normalization of dopamine (DA) and serotonin (5-HT) dysfunction, but produce no

2 significant reinforcement by itself. The following aims were designed to address this hypothesis:

1. To characterize the effects of S-MEPH on preclinical indicators of anxiety and

depression.

2. To characterize the effects of S-MEPH on prime-induced relapse to cocaine

seeking and test for any reinforcing effects of S-MEPH itself.

3. To investigate the effects of S-MEPH administration on DA and 5-HT systems.

This work involves characterizing a new approach to developing an anti-craving and anti- relapse therapeutics for psychostimulant addiction, and developing the pharmacological profile of a novel and prevalent psychostimulant (MDPV) in the context of dependence and addiction.

Cocaine

Currently, cocaine is one of the most widely abused recreational drugs. It can be consumed by multiple routes of administration that include inhalation, nasal insufflation, and intravenous injection. Physiological effects include constricted blood vessels, dilated pupils and increased body temperature, heart rate and blood pressure, vertigo and/or muscle twitches. This can cause a variety of severe medical consequences including neurological effects (stroke, seizure, headaches, and coma), gastrointestinal complications (abdominal pain and ) and cardiovascular complications such as ischemic stroke and myocardial infarction [Pomara et al., 2012, Zimmerman, 2012]. The effects can last between five to ninety minutes depending on the route use to administer and the doses taken [Zimmerman, 2012]. Cocaine abuse leads to acute withdrawal behaviors characterized by restlessness, irritability, anxiety, depression, paranoia,

3 tremors, and development of tolerance from repeated use, due to the uncontrollable pattern of use.

The rewarding and stimulating effects of cocaine are largely due to the effects of cocaine on the brain’s nigrostriatal and mesolimbic dopamine pathways. Normally, dopamine is released into the synapse, where it can bind to a dopamine transporter and then recycled back for later use. However, with cocaine present, dopamine builds up in the synapse. Cocaine blocks the normal DA uptake process and DA activates dopamine receptors [Di Chiara and Imperato, 1988; Tanda et al., 2005]. This contributes to the rewarding and behavioral effects of cocaine. Cocaine also binds to serotonin and norepinephrine transporters, increasing extracellular levels of serotonin and norepinephrine by inhibiting the reuptake of serotonin and norepinephrine as well [Ritz et al., 1990; Andrews and Lucki, 2001]. To date, there are no FDA-approved medications available to treat cocaine addiction, despite many years of researching new approaches to reducing cocaine’s rewarding effects. Currently, the predominant form of treatment is behavioral therapy in combination with medications to treat symptomatic withdrawal effects. Some pharmacotherapeutics that have been tested for cocaine addiction include creating vaccines with cocaine-specific antibodies, agonist-replacement therapies in particular monoamine releasers, and mixed mechanism agents that target multiple neurotransmitters like dopamine, serotonin, norepinephrine, gamma-aminobutyric acid

(GABA), and glutamate [Shorter et al., 2015; Kosten and Domingo, 2013; Cai et al.,

2013]. Consequently, new approaches and targets need to be identified to better treat psychostimulant addiction.

4 Cathinones

In recent years, new psychoactive substances (NPS) have emerged on the international drug market at an alarming rate. NPS is a term used for unregulated substances that mimic the effects of controlled substances [United Nations, 2013]. NPS includes a variety of drug classes, including hallucinogenic drugs, sedatives, synthetic , synthetic opiates, synthetic cathinones, and “other substances.” In 2015, 75 new substances were identified on the international drug market [United Nations, 2016].

Years prior, had the largest number of new substances identified.

However in 2015, synthetic cathinones had the highest number of substances identified

[United Nations, 2016].

Synthetic cathinones are analogues and derivatives of cathinones. Cathinones are the naturally occurring active stimulant in the khat (Catha edulis) plant [De Felice et al.,

2014]. Khat is native to Arabian Peninsula and in some regions of Eastern Africa. The leaves are commonly chewed or brewed in teas for their stimulant effects. The recreational use of khat is common among the native populations in these regions and these practices have been occurring for decades. In recent years, the use of synthetic cathinones has increased dramatically since early 2008. Designer drugs with psychostimulant properties known as “bath salts” became prevalent in America early

2009 [Rosenbaum et al., 2012; Spiller et al., 2011]. The American Association of Poison

Control Centers reported a 20-fold increase in calls reported to poison control centers from 2010 to 2011 regarding adverse effects from “bath salts” exposure [AAPCC, 2013].

Due to this unprecedented rise in synthetic cathinone use, in early September 2011, the

5 U.S. classified three synthetic cathinones and their analogues as Schedule I drugs [D. E.

A. United States Department of Justice, 2011a].

“Bath salts” have gained popularity worldwide with psychostimulant-like effects.

Users report subjective effects similar to those of cocaine, 3,4- methylenedioxymethamphetamine (MDMA, ‘ecstasy’), and methamphetamine (METH)

[Prosser and Nelson, 2012; Spiller et al., 2011]. These compounds were sold through the

Internet, smoke shops, and “head shops” as “plant food” or “bath salts” and labeled “not for human consumption” to evade drug laws. They are often considered legal high alternatives and sold under hundreds of different brand names including “meow meow”,

“bliss”, “blue silk”, “white rush”, “ivory wave”, and “MCAT” [Laurent et al., 2015;

Prosser and Nelson, 2012; Karch, 2015]. They are typically sold in gram quantities in powders, pills, and liquids and are usually snorted, smoked, or injected.

Three synthetic cathinones, mephedrone (MEPH), , and methylenedioxypyrovalerone (MDVP), became particularly widespread and popular in the United States and have been very problematic since early 2009 [Karch, 2015; D. E.

A. United States Department of Justice, 2011b]. Although synthesis of these synthetic cathinones dates back for decades (MEPH in 1929, methylone in 1996, and MDPV in

1967), they were not popularized until the early 2000’s [German et al., 2014]. The use of these drugs have led to many published reports of toxicity, agitation, hyper arousal, psychotic behavior, excited delirium, and death [Karch, 2015; Miotto et al., 2013;

Rosenbaum et al., 2012; Spiller et al., 2011]. After years of consistent prevalence of

MEPH, methylone and MDPV, and reports of adverse effects associated with these drugs, these three compounds were emergency scheduled in late 2011, which eventually

6 led to permanent scheduling (Schedule I) [Spiller et al., 2011; D. E. A. United States

Department of Justice, 2011a]. Scheduling of MEPH, methylone and MDPV has significantly reduced consumption and access of these first generation synthetic cathinones [Stogner and Miller, 2013], however second generation synthetic cathinones have emerged as replacements [Brandt et al., 2010; Marinetti and Antonides, 2013]

(Figure 1). It will be important to develop the pharmacological profile of these prevalent psychostimulants to help elucidate the mechanisms of more synthetic cathinones (i.e. multiple generations) that are likely to emerge as replacements of the first generation synthetic cathinones.

Figure 1.1: Chemical structures of first and second-generation synthetic cathinones. First‐generation synthetic cathinones are mephedrone, methylone and MDPV. Second‐generation synthetic cathinones are 4‐methyl‐N‐ethylcathinone (4‐MEC), 4‐methyl‐α‐pyrrolidinopropiophenone (4-MePPP), , , α‐pyrrolidinovalerophenone (α‐PVP), and α‐pyrrolidinobutiophenone (α‐PBP), which appeared following the scheduling of the first-generation synthetic cathinones. Image from De Felice et al., 2014.

7 Chirality The term “chiral” means asymmetry in such a way that the structure and its mirror image are not superimposable. Frequently used in reference to molecules, tetrahedral carbons with four unalike elements are the molecules most commonly found. Most often these configurations are referred to as being “Right-handed” or “Left-handed”. However, there are a number of ways to designate the configuration of a chiral molecule. These methods are absolute configuration (R/S) and optical rotation (+/-).

(+) and (-) vs R and S

Optical rotation, also known as optical activity or the (+/−) naming convention is one naming system used for enantiomers. The direction polarized light is rotated determines its labeling (+/−). If a compound rotates light in a clockwise direction, it will be labeled

(+). When a compound rotates light in a counter-clockwise direction, it is labeled (-).

Another naming convention utilized to differentiate enantiomers is R/S, which has no relation to the previously described naming system. Absolute configuration, also known as the R/S naming convention, remains one of the better-understood naming conventions for chiral molecules. In this convention, the “R” stands for “Rectus”, the Latin word meaning “Right”, while the “S” stands for “Sinister”, a Latin word meaning “Left”. The chiral center of these molecules, the carbon, is denoted with an “R” for a right-handed configuration, or an “S” for a left-handed configuration, based on each element’s priority

(atomic number) and the clockwise, or counter-clockwise, spin from priority 1 to priority

4 (Figure 2). An easy way to read a tetrahedral carbon for its configuration is to first number the attached elements by priority, then place the lowest priority element (4) facing away from you. In this position, it is easy to connect the elements in a clockwise or counter-clockwise direction. Furthermore, given a “Right-handed” or “Left-handed”

8 chiral molecule, switching any two groups on the molecule in the chiral opposite. The

R/S naming system does not always translate to the (+)/(-) system, and for that reason

R/S is the preferred system used to differentiate between compounds, for consistency in the literature.

Figure 1.2: Mephedrone enantiomers structural composition: Chemical structures of MEPH enantiomers, MEPH chiral center produces asymmetrical orientations (e.g. R- MEPH (left panel) S-MEPH (right panel)). Image from www.Mephedrone_Enantiomers_Structural_Formulae.png

Enantiomer Stereochemistry

Stereospecific differences such as differences in mechanism of action, differences in potencies, or inactivity of one enantiomer versus the other can exist between enantiomers.

Stereospecific differences have been explored investigating behavioral and neurochemical differences of amphetamines and cathinones. Adderall ®, a widely prescribed stimulant for attention deficit disorder, uses a 3:1 ratio of S:R amphetamine salts to both lower the abuse liability and optimize the pharmacokinetic profile [Heal et al., 2013]. Methamphetamine enantiomers have different and therapeutic potentials, where the R-enantiomer is a common component found in the nasal decongestant Vick’s Vapor Inhaler, the S-enantiomer is the more potent and addictive form of the two [Li et al., 2010]. Similarly, in vivo studies conducted with MDMA have

9 shown S-MDMA displays stimulant-like effects, whereas R-MDMA display -like effects in mice [Fantegrossi et al., 2003; Murnane et al., 2009].

Behavioral and physiological studies in mice and rats indicate that the S-enantiomer of

MDPV mediates the biologic effects, including increasing locomotor activation, blood pressure and heart rate; although both enantiomers fully substituted for cocaine [Gannon et al., 2016; Schindler et al., 2016]. Another study showed that S-MDPV is more potent than racemic MDPV and R-MDPV as an inhibitor of DAT and NET, and S-

MDPV produced greater intracranial self-stimulation (ICSS) facilitation than racemic

MDPV which indicates that the abuse-related effects of racemic MDPV reside primarily with its S enantiomer; on the other hand R-MDPV failed to alter ICSS at doses up to 100 times greater than the lowest effective dose of S-MDPV [Kolanos et al., 2015]. Studies with MEPH have reported that although both enantiomers display similar potency as substrates at DA, each enantiomer has a distinct stereospecific mechanism [Gregg et al.,

2015; Vouga et al., 2015]. These examples of varying contributions of enantiomers warrant further study across multiple paradigms to better understand enantiomeric specificity.

MEPH: Mechanism of Action and Behavioral Effects

MEPH was among the three synthetic cathinones that were investigated preclinically for it behavioral and neurochemical effects. In vitro assays have suggested that MEPH acts similar to amphetamine. A study with oocyte expressing hDAT showed that MEPH produce an inward current, a depolarizing effect, acting as a substrate (monoamine releaser) [Cameron et al., 2013]. In vitro synaptosomal release assays revealed that

MEPH acts as a nonselective monoamine transporter substrate with similar potency and

10 selectivity to MDMA [Baumann et al., 2012; Simmler et al., 2013; Opacka-Juffry et al.

2014]. In addition, MEPH readily enters the brain. This was confirmed in vitro, showing

MEPH has very high blood brain barrier (BBB) permeability [Simmler et al., 2013]. In vivo studies by Kehr et al. (2011) showed that MEPH increases extracellular DA levels in nucleus accumbens (NAcc) in rats comparable to amphetamine, whereas the increase in

5-HT levels in NAcc are comparable to MDMA. Other in vivo studies demonstrated that

MEPH increases extracellular DA and 5-HT levels in the rat NAcc, with greater effects observed on 5-HT levels [Baumann et al., 2012].

MEPH produces relatively weak locomotor activation, causes -like effects, induces conditioned place preference (CPP), facilitates ICSS, substitutes for abused drugs, and maintenances self-administration. MEPH produces dose-dependent hyper locomotion in rodents [Baumann et al., 2012; López-Arnau et al., 2012; Marusich et al., 2012; Fantegrossi et al., 2013; Pail et al., 2015]. MEPH treated mice spent significantly more time in the open arms in the elevated plus-maze compared to controls

[Pail et al., 2015]. Preclinical studies investigating the rewarding effects of MEPH report

MEPH’s ability to elicit CPP in rats, mice and invertebrates [Vouga et al., 2015; Gregg et al., 2015; Karlsson et al., 2014; Lisek et al., 2012; Ramoz et al., 2012]. In ICSS studies,

MEPH increases response rates and reduces thresholds [Robinson et al., 2012; Gregg et al., 2015; Nguyen et al., 2016a; Bonano et al., 2014]. There are few published studies investigating the discriminative effects of MEPH. Drug discrimination studies in rodents show that MEPH substitutes for the discriminative stimulus effects of cocaine, MDMA and METH [Gatch et al., 2013; Varner et al., 2013; Harvey and Baker, 2016]. In terms of reinforcing effects, MEPH maintains abuse-related behavioral properties in self-

11 administration under multiple paradigms, such as fixed-ratio schedule and progressive ratio schedule [Hadlock et al., 2011; Aarde et al., 2013b; Nguyen et al., 2016b;

Vandewater et al., 2015; Creehan et al., 2015; Motbey et al., 2013].

MEPH exists as two enantiomers: R-mephedrone (R-MEPH) and S- mephedrone (S-MEPH). Stereochemistry impacts the neuropharmacological profile of

MEPH. Few investigations into the neuropharmacological effects of the individual enantiomers of MEPH exist; however, results are consistent in showing that R-MEPH seems to be the more potent psychostimulant responsible for the addictive effects observed in racemic MEPH [Gregg et al., 2015; Vouga et al., 2015]. The adverse effects of MEPH were separated from potential therapeutic effects in invertebrates. S-MEPH abolished cocaine environmental place conditioning (EPC), S-MEPH attenuated the reduction in motility caused by cocaine withdrawal, racemic MEPH, R-MEPH, and S-

MEPH increase stereotypy; however, S-MEPH is less potent and efficacious than racemic

MEPH and R-MEPH, and S-MEPH does not produce EPC, whereas racemic MEPH and

R-MEPH produced EPC [Vouga et al., 2015]. R-MEPH and S-MEPH in vitro and in vivo studies indicated that both enantiomers have similar potencies for DAT as releasers, however, S-MEPH is 50-fold more selective for SERT in promoting 5-HT release. R-

MEPH produces greater hyper locomotive activity than S-MEPH in rats, and R-MEPH has the ability to produce CPP in rats. Lastly, R-MEPH reduces ICSS thresholds to a greater extent than S-MEPH in rats [Gregg et al., 2015].

The stereochemistry and neuropharmacology of these enantiomers are related to their capacity to influence the rewarding and reinforcing effects observed in racemic

MEPH. Overall, the pharmacological effects of MEPH are similar to established

12 psychostimulants, and understanding the stereospecific properties of MEPH in vitro and in vivo will provide greater insight into the underlying abuse liability associated with racemic MEPH.

MDPV: Mechanism of Action and Behavioral Effects

MDPV is an analogue of pyrovalerone, a with stimulant effects, structurally similar to methcathinone (“Mcat”) and MDMA [Murray et al., 2012].

Although over the past decade MDPV has become a major public health concern, there is only limited information available about the mechanism of action and biologic effects. In vitro assays have suggested that MDPV shares similar functional mechanisms to cocaine.

Like cocaine, MDPV is a monoamine transporter blocker; it does not induce release of monoamines [Cameron et al., 2013; Simmler et al., 2013]. In contrast to cocaine, MDPV is a very potent and selective DAT and NET inhibitor, at least 10 times more potent than cocaine, with weak activity at SERT [Baumann et al., 2013; Simmler et al., 2013]. A study with oocyte expressing hDAT showed that MEPH produces outward currents, a hyperpolarizing effect, acting as an inhibitor (monoamine inhibitory blocker) [Cameron et al., 2013]. MDPV has two primary metabolites that were identified, 3,4- dihydroxypyrovalerone (3,4-catechol-PV) and 4-hydroxy-3-methoxypyrovalerone (4-

OH-3-MeO-PV) using human and rat urine and plasma [Strano-Rossi et al., 2010; Meyer et al., 2010a; Anizan et al., 2014]. Similar to MEPH, MDPV has high BBB permeability

[Simmler et al., 2013]. Consistent with in vitro findings, MDPV is at least 10 times more potent than cocaine at increasing extracellular DA in the NAcc [Baumann et al., 2013;

Schindler et al., 2016].

13 MDPV produces stimulant-like effects in both humans and rodents. When consumed in low doses, MPDV produces euphoria, alertness, increased , and elevated blood pressure. However, when consumed at high doses, MPDV can lead to sympathomimetic toxicity, psychotic episodes, sedation, agitation, violent behavior, hallucinations, tachycardia and even death in humans [Spiller et al., 2011; Murray et al.,

2012; Penders and Gestring 2011; Froberg et al., 2015]. MDPV neurotoxicity may be related to the potent DA and NE effect [Simmler et al., 2013]. In rodents, MDPV produces time-dependent and dose-dependent increases of locomotor activity [Baumann et al., 2012; Fantegrossi et al., 2013; Marusich et al., 2012; Gatch et al., 2013; Nguyen et al., 2016a]. A locomotor study conducted in mice tested MDPV, methylone, mephedrone, naphyrone, and butylone, and found that MDPV and MEPH produce similar locomotor stimulant effects. MDPV effects lasted much longer [Gatch et al., 2013].

Another study in rats, also comparing MDPV, MEPH, methylone, , 3- fluoromethcathinone, 4-fluoromethcathinone, COC, and METH, showed that all the drugs tested induced hyper locomotion, with MDPV having the greatest potency

[Marusich et al., 2012].

MDPV also has potent reinforcing effects in rats [Watterson et al., 2012, 2014;

Aarde et al., 2013a, 2015; Schindler et al., 2016]. 60% of rats exhibit a binge-like pattern

(defined as ≥8 infusions in a given 5-min interval) during the initiation of self- administration [Aarde et al., 2015]. Similar to cocaine, MDPV self-administration was readily acquired, and dose substitution studies displayed an inverted U-shape dose-effect curve similar to cocaine [Schindler et al., 2016]. Self-administration acquisition of MDPV and cocaine were comparable under fixed ratio schedule, while MDPV was

14 approximately 10 times more potent and 3 times more effective at maintaining self- administration in progressive-ratio (PR) schedule [Gannon et al., 2017]. Long access

(6 hours/day) sessions were conducted to assess escalation of drug intake, which revealed that MDPV led to escalation in drug taking similar to escalation patterns of METH

[Watterson et al., 2014]. There is not much literature investigating drug discrimination effects of MDPV. One laboratory found that MDPV, MDMA, and METH fully substituted for the MDPV training dose in mice [Fantegrossi et al., 2013]. MDPV fully substitutes in rats trained to discriminate cocaine and METH [Gatch et al., 2013].

MDPV produces rewarding effects that were evaluated in both ICSS and CPP paradigms. MDPV produces a dose-dependent increase in response rates and reduces thresholds for ICSS in rats [Bonano et al., 2014; Watterson et al., 2014; Kolanos et al.,

2015]. MDPV produces CPP in rats and mice [Karlsson et al., 2014; Gregg et al., 2016;

Hicks et al., 2017; King et al., 2015a, 2015b]. The rewarding effects induced by MDPV were attenuated by pretreatment with ceftriaxone, a GLT-1 activator, N- acetylaspartylglutamate (NAAG), an endogenous mGluR2/3 agonist and 2- phosphonomethyl-pentanedioic acid (2-PMPA), a GCPII (glutamate carboxypeptidase II) inhibitor [Hicks et al., 2017; Gregg et al., 2016]. MDPV has the ability to produce greater

CPP compared to amphetamine and MEPH in mice [Karlsson et al., 2014]. Sex differences did not influence CPP, but did in fact alter conditioned taste avoidance

(CTA). CTA is used to assess the aversive effects of a drug of abuse. Female rats showed a weaker avoidance response compared to male rats despite having similar preferences

[King et al., 2015a]. Age differences can also affect conditioned taste avoidance; adolescent rats have weaker aversions that develop at a slower rate compared to adult rats

15 [Merluzzi et al., 2014]. These studies indicate that MDPV possesses an abuse liability and a potential for addiction and needs to be investigated further to understand the pharmacology of these emerging drugs of abuse.

Dopamine Neurotransmission

DA is a neurotransmitter that is synthesized from the amino acid . Tyrosine is converted to L-3,4-dihydroxyphenylalanine (L-DOPA) by tyrosine hydroxylase (TH), which creates dopamine via decarboxylation of DOPA [Cooper et. al., 2003]. Following synthesis, DA is stored in vesicles then released into the synaptic cleft after a nerve impulse. Once DA is released from the presynaptic neuron, DA binds and activates DA receptors. There are five known DA receptors that exist which are G protein–coupled receptors (GPCRs). They are divided into two major classes, namely

D1- like receptors (D1 and D5) and D2-like receptors (D2, D3 and D4) [Neve et al.,

2010; Beaulieu and Gainetdinov, 2011]. Although similarities exist between the receptors, there are also vast differences, which allows them to propagate unique intracellular signaling events. Distinguishing factors includes neuronal distributions, dopamine affinity, and specificity for G protein signaling and coupling [Neve et al., 2010,

Cooper et al., 2003]. D1-like receptors couple to Gαs/olf family of G-proteins, which stimulate adenylyl cyclase, and cause an increase in cAMP production [Beaulieu and

Gainetdinov, 2011]. In addition to cAMP-regulated signaling, D1 receptors can also couple to Gαq subunits to stimulate phospholipase C, leading to the production of inositol trisphosphate and diacylglycerol; inositol trisphosphate lead to intracellular calcium mobilization and diacylglycerol activate protein kinase C [Beaulieu and Gainetdinov,

2011]. D2-like receptors are coupled to Gαi/o family of G-proteins, inhibit adenylyl

16 cyclase resulting in a reduction of cAMP production [Beaulieu and Gainetdinov, 2011].

D1-like receptors are found exclusively postsynaptically, while D2-like receptors are expressed both postsynaptically on non-dopamine cells and presynaptically on dopamine neurons [Beaulieu and Gainetdinov, 2011]. The D1 receptor is the most highly expressed

DA receptor and high mRNA levels are expressed within the frontal cortex, nucleus accumbens, caudate putamen, substantia nigra, hypothalamus, and olfactory bulb [Rankin et al., 2010]. D2 receptors are expressed in all four DA pathways (nigrostriatal, mesolimbic, mesocortical, and tuberoinfundibular), with the highest mRNA expression in the nucleus accumbens, striatum, and olfactory tubercule [Bouthenet et al., 1991]. In addition, D3 receptor distribution is most abundant in the ventral striatum (nucleus accumbens and the olfactory tubercle) and is restricted to the limbic areas of DA projections [Bouthenet et al., 1991]. The mRNA for this receptor shows a restricted pattern of expression in the central nervous system. Significant levels of mRNA expression of the D4 receptor were found in the olfactory bulb, hypothalamus, thalamus, and frontal cortex [O’Malley et al., 1992]. Lastly, D5 receptors are localized in the substantia nigra, striatum, hypothalamus, cerebral cortex, nucleus accumbens, and olfactory tubercle [Khan et al., 2000].

DA neurons have an essential role in CNS functions such as reward, memory, learning, locomotion, and attention [Beaulieu and Gainetdinov, 2011]. There are four major dopaminergic pathways [Fuxe, 1965]. The four major pathways of DA signaling are: nigrostriatal, mesolimbic, mesocortical, and tuberoinfundibular pathway (Figure 3).

The nigrostriatal pathway is the primary source of DA neurons, where cell bodies originate in the substantia nigra and project to the caudate-putamen. This pathway is

17 involved in locomotor function and loss of innervation of this pathway causes motor deficits, which lead to the symptomatic effects of Parkinson’s disease [Barbeau 1962].

The mesolimbic pathway neurons project from the ventral tegmental area (VTA) mainly to the NAcc, prefrontal cortex, and other limbic areas such as amygdala, septum and olfactory tubercule [Oades and Halliday, 1987]. This pathway plays an important role in reward, motivation, and pleasure. The mesocortical pathway is involved in cognition, emotions, and affect; while dopaminergic neurons also originate in the VTA but instead project to the prefrontal cortex. Finally, dopaminergic neurons in the tuberoinfundibular pathway consist of projections from the periventricular nuclei in the hypothalamus to the median eminence, which controls prolactin release from the anterior pituitary gland

[Weiner and Ganong, 1978]. DA neurons can also be found in the periphery and regulate cardiovascular and renal functions [Beaulieu and Gainetdinov, 2011].

Figure 1.3. Circuitry of dopaminergic pathways. Yellow lines represent mesocortical pathway with dopaminergic projections from the VTA to PFC. Blue lines represent mesolimbic pathway projecting from the VTA to NAcc. Orange lines represent nigrostriatal pathway send DA projections from the substantia nigra to the caudate nucleus and putamen. Red lines represent tuberoinfundibular pathway the periventricular nuclei in the hypothalamus send major outputs to the median eminence. Image from https://www.pinterest.com/pin/477311260487137717/

18 Dopamine and Addiction

Dopamine (DA) has a well-established role in mediating feelings of reward and pleasure. Cocaine increase extracellular DA in the nucleus accumbens by blocking DA reuptake via blockade of dopamine transporter [Di Chiara and Imperato, 1988]. Cocaine acts as a monoamine reuptake inhibitor at DA, norepinephrine (NE) and serotonin (5-HT) transporters [Ritz et al., 1987]. In animal studies, in addition to cocaine, DA reuptake inhibitors such as GBR 12909, mazindol, and nomifensine are self- administered in animal models [Bergman et al., 1989]. In humans, cocaine-induced high correlates with the degree of DAT occupancy [Volkow et al., 1997]. There is an extensive body of literature that suggests the reinforcing effects of drugs of abuse are primarily mediated through the dopamine mesocorticolimbic pathway. The limbic regions (hippocampus, amygdala, and prefrontal cortex) that influence emotion, motivation, cognition, and behavior send glutamatergic outputs to the nucleus accumbens

(NAcc) [Pierce and Kumaresan, 2006].

The mesolimbic DA pathway (VTA to NAcc) is well established as the reward pathway, linking midbrain DA neurons in mediating the rewarding effects of drugs of abuse [Fibiger and Phillips, 1986; Koob, 1992]. In vivo microdialysis assays have been used to characterize the neurochemical effects produced by drugs of abuse measuring monoamine levels in the NAcc, a major output in the mesolimbic pathway. Intracerebral microdialysis, in combination with high performance liquid chromatography (HPLC) and electrochemical detection, can be used to recover and measure endogenous extracellular monoamines from distinct brain regions in animals [Zetterström et al., 1983]. Early microdialysis studies on neurochemical effects of various drugs of abuse reported drugs

19 that are rewarding (e.g., amphetamine, cocaine, opiates, ethanol, nicotine) in both humans and animals increase DA levels in the mesolimbic pathway, specifically in the NAcc [Di

Chiara and Imperato, 1988].

Drug self-administration (IVSA) and intracranial self-stimulation (ICSS) have been used to access the abuse liability of drugs. These assays are sensitive and selective in detecting the abuse potential of abused drugs. In drug self-administration, subjects are implanted with intravenous catheters, and operant responses result in the delivery of a drug. Cocaine is reliably self-administered by animals and when D2-like agonist is substituted for cocaine, animals maintain responding, suggesting D2-like agonist have reinforcing effects [Caine et al., 1999]. In ICSS, experimental subjects are implanted with intracranial electrodes that target specific brain regions, and pulses of electrical brain stimulation maintain lever presses. Abuse-related effects induced by drugs of abuse facilitate low rates of responding or lower threshold for responding [Negus and Miller,

2014]. Amphetamine, a monoamine releaser, facilitate ICSS, an abuse related effect

[Bauer et al., 2013]. Another method to assess the rewarding effects of drugs of abuse is conditioned place preference (CPP). CPP allows rodents to receive multiple pairings of drug or saline in two environmentally distinguishable compartments during the conditioning phase, followed by a preference test where rodents access compartments in a drug-free state. Increased time in the drug-paired compartment may imply a heightened motivation associated with the rewarding effects from the drug pairings [Mueller &

Stewart, 2000]. Systemic administration of D1-like but not D2-like antagonists attenuate cocaine CPP [Nazarian et al., 2004]. Consistent with D1-like antagonist findings, D1-like were able to produce CPP [Abrahams et al., 1998].

20 In addition to CPP and self-administration, cocaine induces locomotor activation and behavioral sensitization. While cocaine induces locomotor activation [Sorg et al.,

2004], D1-like and D2-like antagonist inhibit hyperactivity induced by cocaine [Adams et al., 2001; Chausmer and Katz, 2001]. Behavioral sensitization results from repeated administration of a psychostimulants, abstinence period, and reintroduction of the drug

[Steketee and Kalivas, 2011]. D1-like and D2-like antagonists attenuate the increase in cocaine-induced locomotor activity [Mattingly et al., 1994]. Although D1-like agonists did not produce a sensitized locomotor response to methamphetamine or cocaine treated rats [Ujike et al., 1990], pretreatment with D1-like agonist reversed established cocaine sensitization [Li et al., 2000]. However, D2-like agonists did produce a sensitized locomotor response to methamphetamine or cocaine treated rats [Ujike et al., 1990].

Serotonin Neurotransmission

Serotonin (5-HT), a monoamine neurotransmitter, is primarily found in the (GI tract) and blood platelets, with only about 2% of serotonin found in the central nervous system (CNS) [Hoyer et al., 2002; Cooper et al., 2003]. 5-

HT modulates cognition, mood, emotion, appetite, addiction as well as motor function

[Frazer and Hensler, 1999]. 5-HT is synthesized via conversion of to 5- hydroxytryptophan by tryptophan hydroxylase, which is converted to serotonin via aromatic acid decarboxylase [Cooper et al., 2003]. At least fourteen serotonin receptor subtypes have been identified which are subdivided into seven receptor families. 5-HT receptor families (5-HT1-7) are divided based on molecular, pharmacological and functional diversity. All the 5-HT receptor families are GPCRs, with the exception of 5-

HT3, which is a ligand-gated ion channel [Hoyer et al., 2002]. In the brain, 5-HT neurons

21 are primarily located within the raphe nuclei, and 5-HT neurons project from the raphe nuclei to the forebrain [Steinbusch, 1981]. 5-HT receptor distribution and localization has extensively been studied. 5-HT1 receptor family is comprised of 5 receptor subtypes: 5-

HT1A, 5-HT1B, 5-HT1D, 5-HT1E and 5-HT1F, which share 40–63% sequence homology, and are coupled to Gi/o to inhibit adenylyl cyclase and decrease cAMP formation [Hoyer et al., 1994]. 5-HT2 receptor family is comprised of 3 receptor subtypes: 5-HT2A, 5-HT2B, and 5-HT2C that share 50% sequence homology, and are coupled to Gq/11 stimulation activates inositol triphosphate (IP3) leading to intracellular calcium mobilization. 5-HT2A and 5-HT2C receptors are widely distributed throughout the CNS, whereas 5-HT2B is found mainly in the periphery (Figure 4) [Quesseveur et al.,

2012]. 5-HT3 receptors are localized in the hippocampus, with the CA1 pyramidal cell layer, nucleus of the solitary tract, and the area postrema [Laporte et al., 1992]. Lastly,

HT4, HT6, and HT7 receptors all couple to the Gαs family of G-proteins, resulting in the stimulation of adenylyl cyclase and increase cAMP production.

Figure 1.4: Distribution of the 5-HT2A, 5-HT2B and 5-HT2C receptors in the brain. White dots represent 5-HT2A receptors. Gray dots represent 5-HT2B receptors. Black dots represent 5-HT2C receptors. Abbreviations: AMY: Amygdala; Cx: Cortex; DR: Dorsal raphe; Hi: Hippocampus; HYP: Hypothalamus; LC: Locus coeruleus; NAc: Nucleus accumbens; Th: Thalamus; VTA: Ventral tegmental area. Image from Quesseveur et al., 2012.

22 Functional interactions between various monoamine systems exist (Figure 5). DA and 5-HT systems interact in multiple capacities such as, motor functions [Fink et al.,

1979; Gerson and Baldessarini, 1980], thermoregulation [Grabowska et al., 1973;

Yamawaki et al., 1983], neurochemical effects [Balsara et al., 1979; Carter and Pycock,

1978; Waldmeier and Delini-Stula, 1979], and various disorders [Koob, 1992; Fibiger,

1995]. The specific nature of these interactions is difficult to explain, however 5-HT receptor subtypes may contribute to the excitatory and inhibitory roles of 5-HT on DA release. For example, 5-HT2A receptors facilitate the release of DA upon activation

[Pehek et al., 2001, 2006; Bowers et al., 2000]. 5-HT2A receptors antagonism attenuate

DA release [Pehek et al., 2001, 2006]. Furthermore, 5-HT2C receptors inhibit DA release through the activation of GABAergic interneurones in the VTA [Pessia et al., 1994; De

Deurwaerdère et al., 2004]. 5-HT2C receptors exert tonic inhibitory regulation of DA release primarily through the nucleus accumbens shell [Navailles et al., 2006]. In addition, the involvement of 5-HT2C receptors expressed into both the VTA and nucleus accumbens contribute to the phasic inhibitory control exerted by 5-HT2C receptors within the mesoaccumbens DA pathway [Navailles et al., 2006, 2008]. Taken together, the crosstalk between the 5-HT and DA systems may provide a pharmacological approach to develop new pharmacotherapeutics to treat drug abuse and neuropsychiatric disorders.

23

Figure 1.5: Functional interactions of monoaminergic neurons. Direct and indirect interactions between NE, 5-HT and DA neurons are mediated through various receptor types. Image from Hamon and Blier, 2013.

Serotonin and Addiction

The serotonin system is commonly implicated in the regulation of mood, anxiety, depression, and drug addiction. Many psychostimulants such as cocaine, methamphetamine, and MDMA interact with multiple neurotransmitter systems, including 5-HT. Many substances like cocaine and methamphetamine predominantly exert their effects by increasing dopamine. Both systemic injections and local infusions of cocaine dose-dependently produce increases in extracellular levels of DA and 5-HT levels in the nucleus accumbens, with greater increase in DA [Andrews and

Lucki, 2001]. Cocaine increases extracellular DA in the nucleus accumbens, however 5-

HT uptake inhibitors attenuate the reinforcing effects of cocaine, suggesting elevating 5-

HT can attenuate the reinforcing effects of cocaine [Czoty et al., 2002]. Consequently,

24 cocaine withdrawal significantly decreases levels of extracellular 5-HT in the nucleus accumbens [Parsons et al., 1996]. In contrast, MDMA predominantly increases 5-HT and its empathogenic effects are mediated through the 5-HT system [Hysek et al.,

2012]. MDMA stimulates monoamine release, with preferential release of 5-HT compared to the release of other monoamines though the respective transporters with greater affinity for the serotonin transporter compared to the dopamine transporter

[Verrico et al., 2007]. MDMA and other substances that preferentially increase 5-HT are also associated with toxicity including serotonin syndrome, hyperthermia, hyponatremia, and seizures [Simmler et al., 2011; Liechti et al., 2003, 2005].

MDMA facilitates both low ICSS rates and depressed high ICSS rates, while , a 5-HT-selective releaser, exclusively depresses ICSS [Bauer et al., 2013].

These results elucidate a role to discriminate abuse-related and abuse-limiting drug effects, and selectivity to promote DA vs. 5-HT release correlates with abuse-related effects.

Several 5-HT receptor subtypes that are most implicated in addiction are 5-HT1A,

5-HT1B, 5-HT2A, 5-HT2C and 5-HT3 [Di Matteo et al., 2008]. Blocking or stimulating these receptor subtypes can alter the neurochemical and behavioral effects of a drug.

Specifically, 5-HT2 receptors have been implicated in addiction. For example, several studies indicate that 5-HT2A and 5-HT2C receptors have opposite effects. In rats, 5-

HT2A antagonists or 5-HT2C agonists reduce cocaine self-administration and cue- and cocaine- primed reinstatement of extinguished drug seeking behaviors [Nic Dhonnchadha et al., 2009; Pockros et al., 2011; Fletcher et al., 2003, 2007], whereas 5-HT2C antagonists increase responding for cocaine [Fletcher et al., 2002]. Additionally, 5-HT2A

25 and 5-HT2C receptors also have opposing effects on impulsivity. Infusion of a selective

5-HT2A antagonist into the nucleus accumbens significantly decreased impulsivity. In contrast, a selective 5-HT2C antagonist into the nucleus accumbens dose-dependently increased impulsivity [Robinson et al., 2008]. Another group showed that 5-HT2A receptor antagonists attenuate cocaine-induced hyperactivity, while 5-HT2C receptor antagonists increase cocaine-induced hyperactivity [Fletcher et al., 2002]. Likewise, administration of 5-HT2C receptor agonists attenuated cocaine conditioned place preference (CPP), cocaine-induced hyperactivity and development of behavioral sensitization [Craige and Unterwald, 2013]. Activation of 5-HT2A receptors leads to anxiolytic-like effects [Onaivi et al., 1995; Petit-Demouliere et al., 2009]. Conversely, activation of 5-HT2C receptors leads to anxiogenic-like effects [Fone et al., 1996;

Rodgers et al., 1992]. In rodent studies, blockade of 5-HT2C receptors produce anxiolytic-like effects [Dekeyne et al., 2008, Millan et al., 2005]. Mice withdrawn from chronic cocaine exhibited increased anxiety-like behavior; 5-HT2C ,

SB 242084 attenuated the anxiety-like behavior produced by cocaine withdrawal [Craige et al., 2015]. Overexpression of 5-HT2C receptors in mice leads to elevated anxiety-like behavior [Kimura et al., 2009] but 5-HT2C receptor knockout mice [Heisler et al., 2007].

Taken together, 5-HT2A and 5-HT2C receptors have opposing effects on DA neurotransmission. Stimulation of the 5-HT2A receptor activates the mesolimbic DA pathway whereas the 5-HT2C receptor inhibits the mesolimbic DA pathway.

General Summary of Objectives

Synthetic cathinones have emerged on the drug market in the United States and UK over the past decade at an unprecedented rise. Synthetic cathinones also known as “bath salts”

26 are amphetamine-like or cocaine-like compounds that are found in the khat plant. Two of the more popular “bath salts” that are found on international drug markets are MEPH and

MDPV. MEPH acts as a monoamine releaser similar to amphetamine, while MDPV is a potent monoamine transporter reuptake inhibitor with cocaine-like effects [Novellas et al., 2015; Baumann et al., 2012; Simmler et al., 2013]. Similar to other amphetamines and cathinones, MEPH has a chiral center and two enantiomers exists, R-mephedrone (R-

MEPH) and S-mephedrone (S-MEPH). Stereospecific effects of enantiomers can impact there neuropharmacology. Investigations into the stereochemistry of MEPH are limited.

Gregg et al (2015) have shown S-MEPH has a preferential serotonin component over dopamine compared to R-MEPH, and that R-MEPH produces significantly greater place preference and locomotor activity compared to S-MEPH. “Bath salts” at low doses have been reported to enhance mood and increase alertness, while high doses or chronic use leads to violent behaviors, psychosis, hyperthermia, agitation, tachycardia and sometimes death [Spiller et al., 2011; Dargan et al., 2011; Prosser & Nelson, 2012]. Despite the adverse effects to users and the health risk psychostimulant use poses, there is still no

FDA approved pharmacotherapy to date. Consequently, new approaches and targets need to be identified to treat psychostimulant addiction. Given the emergence of synthetic cathinones and the significant negative impacts on global public health, the studies in this dissertation investigated: (1) the role of S-MEPH as potential template to develop an anti- anxiety and pharmacotherapy during psychostimulant (cocaine and

MDPV) withdrawal, (2) investigate the effect of S-MEPH on relapse to cocaine seeking using the self-administration model, and (3) assess any reinforcing effects of S-MEPH.

This work provides insight into the neurochemical and behavioral profiles of each MEPH

27 enantiomer. Additionally, our investigations will involve characterizing a new approach to developing an anti-craving and anti-relapse therapeutic for psychostimulant addiction and developing the pharmacological profile of a novel and prevalent psychostimulant

(MDPV) in the context of dependence and addiction.

28 CHAPTER 2

SYNTHETIC CATHINONES AND STEREOCHEMISTRY: S ENANTIOMER OF

MEPHEDRONE REDUCES ANXIETY- AND DEPRESSANT-LIKE EFFECTS IN

COCAINE- OR MDPV-ABSTINENT RATS

Helene L. Philogene-Khalid1,2, Callum Hicks1,2, Allen B. Reitz3, Lee-Yuan Liu-Chen1,2 , and Scott M. Rawls1,2

1Department of Pharmacology, Lewis Katz School of Medicine, Temple University, Philadelphia, PA, USA

2Center for Substance Abuse Research, Lewis Katz School of Medicine, Temple University, Philadelphia, PA, USA

3Fox Chase Chemical Diversity Center Inc., Doylestown, PA

Drug and Dependence

29 Introduction

Psychostimulants of the designer cathinone class (i.e., beta-ketone amphetamines) are known colloquially as “bath salts” [Karch et al., 2015; Kerrigan et al., 2016].

Mechanistically, synthetic cathinones have been classified according to two distinct actions on the monoamine system. One class is comprised of substrate-type releasers, such as mephedrone (4-methylmethcathinone, MEPH), that enter the presynaptic neuron and promote transporter-mediated release of dopamine (DA) and serotonin (5-HT)

[Baumann et al., 2012; Eshleman et al., 2013; López-Arnau et al., 2012; Opacka-Juffry et al., 2014]. In vivo microdialysis studies have shown that the substrate action of MEPH increases extracellular DA and 5-HT in the nucleus accumbens of rats [Baumann et al.,

2012; Kehr et al., 2011]. MEPH also increases locomotor activity in rats and mice following acute exposure and produces locomotor sensitization following repeated, intermittent exposure [Gatch et al., 2013; Gregg et al., 2013; López-Arnau et al., 2012;

Motbey et al., 2012; Shortall et al., 2012; Wright et al., 2012]. In preclinical rat models of abuse liability, MEPH facilitates intracranial self-stimulation (ICSS), is self- administered, and produces conditioned place preference (CPP) that is dependent on active dopamine D1 receptors [Bonano et al., 2014; Hadlock et al., 2011; Lisek et al.,

2012; Motbey et al., 2013].

A second class of synthetic cathinones is comprised of monoamine transport blockers, such as 3,4- methylenedioxypyrovalerone (MDPV), that prevent cellular reuptake of monoamines through a cocaine-like mechanism [Baumann et al., 2013].

Relative to cocaine, MDPV is a 10-fold more potent monoamine transport blocker and shows preferential selectivity for dopamine transporters (DAT) and norepinephrine

30 transporters (NET) with weak inhibition at 5-HT transporters (SERT) [Baumann et al.,

2013; Simmler et al., 2013]. MDPV produces cocaine-like behavioral effects, but displays greater potency than cocaine in fixed-ratio and progressive-ratio self- administration paradigms, as well as in locomotor studies [Aarde et al., 2013a; Schindler et al., 2016]. MDPV is short acting, so users have a frequent desire to re-dose (binge-like patterns of consumption) [Novellas et al., 2015]. MDPV and MEPH have provided templates for the design and synthesis of newer generation synthetic cathinone compounds, such as α-PVP, 4-MEC, naphyrone, and pentedrone, which differ in structure from MDPV and MEPH by simple functional group substitutions.

The stereochemistry of enantiomers allows us to separate the addictive and potential therapeutic effects of a drug, as each enantiomer can produce different CNS effects. For example, amphetamine salt (Adderall ®), a widely prescribed drug for attention deficit disorder, uses a 3:1 mixture of S:R amphetamine salts to optimize the pharmacokinetic profile and lower the abuse liability [Heal et al., 2013].

Methamphetamine has enantiomers with different where the S- enantiomer (Desoxyn) is more potent and addictive than the R-enantiomer, while the R- enantiomer is used in the nasal decongestant Vick’s Vapor Inhaler [Li et al., 2010].

Research also shows divergent behavioral and physiological effects with the two enantiomers of MDPV (Gannon et al., 2016; Schindler et al., 2016). Although both R- and S-MDPV fully substituted for cocaine, only the S-enantiomer dose-dependently increased locomotor activity, blood pressure and heart rate [Gannon et al., 2016;

Schindler et al., 2016].

31 Stereochemistry also influences the behavioral and neurochemical profiles of

MEPH [Gregg et al., 2015; Vouga et al., 2015]. MEPH has a chiral center at the α-carbon and exists as two enantiomers (R- and S-), which are sufficiently stable to racemization to allow for their independent in vitro and in vivo evaluation. Gregg et al. (2015) showed that both enantiomers (R- and S-) of MEPH display similar potency as substrates at DAT, however, S-MEPH is about 50-fold more potent than R-MEPH in promoting 5-HT release by its substrate action at SERT. Dose-response experiments using ICSS to compare rewarding effects demonstrated that R-MEPH (1–10 mg/kg) produces greater maximal facilitation of ICSS than equivalent doses of S-MEPH [Gregg et al., 2015]. In

CPP studies, R-MEPH (5, 15 and 30 mg/kg) produces place preference whereas equivalent doses of S-MEPH do not have rewarding effects [Gregg et al., 2015].

Additionally, R-MEPH (5, 10, 20 and 30 mg/kg) induces greater locomotor activity than

S-MEPH (5, 10, 20 and 30 mg/kg).

During the early withdrawal phase, human cocaine abusers report heightened anxiety and depression [Elman et al., 2002; Gawin and Kleber, 1986], both of which are observed in animal studies using validated behavioral assays [Erb et al., 2006; Perrine et al., 2008]. At the neurochemical level, discontinuation of chronic cocaine exposure produces 5-HT deficits, including decreases in extracellular 5-HT in the nucleus accumbens during early withdrawal [Parsons et al., 1996]. Given that S-MEPH is a potent

5-HT releaser and lacks significant rewarding effects in CPP and ICSS assays [Gregg et al., 2015], we hypothesized that it would reduce anxiety- and depression-like behaviors during withdrawal following repeated cocaine or MDPV exposure. We characterized the effects of S-MEPH on withdrawal-induced anxiety- and depression-like behavior using

32 the elevated plus maze (EPM) and forced swim test (FST), respectively. Both are well established and validated rodent tests of emotional behaviors [Walf et al., 2007;

Estanislau et al., 2011], and known (e.g. diazapem) and anti-depressants (e.g. fluoxetine and ) produce significant effects in these models [Paine et al., 2002;

Liu et al., 2010; Jesse et al., 2010]. Although 5-HT release is the primary mechanism of action of S-MEPH, we also screened S-MEPH against a battery of receptors in binding and functional assays to determine the degree of affinity and interaction between S-

MEPH and a range of receptor targets. By correlating the in vitro binding results with the behavioral effects of S-MEPH, we aimed to elucidate a possible secondary mechanism that may contribute to its clinical relevance.

Materials and methods

Animals and compounds

Adult male Sprague-Dawley rats (250–300 g; N=108; Harlan Laboratories, Indianapolis,

IN) were used in all experiments. Animals were housed two per cage in a humidity- controlled vivarium, maintained on a 12-h light/dark cycle with lights turning on daily at

7:00AM. Rats were provided with ad libitum food and water access except during experimental testing. All procedures and animal care was conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and Temple University’s

Institutional Animal Care and Use Committee guidelines.

Cocaine hydrochloride was obtained from Sigma Aldrich (St. Louis, MO, USA, while MDPV and S-MEPH were synthesized according to published methods [Gregg et al., 2015]. All drugs were dissolved in physiological saline (0.9%) and injected

33 intraperitoneally (i.p.) at a volume of 1 ml/kg. Equivalent injections of saline were given for control conditions.

Experimental Procedures

For both experiments, we used a binge-like paradigm in which saline, cocaine (10 mg/kg), or MDPV (1 mg/kg) was administered 3x/day for 10 days in 1-h intervals during the morning hours (~10:00AM-12:00PM) [Anneken et al., 2015; Craige et al., 2015].

Rats were randomized into 9 treatment groups (N=6 per treatment group): 1) Saline control, 2) Saline + S-MEPH 10 mg/kg, 3) Saline + S-MEPH 30 mg/kg, 4) Cocaine +

Saline, 5) Cocaine + S-MEPH 10 mg/kg, 6) Cocaine + S-MEPH 30 mg/kg, 7) MDPV +

Saline, 8) MDPV + S-MEPH 10 mg/kg, and 9) MDPV + S-MEPH 30 mg/kg. The doses of S-MEPH were chosen based on Gregg et al. (2015) who showed that R-MEPH (5, 15 and 30 mg/kg), but not S-MEPH (5, 15 and 30 mg/kg), dose-dependently increased place preference.

Elevated plus maze (EPM) experiments

Twenty-four hours after the last injection of cocaine, MDPV, or saline, rats (N=54) were injected with saline or S-MEPH (10 or 30 mg/kg) 2x/day for 2 days 6-h apart

(~10:00AM-4:00PM) due to the relatively short half-life of racemic MEPH. Twenty-four hours after the last treatment with saline or S-MEPH (10 or 30 mg/kg), anxiety-like behavior was evaluated using the EPM test. EPM testing was conducted in the afternoon

(~1:00PM-5:00PM). The apparatus consisted of four equal-sized runways (19” by 4”) that were elevated 20” off the ground in the shape of a plus sign. Two of the arms were enclosed by walls (13” high) on three sides (“closed arm”), while the other two arms were without walls (“open arm”). Rats were tested using adjusted lighting with the open

34 arm illuminated to 200 lux, while the center was set to 160 lux. Arm entry was considered when a rat’s head, shoulder, and two front limbs moved into the arm. The apparatus was thoroughly cleaned with 70% ethanol and allowed to dry between each session. Individual rats were first placed in the center of the maze and allowed to roam for 10min. The session was recorded, and the time on the open and closed arms was later scored by an experimenter blinded to treatment conditions. The amount of time spent on the open arms was determined and reported as a percentage of the entire session time.

Open arm entries were also calculated.

Forced swim test (FST) experiments

Depression-like behavior was assessed in the FST using a separate cohort of rats (N=54).

Following the 10-day binge injections of cocaine or MDPV, animals were returned to their home cages for 2 days before behavioral testing. Behavioral testing occurred on a varying schedule: day 1 swim sessions were conducted from ~1:00PM-5:00PM, whilst day 2 swim sessions were run from ~1:00PM-3:00PM. Rats were placed individually into a glass cylinder (18” high, 8” diameter) filled with 10” of water (25oC) [El Hage et al.,

2012]. Two swim sessions were performed to test for immobility. A 15 min pretest, during which no behaviors were recorded, served as habituation and was conducted 48-h into withdrawal. This was followed 24-h later by a 5 min test, during which the behaviors were recorded. Immediately after the first test, rats were dried and returned to their home cages and then treated with S-MEPH (10 and 30 mg/kg, i.p.) or saline. Two subsequent treatments were given 4-h and 30 min prior to a second swim session. The dosing paradigm was chosen to achieve the maximal pharmacological effect (Castagne et al.

2010). Time spent in the immobile state was manually scored after testing by an

35 experimenter blinded to treatments. Immobility was defined as no additional movements other than those necessary to keep the rat’s head above water, and was expressed as a percentage of the total session time.

Binding and Functional Assays

Binding and functional assays were performed at the National Institute of Mental Health

Psychoactive Drug Screening Program (PDSP), which is directed by Dr. Bryan Roth at the University of North Carolina. For radioligand binding, S-MEPH was screened at 10

M for binding to the following receptors: 5-HT (5-HT1A,B,D, 5-HT2A-C, 5-HT3, 5-HT6, 5-

HT7); DA (D1, D2, D3, D4, D5); sigma (sigma-1); kappa opioid; adrenergic (α1A, α1B, α1D,

α2A, α2B, α2C, β1, β2, β3); muscarinic (M1-M4); and nicotinic (α2β2; α2β4; α3β2; α3β4; α4β2;

α4β4; α7). 5-HT receptor subtypes were screened because 5-HT receptor activity influences DA transmission during cocaine addiction [Vázquez-Gómez et al, 2014].

Nicotinic receptors were screened because S-MEPH is structurally similar to the cathinone derivative bupropion (Wellbutrin), which inhibits monoamine reuptake and nicotinic receptors [Arias et al., 2009; Filip et al., 2006]. Sigma receptors and sodium channels were examined for comparison to the mechanism of action of cocaine. For targets that had more than 50% inhibition, Ki values were obtained, and then moved to functional assays. Functional assays used measurements of intracellular calcium mobilization for Gq/11-coupled targets performed on serotonin receptors: 5-HT2A, 5-

HT2B, 5-HT2C. For a detailed description of the protocol, refer to the supplementary material.

Statistical analysis

A two-way ANOVA was used to analyze time spent on the open arm and number of open

36 arm entries during EPM testing, as well as immobility time in the FST. The two between- subject factors were “chronic treatment” and “treatment”. “Chronic treatment” was defined as the binge-like paradigm of cocaine, MDPV or saline. “Treatment” referred to the administration of two doses of S-MEPH (10 or 30 mg/kg) or saline following discontinuation of the binge-like paradigm. In the case of a significant overall ANOVA,

Bonferroni post-hoc tests were used to determine group differences. All analyses were conducted using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA) with statistical significance set at P < 0.05.

Results

S-MEPH reduces anxiety-like behavior during psychostimulant withdrawal

(Fig. 2.1) Data are presented as percentage of time spent on the open arms (N=6 per treatment group). A two-way ANOVA indicated a significant overall effect of chronic treatment [F(2, 45) = 3.86, P < 0.05] and treatment [F(2, 45) = 6.76, P < 0.05], as well as a significant interaction [F(4, 45) = 8.04, P < 0.05]. Bonferroni analysis showed that rats withdrawn from chronic cocaine exposure (COC SAL) or MDPV (MDPV SAL) spent less time on the open arms compared to drug-naïve control rats (SAL SAL) (P < 0.05). In rats withdrawn from chronic cocaine exposure, subsequent treatment with 10 mg/kg S-

MEPH (COC SM 10) increased the percentage of time spent on the open arms compared to treatment with saline (COC SAL) (P < 0.05). Similar efficacy for 10 mg/kg of S-

MEPH was observed in rats withdrawn from chronic MDPV exposure, as treatment with

S-MEPH during the drug-free period (MDPV SM 10) enhanced the percentage of time spent on the open arms compared to treatment with saline (MDPV SAL) (P < 0.05).

Treatment with a higher dose of S-MEPH (30 mg/kg) also increased the percentage of

37 time spent on the open arms for rats withdrawn from either cocaine (COC SM 30) (P <

0.05 versus COC SAL) or MDPV (MDPV SM 30) (P < 0.05 versus MDPV SAL); however, treatment with 30 mg/kg S-MEPH by itself (SAL S-MEPH) caused a slight, but significant, decrease in time spent on the open arms compared to drug-naïve control rats

(SAL SAL) (P < 0.05). The mean number of open arm entries for each treatment condition (N=6 per treatment group) are presented in Figure 1B. A two-way ANOVA indicated a significant overall effect of treatment [F(2, 45) = 4.06 , P < 0.05] but not chronic treatment [P > 0.05], as well as a significant interaction effect [F(4, 45) = 5.99 ,

P < 0.05]. Bonferroni post-hoc tests indicated that withdrawal following chronic cocaine

(COC SAL) or MDPV (MDPV SAL) exposure caused a significant reduction in the number of open arm entries compared to drug-naïve control rats (SAL SAL) (P < 0.05).

This effect was reversed after S-MEPH treatment in both cocaine (COC SM 10, 30) (P <

0.05 versus COC SAL) and MDPV (MDPV SM 10, 30) (P < 0.05 versus MDPV SAL) exposed animals.

S-MEPH reduces depression-like behavior during psychostimulant withdrawal

(Fig. 2.2)

Data are presented as the percentage of time spent in the immobile state (N=6 per treatment group, except in group Saline + S-MEPH 10mg/kg with N=5 due to one rat dying during the binge injections). Analysis with two-way ANOVA showed effects of chronic treatment [F(2, 44) = 3.64, P < 0.05] and treatment [F(2, 44) = 72.37, P < 0.05], as well as a significant interaction [F(4, 44) = 5.26, P < 0.05]. Bonferroni analysis indicated that rats withdrawn from chronic cocaine exposure (COC SAL) or MDPV

(MDPV SAL) exposure spent more time in the immobile state compared to drug-naïve

38 control rats (SAL SAL) (P < 0.05). In rats withdrawn from chronic cocaine exposure, subsequent treatment with 10 mg/kg S-MEPH (COC SM 10) reduced the time spent in the immobile state compared to saline treatment (COC SAL) (P < 0.05). Similar efficacy for 10 mg/kg of S-MEPH was observed in rats withdrawn from chronic exposure to

MDPV, as treatment with S-MEPH during the drug-free period (MDPV SM 10) reduced the time spent in the immobile state compared to treatment with saline (MDPV SAL) (P

< 0.05). Treatment with a higher dose of S-MEPH (30 mg/kg) also decreased time spent in the immobile state for rats withdrawn from cocaine (COC SM 30) (P < 0.05 versus

COC SAL) or MDPV (MDPV SM 30) (P < 0.05 versus MDPV SAL); however, treatment with the higher dose of S-MEPH by itself (30 mg/kg) (SAL S-MEPH) reduced time spent in the immobile state compared to drug-naïve control rats (SAL SAL) (P <

0.05).

S-MEPH interacts with 5-HT2 receptors

At 1 M, S-MEPH inhibited radioligand binding to 5-HT2A, 5-HT2B, 5-HT2C, and sigma-

1 receptors by more than 50%. Ki values for receptors were then determined by competitive inhibition of radioligand binding: 5-HT2A (Ki = 1.3 M), 5-HT2B (Ki = 0.857 M), 5-

HT2C (Ki = 3.9 M), and sigma-1 (Ki = 1.5 M). Functional assays using measurements of intracellular calcium mobilization performed on serotonin receptors revealed that there was no stimulation up to 100 µM, suggesting that S-MEPH acted as an antagonist at these receptors. In addition, at a concentration of 1 μM, S-MEPH did not inhibit more than 50% of radioligand binding at the following receptors: 5-HT (5-HT1A,B,D, 5-HT3, 5-

HT6, 5-HT7); DA (D1, D2, D3, D4, D5); kappa opioid; adrenergic (α1A, α1B, α1D, α2A, α2B,

α2C, β1, β2, β3); muscarinic (M1-M4); and nicotinic (α2β2; α2β4; α3β2; α3β4; α4β2; α4β4; α7).

39 Discussion

Synthetic cathinones are noted for their psychoactive effects and categorized as

Schedule I drugs in the United States; however, one older cathinone derivative, bupropion (Wellbutrin), is a frequently prescribed antidepressant and smoking cessation therapy. This paradox between newer synthetic cathinones (i.e., ones appearing within the past decade) and bupropion suggests that cathinones possess adverse and therapeutic properties that are dependent on factors such as structure and stereochemistry [Heal et al.,

2013; Li et al., 2010]. We demonstrated previously that stereochemistry influences the neuropharmacological profile of MEPH and the R-enantiomer is primarily responsible for rewarding properties of MEPH [Gregg et al., 2015; Vouga et al., 2015]. The S- enantiomer is a potent 5-HT-releasing agent (50-fold stronger than R-MEPH) that does not produce rewarding effects in CPP and ICSS studies [Gregg et al., 2015; Vouga et al.,

2015]. S-MEPH is structurally similar but mechanistically distinct from bupropion, with

S-MEPH acting as a monoamine substrate to release 5-HT and bupropion acting as a

DAT/NET blocker with minimal effects at SERT. Our original work with S-MEPH provided rationale for the present study in which we demonstrated efficacy for S-MEPH against psychostimulant withdrawal behaviors. The major findings of the present study were that: 1) MDPV, a synthetic cathinone and monoamine transport blocker, produced cocaine-like withdrawal behaviors in the EPM and FST; 2) S-MEPH, given to rats during the drug-free period following cocaine or MDPV, attenuated anxiety- and depression-like behaviors resulting from psychostimulant withdrawal; and 3) S-MEPH displayed affinity and interactions with 5-HT2 receptors but no agonist activity.

In our behavioral experiments, S-MEPH counteracted anxiety- and depression-

40 like behaviors in rats withdrawn from a chronic cocaine or MDPV regimen when given during the drug-free period following psychostimulant exposure. In both the EPM and

FST, S-MEPH displayed efficacy at 10 and 30 mg/kg. Although neither dose of S-MEPH is rewarding in the rat CPP assay [Gregg et al., 2015; Vouga et al., 2015], both doses produce some locomotor activation, especially ambulation, but not to the magnitude or duration of the R-enantiomer. Evidence that S-MEPH produces locomotor activation has to be considered in the interpretation of its efficacy in EPM and FST experiments. In the case of the EPM results, behavioral testing was conducted 24-h after the last treatment with S-MEPH, well after locomotor activation produced by acute S-MEPH subsides

[Gregg et al., 2015]. Therefore, the ability of S-MEPH to increase time spent on the open arms in cocaine- or MDPV-withdrawn rats is more likely due to an anxiolytic-like effect than to locomotor activation resulting from acute S-MEPH exposure.

In the FST experiments, behavioral testing was conducted 30 min after the last S-

MEPH injection, so hyperlocomotion caused by acute S-MEPH exposure may have exerted a more significant influence on behavioral outcomes. We chose the 30 min interval between the last S-MEPH injection and onset of behavioral testing based on the temporal profile of S-MEPH locomotor activation [Gregg et al., 2015]. In that study, 10 mg/kg of S-MEPH produced an increase in locomotor activity that returned to baseline levels 30-35 min post-injection, which was the time frame of FST experimentation here, and remained at baseline activity levels for the remainder of the 90-min observation period [Gregg et al., 2015]. Since 10 mg/kg of S-MEPH did not decrease time spent in the immobile state in drug-naïve control rats, the most parsimonious explanation for its efficacy in cocaine- or MDPV-abstinent rats is a reduction in depression-like behavior, as

41 opposed to an increase in locomotor activity. However, with 30 mg/kg of S-MEPH, locomotor activation likely contributed to the efficacy of S-MEPH in the FST, as this higher dose decreased time spent in the immobile state in drug-naïve control rats and is associated with a more protracted increase in locomotor activation that persists for at least 90 min post-injection [Gregg et al., 2015]. Nonetheless, since the lower dose of 10 mg/kg was efficacious in the FST, we posit that S-MEPH is capable of reducing depression-like behavior accompanying psychostimulant withdrawal. Future studies will better examine S-MEPH for baseline anxiolytic and antidepressant effects, especially since acute administration of racemic MEPH produces both of these effects [Pail, 2015].

S-MEPH was screened against multiple receptor subtypes (5-HT, DA, adrenergic, kappa opioid, muscarinic, nicotinic, and sigma) in binding assays to provide a measure of interactions and degree of affinity (weak, strong, or no connection) between

S-MEPH and receptor targets. Results showed that S-MEPH has moderate affinity for 5-

HT2 receptors but little to no interaction with the other receptors tested. Functional assays revealed that S-MEPH did not display agonist activity at 5HT2 receptors, but exhibited some antagonist activities. Although identification of mechanism by correlating in vitro binding results with behavioral outcomes and pharmacokinetic data is challenging, it is interesting to note that Ki values obtained in the binding assays for 5-HT2 receptors were in the micromolar range [5-HT2A (Ki = 1.3 M); 5-HT2B (Ki = 0.857 M); 5-HT2C (Ki =

3.9 M)]. Pharmacokinetic data indicate that racemic MEPH freely crosses the blood brain barrier and shows considerable distribution throughout the brain, which is reflected by its brain/plasma ratio of 1.85 [Martínez-Clemente et al., 2013]. A plot of locomotor activity versus racemic MEPH concentrations over time shows a direct concentration–

42 effect relationship [Martínez-Clemente et al., 2013]. This model provided by Martinez-

Clemente and colleagues (2013) reveals a mean in vivo EC50 value of 0.86 µM for MEPH that is in fairly close agreement with in vitro results showing that racemic MEPH inhibits monoamine uptake at concentrations below 1 μM [Gregg et al., 2015; López-Arnau et al.

2012]. According to Martínez-Clemente et al. (2013), the peak plasma concentration of racemic MEPH (HCl salt) after oral administration is 1.55 µM (331.2 ng/ml). In this study, we used i.p. injection of 10 mg/kg and 30 mg/kg S-MEPH. Assuming similar following i.p. and oral administration, S-MEPH at 30 mg/kg (i.p.) would have significant occupancy at 5-HT2A and 5-HT2B and, to a lower extent, 5-HT2C receptors. Therefore, actions of S-MEPH on one or more of these 5-HT2 receptors may have contributed to its in vivo pharmacological effects, even though DAT and SERT are the primary targets of it actions with EC50 values of 70.2 nM and 60.9 nM for releasing

DA and 5-HT, respectively.

Preclinical studies have identified roles for 5-HT2A and 5-HT2C receptors in regulating anxiety-induced cocaine withdrawal. Anxiety-like behavior is blocked by a 5-

HT2A receptor antagonist, MDL 11,939, but enhanced by a 5-HT2A receptor agonist,

TCB-2 [Clinard et al., 2015]. 5-HT2C receptor antagonism attenuates anxiety-like behavior [Craige et al., 2015; Martin et al., 2002] whereas a 5-HT2C receptor agonist produces anxiogenic effects [Cornelio et al., 2007; Moya et al., 2011]. One potential mechanism that contributed to the ability of S-MEPH to reduce anxiety-like behavior during psychostimulant withdrawal is 5-HT2C receptor blockade, which could reduce the activity of GABA interneurons and promote downstream elevation of extracellular DA in the nucleus accumbens. Interestingly, 5-HT2C receptor antagonism is thought to be a

43 reason that fluoxetine (Prozac) does not produce the degree of anxiety during initial dosing that often plagues other selective serotonin reuptake inhibitors (SSRIs) [Pälvimäki et al., 1996]. Thus, S-MEPH may work through a dual 5-HT-based mechanism to mitigate psychostimulant withdrawal, with a 5-HT2C receptor block underlying its anxiolytic efficacy and an increase in 5-HT release through its monoamine substrate actions underlying its antidepressant effect [Baumann et al., 2012; Eshleman et al., 2013;

Gregg et al., 2015; López-Arnau et al., 2012; Opacka-Juffry et al., 2014]. A role for 5-

HT2 receptors in the antidepressant effects of S-MEPH is also possible because 5-HT2A and 5-HT2C receptors facilitate depressive-like behavior in preclinical studies [Diaz and

Maroteaux, 2011; Patel et al., 2004]. Although selective serotonin reuptake inhibitors are used as antidepressants, their effects are not directly related to acute effects on the serotonin system. It takes at least two weeks of repeated administration for the antidepressant effects to be manifested, which has been attributed to upregulation of brain-derived neurotrophic factor (BDNF) and BDNF-associated neurogenesis. It is possible that the behavioral effects of repeated administration of S-MEPH demonstrated in this study are also related to neuroplasticity.

In conclusion, we demonstrate that the S-enantiomer of MEPH, at doses that are not rewarding in CPP and ICSS rat experiments, may attenuate psychostimulant withdrawal, perhaps through both 5-HT transporter and receptor interactions. Our results suggest S-MEPH may be a potential structural and pharmacological template to develop maintenance therapy for psychostimulant dependence. However, it will be important in future studies to compare the therapeutic efficacy of S-MEPH against approved antidepressant and/or anxiolytic drugs such as diazepam, fluoxetine or bupropion. Our

44 current design enabled us to simultaneously compare the effects of multiple doses of S-

MEPH against two psychostimulants using behavioral measures of anxiety- and depression-like behavior, which did not allow for testing against known approved antidepressant and/or anxiolytic drugs. An advantage of the S-MEPH template is a rapid onset of action, whereas a disadvantage is its short duration of action and potential for in vivo racemization. It will be important to further assess the abuse liability of S-MEPH using rat self-administration assays, and to determine if S-MEPH can antagonize deficits in extracellular monoamine levels that occur during psychostimulant withdrawal.

45

Fig 2.1. S-MEPH reduces cocaine and MDPV withdrawal-induced anxiety-like behavior in the EPM assay. Rats withdrawn from chronic treatment with saline (SAL), cocaine (COC) or MDPV were then treated with SAL, S-MEPH (10 mg/kg) (SM 10), or S-MEPH (30 mg/kg) (SM 30) during the drug-free period. A) Data are presented as percentage of time spent on the open arms + S.E.M. (N=6). B) Data are presented as open arm entry + S.E.M. (N=6). *P < 0.05 compared to respective chronic treatment group (SAL, COC or MDPV). +P < 0.05 compared to drug-naïve saline control (SAL SAL).

46

Fig 2.2. S-MEPH reduces cocaine and MDPV withdrawal-induced depression-like behavior in the FST. Rats withdrawn from chronic treatment with saline (SAL), cocaine (COC) or MDPV were then treated with SAL, S-MEPH (10 mg/kg) (SM 10), or S-MEPH (30 mg/kg) (SM 30) during the drug-free period. Data are presented as the percentage of time spent in the immobile state (% immobility) + S.E.M. (N=5-6). *P < 0.05 compared to respective chronic treatment group (SAL, COC or MDPV). +P < 0.05 compared to drug-naïve saline control (SAL SAL).

47 Supplementary Materials

General Procedures: Radioligand binding assays and functional assays were conducted by the Psychoactive Drug Screening Program, which is directed by Dr. Bryan Roth of the University of North Carolina and supported by the National Institute of Mental Health. For more details on the experimental procedure for the radioligand binding and functional assays, refer to the website at https://pdspdb.unc.edu/pdspWeb/?site=assays.

Table 2.1: Inhibition (%) of binding of radioligands to receptors by 10 M S- MEPH. Experiments were performed with a ligand and membranes of cells transfected with its pharmacological target.

Experimental Procedure: A solution of S-MEPH and a positive control was prepared in a 1 mg/ml stock. Radioligand for each receptor was diluted in Standard Binding Buffer. S-MEPH, the positive control, radioligand and membranes were dispensed into a 96-well plate and incubated for 1.5 hours at room temperature and protected from light. The reactions were terminated by rapid filtration, washed 4 times, and the filters allowed to dry overnight. On the following day, scintillant was melted onto filters, and the radioactivity on the filter was counted by scintillation counting [Roth et al., 2012.]. For the pharmacological targets that S-MEPH inhibited more than 50% of radioligand binding, binding was performed with a range of concentrations and Ki values determined.

Radioligand Receptor % Inhibition [ 3H]8-OH-DPAT (0.5 nM) 5-HT1A receptor 19.9 [ 3H]GR127543 (0.3 nM) 5-HT1B, 5-HT1D receptors 41.2, 24.8 [ 3H] (0.5 nM) 5-HT2A receptor 73.7 [ 3H]LSD (1 nM) 5-HT2B receptor 79.9 [ 3H] (0.5 nM) 5-HT2C receptor 55.0 [ 3H]LY278584 (0.3 nM) 5-HT3 receptor -7.1 [ 3H]LSD (1 nM) 5-HT6 receptor 1.9 [ 3H]LSD (1 nM) 5-HT7 receptor 34.7 [ 3H]SCH233930 (0.2 nM) D1, D5 receptors ------[ 3H]N-methylspiperone (0.2 nM) D2, D3, D4 receptors ------[ 3H]U69593 (0.3 nM) kappa opioid receptor ------3H] (1 nM) GABAA receptor 3.6 [ 3H] (0.7 nM) α1A, α1B adrenergic receptors ------[3H] (1 nM) α2A, α2B, α2C adrenergic receptors ------[ 125I]Iodopindolol (0.1 nM) β1, β2, β3 adrenergic receptors ------[ 3H]QNB (0.5 nM) M1, M2, M3, M 4 muscarinic ------receptors [ 3H]Epibatidine (0.5 nM) α2β2, α2β4, α3β2, α3β4, α4β2, α4β4 -1.0, -0.1, 3.9, 9.1, 4.5 nicotinic receptors 3H] (3 nM) sigma-1 receptor 50.6

48 Table 2.2: Functional assays conducted to test for agonist/antagonist activity of S-MEPH

Experimental Procedure: For receptors that S-MEPH showed significant affinities (5-HT2A, 2B, 2C), S-MEPH was tested for agonist and antagonist activity at 10 M. For agonist activity, the positive control was set at 100% and basal activity at 0%. For antagonist activity, the basal activity was set at 100%. We found that S-MEPH at 10 M showed potential antagonist activity. Subsequently, full concentration-response experiments were performed to determine IC50 values against EC50 to EC80 concentrations of a known agonist in the same class.

Targets Receptor Response Antagonist 5-HT EC50 pIC50 Gq/11- 5-HT2A, 5- intracellular -6.5, -5.0, -6.6 -8.5, -8.8, -9.4 Coupled HT2B, 5-HT2C calcium mobilization

49 CHAPTER 3

EFFECTS OF MEHEDRONE ENANTIOMERS ON REINSTATEMENT OF

COCAINE SEEKING IN RATS

Introduction

Synthetic cathinones are psychoactive substances that have gained popularity over recent years, under the assumed name of “bath salts”. Cathinones are chiral molecules that exist as two enantiomers. Mephedrone (4-methylmethcathinone, MEPH) is one of the synthetic cathinones that has been marketed as a ‘legal high’ alternative to bypass law enforcement. The two enantiomers specific to MEPH are R-mephedrone (R-MEPH) and

S-mephedrone (S-MEPH), both exhibiting stereochemistry specific influences on the pharmacological profile of MEPH. Although both R-MEPH and S-MEPH display similar potencies in increasing dopamine (DA) and norepinephrine (NE) release at dopamine transporters (DAT) and norepinephrine transporters (NET), S-MEPH is approximately 50 times more potent than R-MEPH in promoting serotonin (5-HT) release at serotonin transporters (SERT) [Gregg et al, 2015]. S-MEPH was screened against multiple receptors and receptor subtypes using binding assays. The binding assays revealed that S-

MEPH displayed affinity for 5-HT2A, 5-HT2B, and 5-HT2C receptors and showed negligible affinity for dopaminergic, adrenergic and nicotinic receptors. Further, functional assays conducted at 5-HT2A, 5-HT2B, and 5-HT2C receptors showed that S-

MEPH displayed no agonist activity at 5HT2 receptors, however displayed some antagonist activities [Chapter 2]. The rewarding effects of R and S-MEPH enantiomers have been previously examined in rodents using a conditioned place preference (CPP)

50 and intracranial self-stimulation (ICSS) paradigms. In comparative dose-dependent experiments, R-MEPH but not S-MEPH produced CPP compared to saline controls

[Gregg et al, 2015]. Moreover, R-MEPH produced greater ICSS facilitation than S-

MEPH [Gregg et al, 2015]. These findings suggest S-MEPH may exhibit less reinforcing efficacy then the R-enantiomer.

Relapse is common among drug abusers even after prolonged abstinence, and can be triggered by key mediators such as drug re-exposure, drug-associated environmental cues or stressors [Everitt et al, 2002; Grimm et al, 2003; Gipson et al, 2013]. In rodents, the reinforcing effects and abuse liability of drugs are commonly assessed using an intravenous self-administration (IVSA) paradigm [Dworkin et al, 1994; O'Connor et al,

2011]. In this paradigm, various reinstatement tests can be used to measure relapse to drug seeking, which have proven to be useful in identifying potential anti-relapse therapeutics for abused drugs such as alcohol and nicotine [Plosker et al, 2015; Hall et al,

2015; Randall et al, 2015]. In drug-primed reinstatement, a small amount of the previously self-administered drug is injected to elicit drug-seeking behavior.

Mesolimbic DA is important for the reinforcing effects and reinstatement of cocaine [Self and Nestler, 1998; Spealman et al, 1999]. DA receptor agonists and antagonists alter drug-primed reinstatement. Specifically, D1-like agonists and antagonists both attenuate drug-primed reinstatement of cocaine seeking [Self et al,

1996; Alleweireldt et al, 2003; Schenk and Gittings, 2003]. D1, D2, and D3 antagonists have the ability to attenuate cocaine reinstatement [Cervo et al, 2003; Ciccocioppo et al,

2001]. On the other hand, D2-like agonists enhance cocaine reinstatement by mimicking the effects of cocaine [Self et al, 1996; De Vries et al, 2002]. In contrast, D2-like

51 antagonists attenuate cocaine-primed reinstatement [Schenk & Gittings, 2003; Khroyan et al, 2000]. Taken together, these findings indicate that there is dissociation between the effects of D1-like and D2-like receptors upon activation.

5-HT also plays an important role in modulating cocaine-induced reinstatement.

Cocaine blocks reuptake of 5-HT [Warner et al, 1993], and several studies have shown a role for 5-HT receptors in reinstatement of cocaine seeking. The 5-HT system is very complex. There are 7 distinct classes of receptors that have different structure and functional roles in the CNS [Hoyer et al, 2002]. The 5-HT2 receptor has been implicated in cocaine reinstatement. MEPH displays relevant receptor binding at 5-HT2A and 5-HT2C receptors [Simmler et al, 2013]. Antagonism of 5-HT2A receptors suppresses both cue- induced and cocaine-primed reinstatement [Fletcher et al, 2002; Nic Dhonnchadha et al,

2009]. Antagonism of 5-HT2C receptors results in enhanced cocaine-primed reinstatement

[Fletcher et al, 2002; Nic Dhonnchadha et al, 2009], while activation of the 5-

HT2C receptor attenuates both cue-induced and cocaine-primed reinstatement [Fletcher et al, 2002; Neisewander and Acosta, 2007]. Furthermore, 5-HT2A antagonists and 5-HT2C agonists display synergistic suppression of cocaine-induced hyperactivity, impulsivity, as well as cue-induced and cocaine-primed reinstatement of cocaine-seeking [Cunningham et al, 2012]. The results indicate 5-HT2A and 5-HT2C receptor antagonists can have differential effects on cocaine-seeking behavior and may have different therapeutic advantages in suppressing cravings and relapse.

The present study employed a rodent IVSA paradigm to examine R-MEPH and S-

MEPH-induced prime reinstatement to cocaine seeking behavior in rats. Given the role of

5-HT in cocaine relapse, and the strong 5-HT component of S-MEPH, we looked to see

52 whether rats with a history of cocaine exposure would reinstate their drug-seeking behavior in response to a priming injection of S-MEPH. We also tested R-MEPH and cocaine as “positive controls”.

Methods and Materials

Animals

Male Sprague-Dawley rats weighing between 275-300g (N=16; Harlan Laboratories) were initially housed two per cage and allowed to acclimate to the facility conditions prior to surgery. After surgery, rats were individually housed. The room was temperature and humidity-controlled and maintained on a 12-hour reverse light/dark cycle (lights on at 9:00 A.M.). Food and water were available ad libitum, except during experimental sessions. All experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and Temple University Institutional Animal Care and

Use Committee.

Drugs

R-MEPH and S-MEPH were synthesized by Dr. Allen Reitz (Fox Chase Chemical

Diversity Center). Cocaine hydrochloride was provided by the National Institute on Drug

Abuse. All drugs were dissolved in physiological saline (0.9%). Cocaine was delivered through intravenous injection at 50 µL/infusion volume. Body weights were averaged across rats to calculate corrected concentration for cocaine. Following preparation of drug, cocaine was filtered for the self-administration sessions. For prime reinstatement test cocaine, R-MEPH and S-MEPH were administered by intraperitoneal (i.p.) injection at a volume of 1 ml/kg.

53

Catheter Implantation

Catheter implantation was performed as previously described [Simmons et al, 2016].

Briefly, rats were anesthetized using vapor anesthesia (/oxygen mixture) with isoflurane at 5% induction, and maintained at 2–3% during surgery. The rats were prepared for surgery with indwelling jugular vein catheter (Plastics One) and given a preoperative intraperitoneal (i.p.) injection of meloxicam (1 mg/kg, i.p.) analgesia. Rats were given analgesia for 2 days post-surgery. Small incisions were made at the midline of the animal’s back and right side of the animal’s neck to subcutaneously pass the catheter tubing and insert into the jugular vein. Using suture thread, the tubing was secured with a double knot around the jugular vein. All wounds were secured with 9-mm surgical staples. Post-surgery, the rats were individually housed and allowed to recover for 1 week prior to beginning cocaine self-administration. All catheters were flushed daily with 0.3 ml of heparinized saline and a cap was placed over the catheter port.

Cocaine Self-Administration

Rats were trained in sound attenuating operant chambers (Med-Associates) on a fixed ratio-1 (FR-1) schedule of reinforcement to self-administer cocaine (0.375 mg/kg/infusion). The chambers were equipped with a house light, two retractable levers, and a ventilation fan that provided masking noise. At the beginning of each session, rats were placed into the chamber and the infusion tubing, which was enclosed in a spring leash attached to the operant, was attached to the catheter back-mount of each animal.

Animals were trained to lever press for a drug infusion during daily 2-hour sessions for

12 days. During sessions, a response on the active lever resulted in the delivery of one

54 drug infusion and the activation of a cue+tone stimulus complex above the active lever.

There was a 20-s time out period between each infusion to avoid an accidental drug overdose. Responses on the inactive lever were recorded but had no scheduled consequences.

Extinction

Following completion of cocaine self-administration, rats began extinction training.

During extinction sessions, rats were placed in the chambers for 2-hours during which responding on either the active or inactive lever had no scheduled consequences.

Extinction criterion was set to a minimum of seven extinction sessions and rats maintained ≤ 15 presses on the active lever over two consecutive days. Reinstatement of cocaine seeking was conducted the day after rats met the extinction criterion.

Reinstatement Test

A series of drug-primed reinstatement tests, with R-MEPH (10 mg/kg, i.p.) or S-MEPH

(10, 20, 30 mg/kg, i.p.) injection, were used to test reinstatement to drug seeking. The order of the reinstatement tests was counterbalanced using a within subjects Latin square design. All rats (N=9) went through cocaine-primed reinstatement after completing the

Latin square series. The drug-primed injections were given immediately prior to testing.

During reinstatement tests, number of active lever presses (non-reinforced) and inactive lever presses were recorded. Between each reinstatement test, rats were subjected to extinction sessions until they met the extinction criterion as previously described.

Data Analysis

Data were analyzed using GraphPad Prism5 software. Self-administration data for number of active lever pressing during extinction and reinstatement testing were analyzed

55 using two-way within-subjects ANOVA (treatment x session) followed by Bonferonni post hoc secondary analysis. The quantities of each enantiomer were limited. Therefore, animals were excluded prior to the reinstatement test for the following reasons - if stable acquisition was not reached (minimum of 15 infusions for 3 consecutive session) or if

Latin square was not completed. Statistical significance was set at p < 0.05.

Results

Rats readily established active lever responding for cocaine (0.375 mg/kg/infusion) during the 12-day self-administration training. This behavior was extinguished during 7 days of extinction training (Figure 3.1). R-MEPH (10 mg/kg) and cocaine (10 mg/kg) treatment induced cocaine seeking behavior, as reflected by increase in number of lever presses that was previously associated with cocaine infusion (Figure 2). On the other hand, none of the doses of S-MEPH (10, 20 or 30 mg/kg) was able to elicit cocaine seeking (Figure 3.2). Two-way ANOVA showed a significant interaction [F(4, 80) =

13.94, P<0.0001], treatment [F(4, 80) = 12.13, P<0.0001] and session (i.e. extinction vs. reinstatement) [F(1, 80) = 22.43, P<0.0001]. Bonferroni post hoc tests revealed that number of active lever presses were significantly higher for the reinstatement session compared to the last extinction session in rats that were primed with R-MEPH or cocaine, while there were no significant differences in lever pressing in S-MEPH-primed rats between the sessions. Furthermore, responding was significantly higher in the R-MEPH and cocaine-primed reinstatement groups compared to all dose tested with S-MEPH (p <

0.001).

56

Figure 3.1. Cocaine Acquisition and Extinction sessions. Each data point represents mean + S.E.M. of active lever presses (N=9). Rats were trained to self-administer cocaine (0.375mg/kg/ infusion) for 12 sessions for 2 hours daily. Following acquisition, extinction sessions were conducted for minimum of 7 sessions.

57

Figure 3.2 R- and S-MEPH-primed reinstatement. Each bar represents mean + S.E.M. of last extinction session and reinstatement test (N=9). Following successful completion of extinction training rats, previously maintained by cocaine self-administration, underwent R- MEPH-primed (10 mg/kg, i.p.), S-MEPH-primed (10, 20, 30 mg/kg, i.p.), and cocaine-primed (10 mg/kg, i.p.) reinstatement sessions. R-MEPH reinstated cocaine responding in animals with a history of cocaine self-administration. S-MEPH at doses tested failed to reinstate cocaine responding. Significant differences between last extinction session vs. R-MEPH- and cocaine-primed reinstatement session are indicated by *** (p<0.001). Significant differences between reinstatement of R-MEPH group vs. S-MEPH group are indicated by ### (p<0.001). Significant differences between reinstatement of cocaine group vs. S-MEPH group are indicated by $$$ (p<0.001).

Discussion

The purpose of this study was to determine if the S-enantiomer of MEPH has the ability to induce reinstatement to cocaine seeking after extinction of responding. Our studies include the first evaluations of R- and S-enantiomers of MEPH in reinstatement of cocaine seeking behavior. We showed that rats trained to self-administer cocaine (0.375 mg/kg/inf.) under FR-1 schedule of reinforcement readily acquired self-administration.

58 Studies have shown rats reliably reinstated their drug seeking behavior following a period of extinction in response to a cocaine-prime (10 mg/kg) injection. Dose of cocaine was chosen based on previously published data that produced significant reinforcing properties on an FR-1 schedule of reinforcement, as well as published data on cocaine- prime reinstatement [Enman et al, 2015; Nair et al, 2013; Ogbonmwan et al, 2015;

Gabriele et al, 2011; McFarland et al, 2003]. The similar effect was observed with R-

MEPH prime injection. 10 mg/kg R-MEPH reinstated responding similar to that of cocaine-prime, but not higher than cocaine. In contrast, all doses of S-MEPH (10, 20, 30 mg/kg) tested failed to induce reinstatement to cocaine seeking. These data suggest that

R-MEPH is responsible for the rewarding and reinforcing effects seen with the racemic form of MEPH.

DA plays an important role in mediating cocaine-seeking behaviors [Bachtell et al, 2005; Schmidt et al, 2006]. The magnitude of cocaine-induced reinstatement is dependent on the level of increase in DA in the nucleus accumbens [Madayag et al,

2010]. Pretreatment with either selective D1 receptor agonists, antagonists or selective dopamine D2 receptor antagonists attenuate cocaine seeking in animal models elicited by cocaine-prime injections [Alleweireldt et al, 2003; Self et al, 1996; Khroyan et al,

2000; Anderson et al, 2004]. Cocaine (10 mg/kg) reinstates drug seeking behavior, however D1-like receptor agonists, SKF-81297 and SKF-82958, attenuated the drug seeking behavior [Khroyan et al, 2000]. Selective D1-like antagonist, SCH 23390, and

D2-like antagonists, , dose-dependently decreased cocaine-primed reinstatement [Sun et al, 2005]. Despite seemingly conflicting reports of the roles that these subtypes of DA receptors play, activity of DA receptors appears to be a key

59 component in the neural mechanism involved in induction of reinstatement to cocaine seeking.

Similar to cocaine, a priming injection of R-MEPH reinstated drug-seeking behavior in rats with a history of cocaine. However, S-MEPH did not induce reinstatement. This supports the work by Gregg (2015) and Vouga (2015) who compared the stereochemistry and neuropharmacology of MEPH. These studies report that R-

MEPH produced rewarding effects in rats and planarian using environmental place conditioning (EPC), CPP and ICSS paradigms [Gregg et al, 2015; Vouga et al, 2015]. In rats, R-MEPH produced CPP, but S-MEPH was ineffective at all doses (5, 15, 30 mg/kg)

[Gregg et al, 2015]. Furthermore, R-MEPH was greater than S-MEPH in producing facilitation of ICSS [Gregg et al, 2015]. In planarian, racemic MEPH and R-MEPH produce EPC, on the other hand, S-MEPH did not produce significant EPC [Vouga et al,

2015]. Interestingly, others have also found contrasting reinforcing effects with the different MDMA enantiomers. Racemic MDMA and S-MDMA induce reinstatement of responding for amphetamine, whereas priming injections of R-MDMA and fenfluramine did not reinstate amphetamine self-administration in rhesus monkeys [McClung et al,

2010]. Consistent with our findings, priming injections of the selective 5-HT releaser, fenfluramine (1 or 3 mg/kg), did not reinstate extinguished amphetamine responding

[McClung et al, 2010]. The differences in effects observed with these enantiomers may be due to DA mediated effects of S-MDMA. S-MDMA has a greater affinity for DAT compared to R-MDMA and racemic MDMA [Verrico et al, 2007]. Our results are in agreement with other reports showing differentiated effects based on stereochemistry.

60 Cocaine is a triple reuptake inhibitor of DA, 5-HT and NE. Therefore, 5-HT pathways are also involved in altering cocaine reinstatement [Sawyer et al, 2012].

Despite R-MEPH and S–MEPH having similar potency on DA release, S-MEPH’s potency to cause 5-HT release is 50 times greater than R-MEPH [Gregg et al, 2015].

While the surge in DA may have enabled R-MEPH to trigger reinstatement, in the case of

S-MEPH, the concurrent release of 5-HT may have counteracted the effects of S-MEPH triggered DA release. S-MEPH displays relevant receptor binding at 5-HT2A, 5-HT2B and

5-HT2C receptors and has weak antagonist activity. Antagonism of 5-HT2A and 5-HT2C receptors have been shown to give opposite outcomes on cocaine-primed reinstatement

[Nic Dhonnchadha et al, 2009; Fletcher et al, 2002; Pockros et al, 2011; Burmeister et al,

2004; Murnane et al, 2013; Filip et al, 2005]. Antagonism of 5-HT2A receptors has been shown to suppress cocaine-primed reinstatement [Fletcher et al, 2002; Nic Dhonnchadha et al, 2009], while antagonism of 5-HT2C receptors results in enhanced cocaine-primed reinstatement [Fletcher et al, 2002; Nic Dhonnchadha et al, 2009]. A study showed that ketanserin, 5-HT2A/2C receptor antagonist, has the ability to attenuate cue- evoked reinstatement, but failed to attenuate cocaine-prime reinstatement [Burmeister et al, 2004]. Given that 5-HT2A and 5-HT2C receptor antagonists exert opposite effects in cocaine-primed drug-seeking, it may be possible that reinstatement induced by drug- prime is mediated through the 5-HT2A receptor. Acute and chronic SSRI treatment exert different effects on abuse-related effects of cocaine; however they have limited success in clinical trials for cocaine abuse due to the neurobiological changes that occur during chronic administration [Grabowski et al, 1995; Schmitz et al, 2001; Winstanley et al,

2011]. A nonhuman primate study showed suppression of both cocaine induced

61 reinstatement and cocaine elicited DA surge, and proposed 5-HT2A receptor desensitization as the potential mechanism. To further strengthen the utility of 5-HT antagonists as potential treatment for cocaine relapse, they also showed that the effects of chronic treatment lasted after six weeks [Sawyer et al, 2012].

In conclusion, our studies are the first to investigate R-MEPH and S-MEPH in reinstatement of cocaine seeking behavior. We found that R-MEPH prime injection induced reinstatement of cocaine-seeking behavior similar to that elicited by cocaine prime injection, demonstrating abuse potential of R-MEPH. In contrast, S-MEPH had no significant influences on reinstatement of cocaine seeking behavior. MEPH’s dual DA and 5-HT releasing mechanism of action may explain the differences in the profiles of its enantiomers. Although additional studies are needed to assess S-MEPH ability to attenuate cocaine prime-induced reinstatement, it is possible that S-MEPH pretreatment maybe able to reduce the magnitude of cocaine-reinstatement. These enantiomers need to be further investigated for therapeutic efforts for preventing cocaine relapse.

62 CHAPTER 4

STEREOSELECTIVE EFFECTS OF MEPHEDRONE UNDER FIXED RATIO

(FR) AND PROGRESSIVE RATIO (PR) SCHEDULES OF REINFORCEMENT

IN RATS

Introduction

The recreational use of synthetic cathinones (‘bath salts’) has significantly increased worldwide since 2009. Mephedrone (4-methylmethcathinone, MEPH) is among the class of synthetic cathinone (i.e. beta-ketone amphetamines) [Winstock et al, 2011; Simmler et al, 2013]. Although MEPH is now a controlled substance (Schedule I), these compounds rapidly entered the drug market, and were heavily sold via the Internet labeled as ‘not for human consumption’ and sold as “plant food,” “bath salts,” and “research chemicals” aiming to bypass drug laws with new “legal” high alternatives [Laurent et al, 2015;

Valente et al, 2014; Prosser et al, 2012].

Ultrasonic vocalizations (USVs) are vocal calls emitted by rodents that, in addition to their intraspecies communicative function, associate with positive and negative affective states based on emission frequency. Specifically, reward and approach behavior associate with 50-kHz USVs whereas distress and avoidance behavior associated with 22-kHz

USVs [Brudzynski et al, 2005; Covington and Miczek, 2003; Burgdorf et al, 2010].

Drugs of abuse readily elicit 50-kHz USVs in rats whereas 22-kHz USVs are often observed during drug withdrawal [Wright et al, 2010; Panksepp and Burgdorf, 2000;

Wintink and Brudzynski et al, 2001; for review, see Barker, Simmons, West, 2015].

Mesolimbic dopamine (DA) transmission seems to have a key role in the initiation of 50-

63 kHz calls. For example, infusions of amphetamine into the nucleus accumbens (NAcc), a major terminal site of mesolimbic DA projections, induced 50 kHz calls; manipulating

DA transmission also affects call rates elicited by amphetamine [Thompson et al,

2006]. Further, optical stimulation of DA terminals within NAcc from VTA projection neurons transiently evokes 50-kHz USVs [Scardochio et al, 2015]. Thus, USVs serve as a measure of subjective affective state in models of substance use disorders, and mesolimbic DA transmission contributes to the production of positively-valenced 50-kHz

USVs.

MEPH produces empathogenic effects in humans, analogous to those reported in 3,4- methylenedioxymethamphetamine (MDMA, ‘Ecstasy’) users. This similarity may be due to increased levels of serotonin (5-HT) over DA in the nucleus accumbens [Baumann et al, 2012; Kehr et al, 2011]. Preclinical studies in rats using conditioned place preference

(CPP) and self-administration paradigms have shown that MEPH produces rewarding and reinforcing effects by enhancing monoamine transmission [Lisek et al, 2012; Hadlock et al, 2011]. Pharmacologically, MEPH is also similar to MDMA and acts as a non-selective monoamine transporter substrate and monoamine releaser of DA, 5-HT and norepinephrine (NE) [Baumann et al, 2012; Eshleman et al, 2013; Simmler et al, 2013].

Moreover, MEPH has a chemical composition that includes R and S enantiomers, which are found in other controlled substances including methamphetamine (METH), cocaine and MDMA. S-MEPH potency and selectivity for the serotonin transporter (SERT) is greater than R-MEPH, however, both R-MEPH and S–MEPH are similar in potency for the dopamine transporter (DAT) [Gregg et al, 2015]. In one study, S-METH was 25-fold more potent than R-METH in rats trained in a drug discrimination paradigm [Bondareva

64 et al, 2002]. Similarly, the cocaine enantiomer S-methcathinone has been shown to have a three-fold greater potency for substituting for a cocaine discriminative stimulus compared to R-methcathinone in rats [Glennon et al, 1995] and S-MDMA and racemic MDMA are more consistent positive reinforcers than R-MDMA in a rhesus monkey model of self- administration [Wang et al, 2007].

Somewhat surprisingly, the stereospecific effects of MEPH on self-administration have not been investigated. Racemic MEPH has been shown to maintain self- administration behavior under multiple paradigms, including the fixed-ratio (FR) schedule and progressive-ratio (PR) schedules of reinforcement [Hadlock et al, 2011;

Aarde et al, 2013]. The present study examined the stereospecific reinforcing effects of

MEPH in a rat model of self-administration. These findings will expand our understanding of the comparative reinforcing effects exhibited by psychostimulants that are structurally similar. Understanding this phenomenon of stereospecific reinforcing abuse liability will help determine the relative risks compared with other psychostimulants.

Methods and Materials

Animals

Male Sprague-Dawley rats (initial weight 275-300 g, N=48) from Harlan Laboratories

(Indianapolis, IN) were housed two per cage and maintained in a controlled environment on a reverse 12-h light/dark cycle (lights on at 9:00AM). Animals were individually housed after catheter implantation. Food and water were provided ad libitum, except during experimental procedures (drug self-administration, extinction, and reinstatement testing). All animal procedures were conducted in accordance with the NIH Guide for the

65 Care and Use of Laboratory Animals and Temple University guidelines for Animal Care and Use.

Drugs

Racemic MEPH and (R/S)-MEPH were synthesized by Dr. Allen Reitz (Fox Chase

Chemical Diversity Center, Doylestown, PA). Racemic MEPH and (R/S)-MEPH were dissolved in physiological saline (0.9%). Drugs were delivered by intravenous injection at a volume of 50 µL/infusion. Body weights were averaged across rats to calculate required concentration for drug solution.

Surgery

Rats were anesthetized with isoflurane/oxygen vapor mixture (isoflurane 5 percent induction, 2–3 percent maintenance) and prepared for surgery with indwelling jugular vein catheter (Plastics One). Prior to surgery, rats were injected with an analgesic

(meloxicam, 1 mg/kg). The catheter tubing was subcutaneously passed from the back to the right shoulder of the animal, then inserted into the right jugular vein. The tubing was secured with tied suture thread. 9-mm surgical staples were used to close the incisions.

Following surgery, rats were placed into individual cages to recover, then transferred to a clean home cage where they remained individually housed. Rats had ~7 days of recovery before self-administration sessions began. Catheters were flushed with 0.3 ml of heparinized saline daily to maintain catheter patency.

MEPH Self-Administration

Acquisition

Sound-attenuating rat operant chambers (Med-Associates) were used to conduct all drug self-administration experiments. The rat was placed in the chamber, and the catheter

66 located on the animal’s back was connected to the tubing enclosed in the protective spring leash attached to the operant chamber. To begin each session, house lights were turned on and two retractable levers extended into the chamber. Rats were trained on a fixed ratio-1 (FR-1) schedule to lever press for drug infusions during daily 2-hour sessions. During the sessions, a response on the right lever (active lever) resulted in a drug infusion and simultaneous light cue+tone stimulus complex. Responses on the left lever (inactive lever) had no consequences, but were recorded to monitor for non-specific motor behavior. A 20 second time out period followed each infusion to limit the possibility of overdose. Data collection and drug delivery were controlled from MED-PC

IV software (Med Associates). The first experiment used an established dose of racemic

MEPH (N=16, 0.5 mg/kg/inf) as a training dose for 14 days that could reliably support self-administration (Hadlock, 2011). Upon completion of acquisition, MEPH trained-rats received either R-MEPH (0.5 mg/kg/inf) or S-MEPH (0.25, 0.5, 2.0 mg/kg/inf) varied across animals using a within-subjects latin square design. Doses were given in blocks of

6 consecutive sessions. For the second experiment (N=32), we evaluated using three doses of S- MEPH (0.25, 0.5, 2.0 mg/kg/inf) and a comparative dose of R-MEPH (0.5 mg/kg/inf) during daily 2-hour sessions for 10 days to establish enantiomeric MEPH self- administration.

Progressive-Ratio Schedule

Following acquisition of R- and S-MEPH self-administration (as described in experiment

2), the paradigm for drug infusion changed to a Progressive-Ratio (PR) schedule of reinforcement for each enantiomer. The rats in all four groups remained on the same dose schedule they were subjected to during acquisition. PR sessions, consisting of 4

67 hours/session, terminated after 30 min of inactivity, were conducted for 10 days.

Following the first drug infusion, all subsequent infusions were contingent upon the progressively increasing response requirement after each drug-paired lever response; the sequence of increasing response ratio is described by Richardson and Roberts

[Richardson, 1996]. Breakpoint was defined as the final ratio completed to obtain a reinforcement before the session ended.

Ultrasonic Vocalization (USV) Recording and Analysis

Ultrasonic emissions were detected via UltraMic200K aluminum condenser microphones (Dodotronic; Italy) with integrated digital-to-analog conversion which suspended above Plexiglas self-administration chambers. Audio was sampled at 192-kHz and was recorded in “.wav” file format using RavenPro software (Cornell Lab of

Ornithology, Bioacoustics Research Program; Ithaca, NY, USA) on a nearby personal computer. Recording sessions were initiated on single days during the first (days 3-5) and second (days 8-10) weeks of fixed-ratio self-administration. Analysis was conducted offline by trained experimenters, and putative USVs were collected after verification based on spectrographic appearance and audio playback. Only USVs with mean frequency between 38- and 80-kHz and with duration > 15 ms were included in analysis.

Data Analysis

All statistical analyses were conducted using GraphPad Prism5. Data are expressed as mean ± SEM. Analysis was conducted on number of infusions, number of correct lever presses and breakpoints. In experiment 1, cumulative data was analyzed using a one-way

ANOVA. For experiment 2, acquisition data was analyzed using two-way ANOVA with session and dose as within-subject factors. Breakpoints were analyzed using two-way

68 ANOVA, with the enantiomer and drug dose as factors. Significance was considered p <

0.05 and main effects were followed by post hoc analysis using Bonferroni or Dunnett’s comparisons. Total 50-kHz ultrasonic vocalizations (USVs) across two-hour self- administration sessions were analyzed using one-way ANOVAs with Drug Group as independent variable (R-MEPH, S-MEPH 0.25, S-MEPH 0.50, S-MEPH 2.00) for Week

1 and Week 2. When main effects were observed, Bonferroni-corrected contrasts comparing R-MEPH to all doses of S-MEPH were conducted. Familywise was established at 0.05.

Results

Experiment 1: Acquisition of racemic MEPH & Dose-response of enantiomers

Acquisition

Rats were trained with an initial dose of 0.5 mg/kg/inf MEPH for 14 days. The mean number of infusions, active lever presses, and inactive lever presses from the training phase are presented in Figure 4.1. Two-way ANOVA analysis of lever pressing indicated that there was a significant main effect of interaction [F(13, 420) = 16.58, P < 0.001]. The increase in drug infusion (Fig 4.1A) and active lever responses (Fig 4.1B) indicate that all animals acquired MEPH self-administration. There was a significant effect examining active lever presses and inactive lever presses with MEPH training dose which revealed a significant main effect of number of lever presses [F(1, 420) = 629.4, P<0.0001]. There was also a significant effect of session [F(13, 420) = 14.88, P<0.0001]. Bonferroni post hoc analysis confirmed that active lever presses was significantly higher than inactive lever presses for sessions 4-14.

69

A B

Figure 4.1. Acquisition of racemic MEPH Rats were trained to self-administer racemic MEPH (0.5 mg/kg/inf) during 2-hour daily sessions (N =16) for 14 days. Data shows the number of infusions (A) and active vs inactive lever presses (B). Data represents mean + S.E.M. during initial 14 days of acquisition. *P< 0.05, **P<0.01, ***P<0.001 compared to inactive levers.

R and S-MEPH Dose-substitution

Following acquisition of racemic MEPH, dose-substitution studies were conducted using a with-in subject latin square design to test R-MEPH (0.5 mg/kg/inf) and S-MEPH (0.25,

0.5, 2.0 mg/kg/inf). These data are represented in Figure 4.2. Results indicated that there was no significant interaction effect between the four drugs, and that there was no significant effect of session. However, there was a significant effect of treatment group

70 [F(5, 450) = 68.83, P<0.0001]. Post hoc comparisons confirmed there were dose-related differences associated with the number of infusions. Specifically, rats administered S-

MEPH 0.25 and 0.5 mg/kg/inf. had a greater number of infusions compared to those given S-MEPH 2.0 mg/kg/inf. Furthermore, responding was significantly higher in the S-

MEPH 0.25 and 0.5 mg/kg/inf. groups compared to R-MEPH (p < 0.001).

Figure 4.2. R and S-MEPH Dose-substitution Rats previously trained to self-administer racemic MEPH (0.5 mg/kg/inf) (N =16) underwent dose substitution with R and S-MEPH. The number of infusions during dose substitution are graphed. Infusions one week prior to dose-substitution and one week after dose-substitution sessions are also included for comparison. All data are represented as mean + S.E.M. **P<0.01, ***P<0.001 compared to MEPH number of infusions.

Experiment 2: Dose-response on fixed-ratio & progressive-ratio schedule of reinforcement

Fixed-ratio 1

The mean number of infusions, active lever presses, and inactive lever presses for rats trained on R-MEPH (0.5 mg/kg/inf, N=8) or S-MEPH (0.25, 0.5, 2.0 mg/kg/inf,

71 N=8/group) for 10 days is shown in Figure 4.3. Two-way ANOVA confirmed there was a significant effect of treatment group [F(3, 280) = 20.10, P<0.0001] and session day [F(9,

280) = 20.26, P<0.0001] for infusions acquired (Fig 4.3A). There was no significant effect of interaction. S-MEPH produced an inverted U-shaped curve at the doses tested.

Analysis of active lever presses during acquisition (Fig 4.3B) revealed there was a significant main effect of treatment group [F(3, 280) = 1.389, P<0.0001] and day of sessions [F(9, 280) = 15.05, P<0.0001]. Post hoc comparisons confirmed there were dose-related differences associated with active lever presses. A low and high dose of S-

MEPH (0.25 and 2.0 mg/kg/inf) decreased the number of active lever presses (P<0.05)

Compared to moderate dose of S-MEPH (0.5 mg/kg/inf). A moderate dose of S-MEPH

(0.5 mg/kg/inf) increased the number of active lever presses. Inactive lever presses (Fig

4.3C) results showed there was a significant effect of treatment group [F(3, 280) = 10.20,

P<0.0001]. There was no significant difference between days of session and there was no significant interaction of treatment group and day of session.

72 A B C

Figure 4.3. Acquisition of R- and S-MEPH Rats were trained to self-administer either R-MEPH (0.5 mg/kg/inf) or S-MEPH (0.25, 0.5, 2.0 mg/kg/inf) during 2-hour daily sessions (N = 8). The number of infusions (A), active lever presses (B), and inactive lever presses (C) are shown. Data represents mean + S.E.M. during 10 days of acquisition. Significant differences between S-MEPH (0.25 mg/kg/inf) and S-MEPH (0.5 mg/kg/inf) groups vs. S-MEPH (2.0 mg/kg/inf) group are indicated by ** (p<0.01), *** (p<0.001). Significant differences between S-MEPH (0.25 mg/kg/inf) and S-MEPH (2.0 mg/kg/inf) groups vs. R-MEPH (0.5 mg/kg/inf) group are indicated by # (p<0.05), ## (p<0.01), ### (p<0.001).

Progressive-ratio with R and S-MEPH

Following acquisition of R-MEPH (0.5 mg/kg/inf) and S-MEPH (0.25, 0.5, 2.0 mg/kg/inf), rats were tested on PR schedule of reinforcement for 10 days (Fig 4.4).

Analysis of the active lever presses (Fig 4.4A) confirmed there were significant differences between the treatment groups [F(3, 280) = 23.64, P<0.0001]. Post hoc comparisons of the treatment groups revealed S-MEPH (low dose, days 4, 7, 8, 10, p<0.01; moderate dose, days 7, 8, 10, p<0.05) active lever presses significantly decreased compared to R-MEPH. Analysis of the inactive lever presses (Fig 4.4B) confirmed there

73 were significant differences between the treatment groups [F(3, 280) = 34.44, P<0.0001], day of session [F(9, 280) = 2.721, P=0.0047], and treatment by session interaction [F(27,

280) = 3.510, P<0.0001]. Post hoc comparisons of the treatment groups revealed S-

MEPH (low dose, day 3, p<0.001; moderate dose, days 3, 6, p<0.05; high dose, days 3, 5, p<0.05) inactive lever presses significantly decreased compared to R-MEPH. Breakpoints achieved under a PR schedule were also analyzed (Fig 4.4C). Two-way ANOVA confirmed significant effects of the treatment group [F (3, 280) = 26.50, P < 0.0001]. Post hoc analysis confirmed a significantly higher breakpoint was achieved for R-MEPH (0.5 mg/kg/inf) compared to S-MEPH (low dose, days 4, 6, 7, 8, 10, p<0.05; moderate dose, days 4, 6, 7, 8, p<0.05).

A B C

Figure 4.4. Progressive Ratio with R and S-MEPH The reinforcing effects of R and S-MEPH were evaluated using a progressive ratio paradigm. The data shows the number of active lever presses (A), inactive lever presses (B), and the breakpoint (C). Data represents mean + S.E.M. during 10days of progressive ratio schedule of reinforcement. Significant differences between S-MEPH groups vs. R- MEPH group are indicated by * (p<0.05), ** (p<0.01), *** (p<0.001). Self-administration of R-MEPH elicits greater 50-kHz USVs compared to S-MEPH

74 A one-way ANOVA examining total 50-kHz USVs by Drug Group during Week 1 of fixed-ratio self-administration did not reach statisticalsignificance [F(3, 13) = 2.83, p =

0.08] (Figure 4.5, upper panel). During Week 2, a significant main effect of Drug Group was observed [F(3, 13) = 4.54, p < 0.05] and contrasts revealed that R-MEPH 0.50 elicited significantly more 50-kHz USVs compared to S-MEPH 0.50 [|t(7)| = 3.33, p <

0.05] and S-MEPH 2.00 [|t(5)| = 3.10, p < 0.05] (Figure 5, lower panel).

Figure 4.5. Characterization of 50-kHz USVs during self-administration of S- and R- MEPH. Session sums are indicated in left panels and USVs across 2-hour sessions indicated in right panels for Weeks 1 (upper) and 2 (lower). * p < 0.05 compared to R- MEPH.

75 Discussion

Rats with a history of racemic MEPH (0.5 mg/kg/inf) acquired consistent levels of drug infusions and high levels of active lever presses. R-MEPH (0.5 mg/kg/inf) drug intake was comparable to racemic MEPH. S-MEPH (0.25, 0.5, 2.0 mg/kg/inf) dose- dependently maintained self-administration behavior that was greater than racemic

MEPH, and R-MEPH (figure 2). Escalation of S-MEPH intake occurred in rats with previous exposure to racemic MEPH. These findings suggest rats with previous exposure to MEPH may escalate drug intake to a greater magnitude when self-administering S-

MEPH to compensate for a decrease in reinforcement related to that specific enantiomer.

These findings suggest rats with previous exposure to MEPH may escalate drug intake because the dose of S-MEPH is less rewarding than the dose of racemic MEPH rats were previously trained up on. An alternative explanation for the escalation of S-MEPH intake is behavioral sensitization. A drug that produces behavioral sensitization after repeated administration causes an increase in response greater than the initial exposure [Steketee and Kalivas, 2011]. Repeated exposure to drug of abuse leads to further drug use, which evolves into compulsive drug-seeking and drug-taking, resulting in the loss of control, and emerges into addiction. Previous psychostimulant exposure and dose have been shown to enhance acquisition of self-administration. For example, rats that have been previously sensitized to cocaine acquire cocaine self-administration faster and took more infusions compared to saline controls [Zhao et al, 2010]. In rats, pretreatment with amphetamine produced sensitization and accelerated escalation of cocaine intake during self-administration [Ferrario et al, 2007]. Additionally, pre-exposure to cocaine promotes faster cocaine self-administration in rats [Childs et al, 2006]. Our results are consistent

76 with previous studies demonstrating psychostimulant exposure promotes acquisition of drug intake.

Similar to MDMA, no differences in acquisition of self-administration were observed in equivalent doses of MEPH enantiomers [Fantegrossi et al, 2002, 2008]. Here, we found no particular distinctions in reinforcing effects between MEPH enantiomers following 10 days of self-administration. In nonhuman primates, racemic MDMA and its enantiomers show no differences in responding during the early phase of intermittent

MDMA exposure [Fantegrossi, 2008]. USV recordings were conducted to further characterize the subjective effects following self-administration of MEPH enantiomers and are the first demonstration that positive affect is experienced following MEPH self- administration. Positively-valenced USVs (emitted around 50-kHz) were observed following self-administration of R- and S-MEPH with significantly greater abundance observed following R-MEPH infusions. Our findings are consistent with other groups which have shown the elicitation of 50-kHz USVs following systemic and self- administration of cocaine and amphetamine [Wintink and Brudzynski, 2001; Mu et al,

2009; Ahrens et al, 2009; Barker et al, 2014]. Recently, our team demonstrated that the synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV), which blocks DA and NE uptake with approximately 10-fold greater potency compared to cocaine yet has negligible affinity at SERT, elicits a strong bout of 50-kHz USVs following self- administration [Simmons et al, 2016]. Additionally, Simmons and colleagues (2016) reported that MDPV-elicited 50-kHz USVs were more persistent relative to cocaine- elicited USVs, but that USVs decreased to near-zero levels following load-up (i.e. initial infusions in beginning of session) of either compound. Results from the present study

77 support that positive affect is experienced following initial infusions of MEPH but, similarly to other psychostimulants, is relatively sparse after rats titrate to high drug levels. Our results additionally support the idea that augmented DA transmission from self-administered R-MEPH likely underlies the positive subjective response experienced during load-up. Self-administration of S-MEPH, which more strongly blocks 5-HT reuptake, elicited fewer 50-kHz USVs most notably at higher doses. Taken together, these findings suggest that the 5-HT system may have minor influence on drug taking behavior, and that DA transmission underlies in larger part drug-seeking behavior and the experience of positive affect following reward receipt. Future studies should address whether augmented 5-HT transmission, as is observed most closely with S-MEPH self- administration, may dampen the efficacy of DAT blockade and in turn diminish positive affect and attenuate drug-seeking behavior.

In PR studies, R-MEPH produced significantly greater active lever presses and breakpoints compared to S-MEPH. Given that R-MEPH induces CPP in rats, and S-

MEPH did not induce CPP at a dose as high as 30 mg/kg [Gregg et al, 2015], it was not surprising we observed significantly higher breakpoints to further support evidence of R-

MEPH being more rewarding. Interestingly, our finding parallels published enantiomeric effects that are seen with MDMA. For example, a study with Rhesus monkeys examined

MDMA and its enantiomers using PR schedule. It was found that reinforcing strength of racemic MDMA and S-MDMA was substantially higher than R-MDMA [Wang and

Woolverton, 2007]. In addition, S-MEPH had a dose-dependent increase in motivation to work for infusions during PR testing but not greater than R-MEPH. Studies suggest there is a negative relationship between the activity and reinforcing effects of

78 psychostimulants [Wee et al, 2005; Roberts et al, 1999]. Wee (2005) examined if 5-HT activity affect reinforcing efficacy of amphetamine analogs that had similar potencies in vitro as DA releasers but varied in 5-HT releasing potency. Under a PR schedule of reinforcement, PAL 313, PAL 314, PAL 303, PAL 353, d-amphetamine were positive reinforcers. PAL 313, the most potent 5-HT releaser, was still less potent than d- amphetamine, the least potent 5-HT releaser. Taken together, there is a correlation between the DA/5-HT releasing potency and reinforcing potency, suggesting that the dual

DA/5-HT actions of a compound determines its potency as a reinforcer. Consistent with our findings, the decrease in responding observed with the S-enantiomer may be due to 5-

HT releasing potency greater than DA. Interestingly, S-MEPH produce greater 5-HT release compared to R-MEPH, S-MEPH displays a 50-fold greater preference for the 5-

HT system than R-MEPH [Gregg et al, 2015]. Furthermore, S-MEPH up to a dose of 30 mg/kg were inactive in the CPP assay, whereas R-MEPH produces CPP [Gregg et al,

2015]. Based on these findings, it is reasonable to conclude that R-MEPH is mediating the reinforcing strength of racemic MEPH.

In summary, our results show that pre-exposure to a psychostimulant can have different effects on acquisition of self-administration. Rats pre-exposed to racemic

MEPH caused an increase in S-MEPH consumption, where no prior training acquisition of S-MEPH generated a typical inverted U-shaped curve. This study indicates the importance of environmental factors and provides evidence supporting the notion that behavioral sensitization is involved during the early stages of drug addiction and provide insight into the rapid progression of addiction. Furthermore, progressive-ratio revealed that R-MEPH-trained rats motivation to work for reinforcer was significantly greater than

79 S-MEPH-trained rats at any dose of S-MEPH tested. It is possible that R-MEPH is responsible for the rewarding and reinforcing effects observed in racemic MEPH. This phenomenon of stereospecific reinforcing properties provides greater insight into the addictive properties of MEPH.

80 CHAPTER 5

GENERAL DISCUSSION

The overall goal of this dissertation was to characterize the behavioral and neurochemical effects of S-MEPH in animal models of cocaine-induced and MDPV-induced anxiety, depression, and relapse. Although S-MEPH does not produce conditioned place preference (CPP), which suggests that it is not rewarding, we also investigated its potential abuse liability using contingent rat self-administration studies. Three specific aims were investigated in this dissertation. Aim 1 was to characterize the effects of S-

MEPH on preclinical indicators of anxiety and depression. Aim 2 was to determine the in vitro receptor pharmacology of S-MEPH, specifically on the 5-HT receptors. Aim 3 was to determine the reinforcing properties of MEPH enantiomers by rat intravenous self- administration and characterize the effects of S-MEPH on reinstatement to cocaine seeking. The specific aims were addressed by implementing various in vitro and in vivo experiments. In vitro radioligand binding and functional assays were conducted to examine the receptor pharmacology of S-MEPH. In vivo behavioral studies comprised of anxiety models such as the elevated plus-maze (EPM), depression models such as the forced swim test (FST), and addictive-related models using intravenous self- administration and drug-primed reinstatement paradigms.

EPM and FST serve as preclinical assays to test for anxiety and depression. Both are validated rodent models to test for anxiety-like and depression-like behaviors [Walf et al., 2007; Estanislau et al., 2011]. Self-administration assays are used to model human addiction in animals to assess the abuse liability and reinforcing strength of drugs. Self-

81 administration is the “gold standard” to model addiction because animals can mimic addictive behaviors observed in users through contingent delivery of a drug. Fixed ratio schedule of reinforcement is used to evaluate drug taking behavior and progressive ratio schedule of reinforcement is used to evaluate the reinforcing strength of the drug.

Reinstatement test can be used to measure relapse to drug seeking behavior.

Major Findings

The studies in this dissertation have provided a comprehensive in vitro receptor pharmacological profile of S-MEPH and expand on the impact stereochemistry has on the neuropharmacological profile of the individual MEPH enantiomers. Furthermore, based on our findings, S-MEPH is a structural and pharmacological template that may lead to the development of pharmacotherapies aimed to prevent cocaine and MDPV withdrawal.

The major findings from each chapter are summarized below. Rats withdrawn from chronic cocaine or MDPV showed heightened anxiety- and depression-like behaviors that was antagonized by treatment with S-MEPH (10, 30 mg/kg). The present study is the first to evaluate if the reinforcing effects of MEPH are stereospecific. We showed that rats readily acquire self-administration of both enantiomers of MEPH when a history of racemic MEPH administration is already established. Drug naïve rats also show consistent self-administration of MEPH enantiomers, although the reinforcing strength of

R-MEPH is significantly greater than S-MEPH. Self-administered R-MEPH elicited greater rates of 50-kHz USVs versus S-MEPH. Breakpoints for R-MEPH were significantly higher than S-MEPH using a progressive-ratio schedule of reinforcement indicating an increase in motivation to work for R-MEPH. The data suggest similarities and differences in the effects of R-MEPH and S-MEPH on acquisition and motivation of

82 drug intake. Furthermore, S-MEPH does not appear to be reinforcing due to the lack of motivation to work for the S-enantiomer.

Stereochemistry is a useful technique to separate the pharmacological and behavioral effects of a compound. Stereochemistry plays an important role in the neuropharmacology of a compound. Specific enantiomeric proportions can be used to separate potential therapeutic effects. For example, to minimize potential side effects, amphetamine salt (Adderall ®), a widely prescribed medication for attention deficit disorder, uses a 3:1 mixture of S: R amphetamine salts to optimize the pharmacokinetic profile and to lower the abuse liability [Heal et al., 2013]. An example of a drug with different pharmacokinetic profiles is methamphetamine. The S-enantiomer (Desoxyn ®) is the more potent and addictive enantiomer, and the R-enantiomer is used in Vick’s

Vapor Inhaler [Li et al., 2010]. From a drug discovery perspective, separation of specific enantiomers may be useful in identifying novel replacement therapies.

Amphetamine, a monoamine releaser, increase monoamine concentrations in the synaptic cleft through drug-evoked neurotransmitter release into the synapse. MEPH function similarly to amphetamine as a monoamine releaser [De Felice et al., 2014].

Amphetamine and methcathinone are selective monoamine releasers at DAT and NET, whereas MEPH and MDMA are non-selective monoamine releasers [Baumann et al.,

2013]. MEPH releasing capability is similar at DAT, NET, and SERT [Baumann et al.,

2013]. Cocaine, a monoamine reuptake inhibitor, increase monoamine concentrations in the synaptic cleft by competitively binding to the transporters. MPDV function similarly to cocaine as a reuptake inhibitor [De Felice et al., 2014]. When compared to cocaine,

MDPV was 50 times more potent at DAT, 10 times more potent at NET, and 10 times

83 less potent at SERT as a transporter blocker [Araujo et al., 2015]. In contrast to MEPH,

MDPV is a potent DAT and NET inhibitor with very low affinity for SERT.

Stereospecific effects of amphetamines and cathinones structurally similar to

MEPH have been explored. Our lab has previously shown stereoselective effects within the enantiomers of MEPH. S-MEPH is a more potent releaser of 5-HT at SERT compared to R-MEPH and S-MEPH did not produce place preference, whereas R-MEPH produces place preference and sensitization of repetitive movements [Gregg et al., 2015].

S-methcathinone is about four times more potent than R-methcathinone in producing locomotor hyperactivity in mice; in enantiomers substitute for cocaine and amphetamine, however, S-methcathinone was three times more potent than R-methcathinone [Glennon et al., 1995]. Furthermore, R-methcathinone and S-methcathinone produce neurotoxic effects in DA neurons but only S-methcathinone produce neurotoxic effects in

5-HT neurons [Sparago et al., 1996]. No differences between R-MDMA and S-MDMA to maintain self-administration behavior, but there were differences in capacity for each enantiomer to induce reinstatement of responding previously maintained by self- administration [Fantegrossi, 2008; McClung et al., 2010]. Priming injections of S-

MDMA induced amphetamine seeking behavior, while R-MDMA failed to reinstate drug-seeking behavior [McClung et al., 2010]. S- MDPV is three times more potent than

R- MDPV as a DAT and NET inhibitor, the S-enantiomer cause greater increases in locomotor activation, blood pressure and heart rate compared to R-MDPV [Gannon et al.,

2016; Schindler et al., 2016; Kolanos et al., 2015].

84 Chapter 2: S-mephedrone reduces anxiety- and depressant-like effects in cocaine- or

MDPV-abstinent rats

In Chapter 2, the effects of S-MEPH were tested in preclinical models of psychostimulant withdrawal using EPM and FST by examining anxiety-like and depression-like behaviors associated with psychostimulant withdrawal. We found that MDPV produces a cocaine- like decrease in percent time in the open arms, which indicates a degree of anxiety produced by psychostimulant withdrawal. When S-MEPH was administered following the discontinuation of chronic drug administration (cocaine and MPDV), the anxiety-like effects were reversed. In the FST, time spent immobile can be used as an index of despair

[Estanislau et al., 2011]. The data show MDPV or cocaine withdrawn animals displayed depression-like behavior. When S-MEPH is administered, we are able to antagonize the depression-like effects indicating S-MEPH has antidepressant effects during psychostimulant withdrawal. Similar to fluoxetine and bupropion, S-MEPH significantly reduced immobility time in FST [Roni et al., 2015; Jesse et al., 2010].

Based on evidence showing 5-HT2A and/or 5-HT2C can have anxiolytic and antidepressant activity, binding and functional receptor screening of S-MEPH was essential to expound on a possible secondary mechanism that could contribute to its clinical relevance. By screening S-MEPH against a battery of receptors, we found that S-

MEPH has a binding affinity for 5-HT 2 receptors (2A, 2B, 2C) and has minimal affinity for DA, adrenergic and nicotinic receptors. Functional assays revealed S-MEPH has no agonist activity. Taken together, S-MEPH can act as both a 5-HT antagonist and a 5-HT releaser, with a blockade of the 5-HT2C receptor underlying its anxiolytic efficacy and an increase in 5-HT release through its monoamine substrate actions underlying its

85 antidepressant effect. Similar to fluoxetine, an antidepressant, it has a dual mechanism of action, and not only increases 5-HT levels, but also blocks 5-HT2C receptors that help to attenuate the initial onset of anxiety reported by patients. These effects may explain the therapeutic potential we observed in EPM and FST. Psychostimulant withdrawal is causing deficits in 5-HT levels and S-MEPH has the ability to normalize those deficits by increasing 5-HT levels.

Chapters 3 and 4: Reinforcing effects of MEPH enantiomers and the effects of

S-MEPH on drug-induced relapse to cocaine

There is limited data regarding the reinforcing effects of stereoisomers of synthetic cathinones. It has been shown that rats will readily self-administer MEPH (0.125-

2.5 mg/kg/infusion) [Hadlock et al., 2011; Nguyen et al., 2016b; Vandewater et al., 2015;

Creehan et al., 2015; Aarde et al., 2013b; Motbey et al., 2013]. While MEPH reinforcing effects has been investigated, the contribution of each enantiomer has not been determined. As described in chapters 3 and 4, we showed first evaluations into the reinforcing effects of R- and S-MEPH enantiomers. Non-contingent priming injections of

S-MEPH failed to reinstate cocaine responding; in contrast R-MEPH priming injections significantly reinstated responding (Chapter 3) and both R- and S-MEPH are effective reinforcers, however, R-MEPH’s reinforcing strength was significantly greater than S-

MEPH’s (Chapter 4). The results suggest that MEPH’s rewarding and reinforcing effects are mediated by R-MEPH.

Chapter 3 investigated whether or not S- MEPH induces reinstatement to cocaine seeking after extinction. Results showed that S-MEPH (10, 20, 30 mg/kg) priming injection failed to reinstate cocaine-seeking behavior. In contrast, cocaine (10 mg/kg)

86 priming injection induced reinstatement of cocaine-seeking behavior. Similar effects were observed with R-MEPH prime injection. Chapter 4 determined if rats exposed to intravenous R-MEPH and S-MEPH will acquire self-administration behavior and if responding will differentiate under a progressive ratio schedule of reinforcement. The results indicated rats acquire R-MEPH and S-MEPH self-administration behavior, and during acquisition no significant difference exists between the enantiomer at equivalent doses. Despite no observed differences between the enantiomers to maintain self- administration behavior, R-MEPH elicited significantly greater rates of 50-kHz USVs compared to S-MEPH. Moreover, S-MEPH trained rats responded significantly less than

R-MEPH trained rats under a progressive ratio schedule of reinforcement. The data suggest there are similarities and differences in the effects of R-MEPH and S-MEPH on acquisition and motivation of drug intake, and that R-MEPH is responsible for the rewarding and reinforcing effects seen with racemic MEPH.

Implications

Despite many years of research, to date there is no FDA-approved drug to treat psychostimulant addiction. There has been much effort to identify and develop medications, with several promising leads including, vaccines, monoamine modulators, and drugs with dual mechanisms of action to target multiple neurotransmitters like serotonin, norepinephrine, gamma-aminobutyric acid (GABA), and glutamate [Shorter et al., 2015; Kosten and Domingo, 2013; Cai et al., 2013; Kimishima et al., 2016].

Immunotherapy using active vaccines to treat cocaine addiction has been promising in clinical trials. Clinical data demonstrates that following vaccination with cocaine vaccine

(TA-CD), participants reported blunted subjective and euphoric effects of smoked

87 cocaine; the production of cocaine-specific antibodies attenuated the rewarding effects of cocaine [Martell et al., 2005; Haney et al., 2010]. Vaccines require high binding affinity for the drug and some cocaine abusers develop low affinity antibodies, which is a limitation to the effectiveness of vaccinations and likely to limit their efficacy in the clinic. Antiepileptic drugs that increase GABA activity have shown promise because

GABA inhibitory effects inhibit dopamine release. Tiagabine, an antiepileptic drug, increases cocaine-free urine samples in cocaine-dependent methadone-treated patients compared to placebo patients [Gonzalez et al., 2003]. Another study using topiramate, antiepileptic drug, found that topiramate-treated patients had more cocaine-free urine samples, and had greater improvement rates to abstain from cocaine than placebo

[Kampman et al., 2004]. Serotonin also inhibits DA transmission and may be a possible target to reduce cocaine dependence. A clinical trial study found that depressed cocaine- dependent patients taking sertraline, a SSRI for anxiety and depression, had significantly less cocaine-positive urine samples than those taking placebo [Mancino et al., 2014].

Although there are promising therapeutic candidates for cocaine dependence, none have received regulatory approval for treatment.

Due to the lack of pharmacological interventions for psychostimulant abuse, new approaches need to be identified. Rapidly acting therapeutic effects is desirable for the development of a medication to manage acute cocaine withdrawal. S-MEPH would be an advantage template for the development of maintenance therapies due to its rapid onset of action versus SERT blockers, such as the SSRIs, which have a delayed onset of antidepressant effects. Traditional SSRIs take several weeks to months to achieve their antidepressant effects. Additionally, S-MEPH can act as both a 5-HT antagonist and a 5-

88 HT releaser. By blocking the 5-HT2C receptors, it limits the onset of anxiety upon initial dosing and reduce anxiety during psychostimulant withdrawal. Another potential advantage is S-MEPH could lead to the development of a medication that lacks the abuse liability associated with agonist-replacement medications for opioid dependence. The characterization of S-MEPH should be used as a foundation for future studies to perform comprehensive in vitro and in vivo characterization of the newer “second-generation” analogs.

Future Directions and Limitations

Relapse is common among drug abusers even after prolonged abstinence. The fact that relapse is caused by multiple triggers, including presentation of the drug itself, a cue previously associated with the drug, and stress, complicate the development of efficacious pharmacotherapies. Stress is a key major trigger for relapse. The mechanisms and brain regions involved in causing anxiety and depression following abstinence from illicit drugs still need to be understood. Withdrawal from chronic cocaine exposure produces 5-HT deficits, including a decrease in extracellular 5-HT in the nucleus accumbens during early withdrawal [Parsons et al., 1996]. Chronic SSRI treatment have limited success in clinical trials for cocaine abuse due to the neurobiological changes that occur during chronic administration [Grabowski et al, 1995; Schmitz et al, 2001;

Winstanley et al., 2011]. Chronic S-MEPH treatment may also cause neurobiological changes. Furthermore, due to the strong 5-HT releasing component, serotonin syndrome may occur. Acute and repeated administration of MEPH was tested in rats and both regimens showed no significant differences in extracellular DA and 5-HT in the striatum, prefrontal cortex and hippocampus [Shortall et al., 2013]. Given that S-MEPH increases

89 extracellular 5-HT, it may be capable of decreasing anxiety and depression during cocaine or MDPV withdrawal by blocking cocaine and MDPV actions. Clinical use of S-

MEPH will be limited despite its therapeutic potential due to MEPH currently listed as a

Schedule I drug. However, there are drugs including amphetamine and buprenorphine that have been rescheduled and research is currently be conducted using to treat cocaine, alcohol, and nicotine addiction [Bogenschutz et al., 2015; Dos Santos et al.,

2016; Johnson et al., 2017; de Veen et al., 2017; D. E. A. United States Department of

Justice, 2017].

Reduction in 5-HT neurotransmission may contribute to the dysfunction of 5-HT receptors leading to anxiety and depression. Several studies have demonstrated a role for

5-HT2 receptors in anxiety and depression. There is supporting preclinical evidence implicating a role for 5-HT2A and/or 5-HT2C receptor antagonists as potential effective anxiolytics and antidepressants [Giorgetti and Tecott, 2004; Millan, 2005; Di Giovanni et al., 2006]. For example, in the mouse, (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2- aminopropane, (DOI), a 5-HT2A agonist produced anxiolytic-like effects using EPM

[Onaivi et al., 1995], while DOI in the FST assay produced depressive-like behaviors

[Diaz and Maroteaux, 2011]. This suggests that 5-HT2A receptor antagonists might produce antidepressant-like activity. Hence, EMD281014, a 5-HT2A receptor antagonist decreases time spent immobile in FST [Patel et al., 2004]. SB-242084, 5-HT2C receptor antagonist, produced anxiolytic-like activity [Martin et al., 2002]. Moreover, 5-HT2C receptor antagonist, S32006 produced antidepressant-like effects [Dekeyne et al., 2008].

S-MEPH’s ability to attenuate cocaine and MDPV withdrawal-induced anxiety and depression indicates that it may be a potential structural and pharmacological

90 template to develop maintenance therapy for psychostimulant dependence. It will be important in future studies to correlate the potency and efficacy of S-MEPH with clinically available antidepressant and anxiolytics such as diazepam, fluoxetine, and bupropion (structurally similar cathinone, used for smoking cessation and depression).

Inclusion of known anxiolytics or antidepressants would provide additional information about the therapeutic efficacy and potential of S-MEPH. While EPM and FST are well- established and widely used assays of anxiety- and depression-like behavior [Walf et al.,

2007; Estanislau et al., 2011], future studies will examine the anxiety- and antidepressant-like effects of S-MEPH using other established models such as the open field test, social interaction test, sucrose preference test, and novelty-induced hypophagia to gain more insight into the mechanism of S-MEPH.

Both clinical and rodent studies reveal notable sex differences in cocaine abuse.

Women acquire cocaine dependence earlier and more rapidly, show increased craving and severity of cocaine use, and relapse to cocaine more rapidly as compared with men

[Kosten et al., 1993; Robbins et al., 1999; Becker and Hu, 2008]. In addition, women report more depression and anxiety disorders after prolonged cocaine use [Griffin et al.,

1989]. Several measures of cocaine dependence are greater in female rodents, including self-administration, drug seeking, higher progressive ratio breakpoints, and reinstatement of extinguished cocaine seeking [Lynch and Taylor, 2004; Kippin et al., 2005; Feltenstein et al., 2011; Zhou et al., 2012]. The biological mechanisms for these sex differences remain unclear. Sex studies need to be conducted to examine if S-MEPH efficacy will vary between sexes. A recent study found sex differences in conditioned taste avoidance assay but not in CPP assay. MDPV produced CPP in both males and females with no

91 significant difference; however, females displayed a weaker avoidance than males in the conditioned taste avoidance assay [King et al., 2015a]. Sex differences may serve as a predictive measure in determining a particular gender’s susceptibility to abuse a particular substance.

Multiple brain circuits are modulated which promote behavioral changes following cocaine and most likely MDPV use. The in vitro binding and functional assays provided a basis for a possible secondary mechanism that may contribute to S-MEPH’s clinical relevance. Follow up studies using western blot analysis should be conducted to detect what chronic psychostimulant exposure will do to DA and 5-HT systems. Up or downward trends in expression of monoamine transporter protein levels can elucidate how hypodopaminergic and hyposerotonergic conditions associated with drug administration affect the overall homeostasis of the DA/5-HT systems. We hypothesize that S-MEPH will increase 5-HT and restore the tone of the receptors during early withdrawal. By identifying the role of 5-HT receptors in regulating dopamine and serotonin systems, which leads to anxiety during MDPV withdrawal, this will be a potential target to intervene during withdrawal- induced anxiety and depression.

Studies on the pharmacokinetics of synthetic cathinones is limited.

Pharmacokinetic data indicate that MEPH freely crosses the blood brain barrier and widely distributes throughout the brain [Martínez-Clemente et al., 2013]. Six phase I and four phase II MEPH metabolites were present when tested in humans and rats but none are active [Meyer et al., 2010b; Khreit et al., 2013; Martínez-Clemente et al.,

2013; Pedersen et al., 2013]. MDPV has two phase I metabolites, and the metabolites may be inactive and cannot cross the blood-brain barrier [Anizan et al., 2016]. The

92 inactive metabolites may explain the relatively short half-life and duration of action of

MEPH and MDPV. In contrast, fluoxetine a widely used SSRI has an active metabolite norfluoxetine, which contributes to its therapeutic effects [Fuller et al., 1992].

Both fluoxetine and norfluoxetine freely cross the blood-brain barrier [Qu et al., 2009].

Norfluoxetine extend fluoxetine efficacy, fluoxetine half-life is between 1–3 days [Qu et al., 2009]. The cathinones lack of active metabolites may be an indication into their short duration of action.

It will be important to measure 5-HT release patterns in brain reward regions.

Psychostimulants elevate levels of extracellular DA and 5-HT in the nucleus accumbens

[Andrews and Lucki, 2001; Di Chiara and Imperato, 1988]. Analyzing extracellular levels of DA and 5-HT in brain reward circuits following acute and chronic R- and S-

MEPH treatments warrant further studies. Based on previously published literature with

R-MEPH having a greater DA/5-HT ratio compared to S-MEPH [Gregg et al., 2015], we hypothesize that S-MEPH will significantly elevate levels of extracellular 5-HT greater than R-MEPH, while R-MEPH will significantly elevate levels of extracellular DA greater than S-MEPH. These studies are necessary to identify a potential neurochemical mechanism of action for each enantiomer that can be compared to racemic MEPH.

Finally, there are many factors (stress, environment, and drug) that play a role in relapse.

Future studies should test S-MEPH against a variety of relapse triggers, such as, cue- induced reinstatement (re-exposure to drug-associated cues) and stress-induced reinstatement. One limitation of our studies is that we were unsuccessful in our efforts to induce stress following cocaine and MDPV self-administration. Therefore, we were not able to investigate the effects of S-MEPH on stress-induced reinstatement following

93 cocaine and MDPV self-administration. To investigate the effect of S-MEPH on drug prime reinstatement to cocaine or MDPV, rats will be trained to acquire 0.375 mg/kg/inf cocaine or 0.0375 mg/kg/inf MDPV under a fixed ratio (FR-1) schedule of reinforcement, then subjected to extinction sessions. Once extinction criteria are reached, rats will be injected with saline or S-MEPH 2x/day for 2 days 6-h apart to maintain dosing consistency with EPM experiments. Reinstatement test will be conducted the day after last S-MEPH injection. For drug-primed reinstatement test, cocaine (10 mg/kg, i.p.) or

MDPV (10 mg/kg, i.p.) will be used to test reinstatement to drug seeking. The drug- primed injections will be given immediately prior to testing. We hypothesize that rats pretreated with saline will lead to robust responding on the previously reinforced lever, while pretreatment of S-MEPH will attenuate prime-induced reinstatement of drug seeking.

Conclusion

New therapeutic interventions for psychostimulant addiction are desperately needed.

Cathinones that are structurally analogous to the most popularly abused synthetic cathinones today have previously displayed stereoselective actions. We evaluated

MEPH’s stereoselectivity, and found both enantiomers acquire self-administration behavior, but R-MEPH has significantly greater reinforcing strength compared to S-

MEPH. Furthermore, self-administered R-MEPH elicited greater rates of 50-kHz USVs versus S-MEPH. Notably, S-MEPH displayed therapeutic potential in EPM and FST assays.

We provided a strong foundation into the individual enantiomers of MEPH, to better understand the abuse liability and clinical implications of these drugs. S-MEPH enhances

94 the release of 5-HT and was efficacious during acute cocaine and MDPV withdrawal that caused anxiety and depression. S-MEPH may be useful in developing pharmacotherapies that enhance release of 5-HT, DA and/or NE, similar to the cathinone bupropion. We provide a comprehensive assessment of the impact of S-MEPH on cocaine- and MDPV- induced withdrawal. Our studies developed the pharmacological profile of a novel and prevalent psychostimulant (MDPV) in the context of dependence and addiction relative to cocaine. Our research findings serve as a pharmacological template to characterize the newer “second-generation” synthetic cathinones. In conclusion, our studies identify a favorable structural and pharmacological profile to develop an effective treatment to reduce or prevent anxiety and depression during early cocaine or MDPV withdrawal.

95 REFERENCES

Aarde, S. M., Huang, P. K., Creehan, K. M., Dickerson, T. J. & Taffe, M. A. The novel recreational drug 3,4-methylenedioxypyrovalerone (MDPV) is a potent psychomotor stimulant: self-administration and locomotor activity in rats. Neuropharmacology 71, 130–140 (2013a).

Aarde, S. M. et al. Mephedrone (4-methylmethcathinone) supports intravenous self- administration in Sprague-Dawley and Wistar rats. Addict. Biol. 18, 786–799 (2013b).

Aarde, S. M., Huang, P. K., Dickerson, T. J. & Taffe, M. A. Binge-like acquisition of 3,4-methylenedioxypyrovalerone (MDPV) self-administration and wheel activity in rats. Psychopharmacology (Berl). 232, 1867–1877 (2015).

Abrahams, B. S., Rutherford, J. D., Mallet, P. E. & Beninger, R. J. Place conditioning with the dopamine D1-like receptor agonist SKF 82958 but not SKF 81297 or SKF 77434. Eur. J. Pharmacol. 343, 111–118 (1998).

Adams, J. U., Careri, J. M., Efferen, T. R. & Rotrosen, J. Differential effects of dopamine antagonists on locomotor activity, conditioned activity and conditioned place preference induced by cocaine in rats. Behav. Pharmacol. 12, 603–611 (2001).

Ahrens, A. M., Ma, S. T., Maier, E. Y., Duvauchelle, C. L. & Schallert, T. Repeated intravenous amphetamine exposure: rapid and persistent sensitization of 50-kHz ultrasonic trill calls in rats. Behav. Brain Res. 197, 205–209 (2009).

Alleweireldt, A. T., Kirschner, K. F., Blake, C. B. & Neisewander, J. L. D1-receptor drugs and cocaine-seeking behavior: investigation of receptor mediation and behavioral disruption in rats. Psychopharmacology (Berl). 168, 109–117 (2003).

American Association of Poison Control Centers (AAPCC), Bath Salt Data (2013). Retrieved from https://aapcc.s3.amazonaws.com/files/library/Bath_Salts_Data_for _Website_2.282013.pdf.

Andrews, C. M. & Lucki, I. Effects of cocaine on extracellular dopamine and serotonin levels in the nucleus accumbens. Psychopharmacology (Berl). 155, 221–229 (2001).

Anizan, S. et al. 3,4-Methylenedioxypyrovalerone (MDPV) and metabolites quantification in human and rat plasma by liquid chromatography-high resolution mass spectrometry. Anal Chim Acta 827, 54–63 (2014).

96 Anizan, S. et al. Linear pharmacokinetics of 3,4-methylenedioxypyrovalerone (MDPV) and its metabolites in the rat: Relationship to pharmacodynamic effects. Addict. Biol. 21, 339–347 (2016).

Anneken, J. H., Angoa-Perez, M. & Kuhn, D. M. 3,4-Methylenedioxypyrovalerone prevents while methylone enhances methamphetamine-induced damage to dopamine nerve endings: beta-ketoamphetamine modulation of neurotoxicity by the dopamine transporter. J. Neurochem. 133, 211–222 (2015).

Araujo, A. M. et al. Raising awareness of new psychoactive substances: chemical analysis and in vitro toxicity screening of ‘legal high’ packages containing synthetic cathinones. Arch. Toxicol. 89, 757–771 (2015).

Arias, H. R. Is the inhibition of nicotinic acetylcholine receptors by bupropion involved in its clinical actions? Int. J. Biochem. Cell Biol. 41, 2098–2108 (2009).

Bachtell, R. K., Whisler, K., Karanian, D. & Self, D. W. Effects of intra-nucleus accumbens shell administration of dopamine agonists and antagonists on cocaine- taking and cocaine-seeking behaviors in the rat. Psychopharmacology (Berl). 183, 41–53 (2005).

Ball, K. T. & Slane, M. Tolerance to the locomotor-activating effects of 3,4- methylenedioxymethamphetamine (MDMA) predicts escalation of MDMA self- administration and cue-induced reinstatement of MDMA seeking in rats. Behav. Brain Res. 274, 143–148 (2014).

Balsara, J. J., Jadhav, J. H. & Chandorkar, A. G. Effect of drugs influencing central serotonergic mechanisms on -induced catalepsy. Psychopharmacology (Berl). 62, 67–69 (1979).

Barbeau, A. The pathogenesis of Parkinson’s disease: a new hypothesis. Can. Med. Assoc. J. 87, 802–807 (1962).

Barker, D. J. et al. Ultrasonic vocalizations: evidence for an affective opponent process during cocaine self-administration. Psychopharmacology (Berl). 231, 909–918 (2014).

Barker, D. J., Simmons, S. J. & West, M. O. Ultrasonic Vocalizations as a Measure of Affect in Preclinical Models of Drug Abuse: A Review of Current Findings. Curr. Neuropharmacol. 13, 193–210 (2015).

Bauer, C. T., Banks, M. L., Blough, B. E. & Negus, S. S. Use of intracranial self- stimulation to evaluate abuse-related and abuse-limiting effects of monoamine releasers in rats. Br. J. Pharmacol. 168, 850–862 (2013).

97 Baumann, M. H., Clark, R. D. & Rothman, R. B. Locomotor stimulation produced by 3,4-methylenedioxymethamphetamine (MDMA) is correlated with dialysate levels of serotonin and dopamine in rat brain. Pharmacol. Biochem. Behav. 90, 208–217 (2008).

Baumann, M. H. et al. The designer methcathinone analogs, mephedrone and methylone, are substrates for monoamine transporters in brain tissue. Neuropsychopharmacology 37, 1192–1203 (2012).

Baumann, M. H. et al. Powerful cocaine-like actions of 3,4-methylenedioxypyrovalerone (MDPV), a principal constituent of psychoactive ‘bath salts’ products. Neuropsychopharmacology 38, 552–562 (2013a).

Baumann, M. H., Partilla, J. S. & Lehner, K. R. Psychoactive ‘bath salts’: Not so soothing. European Journal of Pharmacology 698, 1–5 (2013b).

Baumann, M. H. Awash in a sea of ‘bath salts’: implications for biomedical research and public health. Addiction (Abingdon, England) 109, 1577–1579 (2014).

Beardsley, P. M. & Shelton, K. L. Prime-, stress-, and cue-induced reinstatement of extinguished drug-reinforced responding in rats: cocaine as the prototypical drug of abuse. Curr. Protoc. Neurosci. Chapter 9, Unit 9.39 (2012).

Beaulieu, J.M., Gainetdinov, R.R. The physiology, signaling, and pharmacology of dopamine receptors. Pharmacol Rev. 63, 182-217 (2011).

Becker, J. B. & Hu, M. Sex differences in drug abuse. Front. Neuroendocrinol. 29, 36–47 (2008).

Bergman, J., Madras, B. K., Johnson, S. E. & Spealman, R. D. Effects of cocaine and related drugs in nonhuman primates. III. Self-administration by squirrel monkeys. J. Pharmacol. Exp. Ther. 251, 150–155 (1989).

Besnard, J. et al. Automated design of ligands to polypharmacological profiles. Nature 492, 215–220 (2012).

Bogenschutz, M. P. et al. -assisted treatment for alcohol dependence: a proof- of-concept study. J. Psychopharmacol. 29, 289–299 (2015).

Bonano, J. S., Glennon, R. A., De Felice, L. J., Banks, M. L. & Negus, S. S. Abuse- related and abuse-limiting effects of methcathinone and the synthetic ‘bath salts’ cathinone analogs methylenedioxypyrovalerone (MDPV), methylone and mephedrone on intracranial self-stimulation in rats. Psychopharmacology (Berl). 231, 199–207 (2014).

98 Bondareva, T. S., Young, R. & Glennon, R. A. Central stimulants as discriminative stimuli. Asymmetric generalization between (-) and S(+)methamphetamine. Pharmacol. Biochem. Behav. 74, 157–162 (2002).

Bouthenet, M. L. et al. Localization of dopamine D3 receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D2 receptor mRNA. Brain Res. 564, 203–219 (1991).

Bowers, B. J., Henry, M. B., Thielen, R. J. & McBride, W. J. Serotonin 5-HT(2) receptor stimulation of dopamine release in the posterior but not anterior nucleus accumbens of the rat. J. Neurochem. 75, 1625–1633 (2000).

Brandt, S. D., Sumnall, H. R., Measham, F. & Cole, J. Analyses of second-generation ‘legal highs’ in the UK: Initial findings. Drug Test. Anal. 2, 377–382 (2010).

Brudzynski, S. M. Principles of rat communication: quantitative parameters of ultrasonic calls in rats. Behav. Genet. 35, 85–92 (2005).

Burgdorf, J., Panksepp, J. & Moskal, J. R. Frequency-modulated 50 kHz ultrasonic vocalizations: a tool for uncovering the molecular substrates of positive affect. Neurosci. Biobehav. Rev. 35, 1831–1836 (2011).

Cai, X., Tsuchikama, K. & Janda, K. D. Modulating cocaine vaccine potency through hapten fluorination. J. Am. Chem. Soc. 135, 2971–2974 (2013).

Caine, S. B. & Koob, G. F. Effects of dopamine D-1 and D-2 antagonists on cocaine self- administration under different schedules of reinforcement in the rat. J. Pharmacol. Exp. Ther. 270, 209–218 (1994).

Caine, S. B., Negus, S. S., Mello, N. K. & Bergman, J. Effects of dopamine D(1-like) and D(2-like) agonists in rats that self-administer cocaine. J. Pharmacol. Exp. Ther. 291, 353–360 (1999).

Cameron, K. N., Kolanos, R., Solis, E., Glennon, R. A. & De Felice, L. J. Bath salts components mephedrone and methylenedioxypyrovalerone (MDPV) act synergistically at the human dopamine transporter. Br. J. Pharmacol. 168, 1750– 1757 (2013).

Carter, C. J. & Pycock, C. J. A study of the sites of interaction between dopamine and 5- hydroxytryptamine for the production of -induced catalepsy. Naunyn. Schmiedebergs. Arch. Pharmacol. 304, 135–139 (1978).

Castagne, V., Moser, P., Roux, S. & Porsolt, R. D. Rodent models of depression: forced swim and tail suspension behavioral despair tests in rats and mice. Curr. Protoc. Pharmacol. Chapter 5, Unit 5.8 (2010).

99 Cervo, L., Carnovali, F., Stark, J. A. & Mennini, T. Cocaine-seeking behavior in response to drug-associated stimuli in rats: involvement of D3 and D2 dopamine receptors. Neuropsychopharmacology 28, 1150–1159 (2003).

Chausmer, A. L. & Katz, J. L. The role of D2-like dopamine receptors in the locomotor stimulant effects of cocaine in mice. Psychopharmacology (Berl). 155, 69–77 (2001).

Childs, E., Shoaib, M. & Stolerman, I. P. Cocaine self-administration in rats with histories of cocaine exposure and discrimination. Psychopharmacology (Berl). 186, 168–176 (2006).

Ciccocioppo, R., Sanna, P. P. & Weiss, F. Cocaine-predictive stimulus induces drug- seeking behavior and neural activation in limbic brain regions after multiple months of abstinence: reversal by D(1) antagonists. Proc. Natl. Acad. Sci. U. S. A. 98, 1976–1981 (2001).

Clemens, K. J., Cornish, J. L., Hunt, G. E. & McGregor, I. S. Intravenous methamphetamine self-administration in rats: effects of intravenous or intraperitoneal MDMA co-administration. Pharmacol. Biochem. Behav. 85, 454– 463 (2006).

Clinard, C. T., Bader, L. R., Sullivan, M. A. & Cooper, M. A. Activation of 5-HT2a receptors in the basolateral amygdala promotes defeat-induced anxiety and the acquisition of conditioned defeat in Syrian hamsters. Neuropharmacology 90, 102–112 (2015).

Cooper, J. R., Bloom, F. E., Roth, R. H. The Biochemical Basis of Neuropharmacology. Oxford University Press, New York. Cooper: Chapter 9, 10. (2003)

Cornelio, A. M. & Nunes-de-Souza, R. L. Anxiogenic-like effects of mCPP microinfusions into the amygdala (but not dorsal or ventral hippocampus) in mice exposed to elevated plus-maze. Behav. Brain Res. 178, 82–89 (2007).

Cornish, J. L. et al. Heat increases 3,4-methylenedioxymethamphetamine self- administration and social effects in rats. Eur. J. Pharmacol. 482, 339–341 (2003).

Covington, H. E. 3rd & Miczek, K. A. Vocalizations during withdrawal from opiates and cocaine: possible expressions of affective distress. Eur. J. Pharmacol. 467, 1–13 (2003).

Craige, C. P. & Unterwald, E. M. Serotonin (2C) receptor regulation of cocaine-induced conditioned place preference and locomotor sensitization. Behav. Brain Res. 238, 206–210 (2013).

100 Craige, C. P., Lewandowski, S., Kirby, L. G. & Unterwald, E. M. Dorsal raphe 5-HT2C receptor and GABA networks regulate anxiety produced by cocaine withdrawal. Neuropharmacology 93, 41–51 (2015).

Creehan, K. M., Vandewater, S. A. & Taffe, M. A. Intravenous self-administration of mephedrone, methylone and MDMA in female rats. Neuropharmacology 92, 90– 97 (2015).

Cunningham, K. A. et al. Synergism between a serotonin 5-HT2A receptor (5-HT2AR) antagonist and 5-HT2CR agonist suggests new pharmacotherapeutics for cocaine addiction. ACS Chem. Neurosci. 4, 110–121 (2013).

Czoty, P. W., Ginsburg, B. C. & Howell, L. L. Serotonergic attenuation of the reinforcing and neurochemical effects of cocaine in squirrel monkeys. J. Pharmacol. Exp. Ther. 300, 831–837 (2002).

Dargan, P. I., Sedefov, R., Gallegos, A. & Wood, D. M. The pharmacology and toxicology of the synthetic cathinone mephedrone (4-methylmethcathinone). Drug Test. Anal. 3, 454–463 (2011).

De Deurwaerdere, P., Navailles, S., Berg, K. A., Clarke, W. P. & Spampinato, U. Constitutive activity of the serotonin2C receptor inhibits in vivo dopamine release in the rat striatum and nucleus accumbens. J. Neurosci. 24, 3235–3241 (2004).

De Felice, L. J., Glennon, R. A. & Negus, S. S. Synthetic cathinones: Chemical phylogeny, physiology, and neuropharmacology. Life Sciences 97, 20–26 (2014). de Veen, B. T. H., Schellekens, A. F. A., Verheij, M. M. M. & Homberg, J. R. Psilocybin for treating substance use disorders? Expert Rev. Neurother. 17, 203–212 (2017).

De Vries, T. J., Schoffelmeer, A. N. M., Binnekade, R., Raaso, H. & Vanderschuren, L. J. M. J. Relapse to cocaine- and -seeking behavior mediated by dopamine D2 receptors is time-dependent and associated with behavioral sensitization. Neuropsychopharmacology 26, 18–26 (2002).

Dekeyne, A. et al. S32006, a novel 5-HT2C receptor antagonist displaying broad-based antidepressant and anxiolytic properties in rodent models. Psychopharmacology (Berl). 199, 549–568 (2008).

Di Chiara, G. & Imperato, A. Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc. Natl. Acad. Sci. U. S. A. 85, 5274–5278 (1988).

Di Giovanni, G., Di Matteo, V., Pierucci, M., Benigno, A. & Esposito, E. Central serotonin2C receptor: from physiology to pathology. Curr. Top. Med. Chem. 6, 1909–1925 (2006).

101 Di Matteo, V., Di Giovanni, G., Pierucci, M. & Esposito, E. Serotonin control of central dopaminergic function: focus on in vivo microdialysis studies. Prog. Brain Res. 172, 7–44 (2008).

Diaz, S. L. & Maroteaux, L. Implication of 5-HT(2B) receptors in the serotonin syndrome. Neuropharmacology 61, 495–502 (2011).

Dos Santos, R. G. et al. Antidepressive, anxiolytic, and antiaddictive effects of , psilocybin and lysergic acid diethylamide (LSD): a systematic review of clinical trials published in the last 25 years. Ther. Adv. Psychopharmacol. 6, 193–213 (2016).

Dworkin, S. I. & Pitts, R. C. Use of rodent self-administration models to develop pharmacotherapies for cocaine abuse. NIDA Res. Monogr. 145, 88–112 (1994).

El Hage, C. et al. Enhanced anxiety observed in cocaine withdrawn rats is associated with altered reactivity of the dorsomedial prefrontal cortex. PLoS One 7, e43535 (2012).

Elman, I., Karlsgodt, K. H., Gastfriend, D. R., Chabris, C. F. & Breiter, H. C. Cocaine- primed craving and its relationship to depressive symptomatology in individuals with cocaine dependence. J. Psychopharmacol. 16, 163–167 (2002).

Erb, S., Shaham, Y. & Stewart, J. Stress reinstates cocaine-seeking behavior after prolonged extinction and a drug-free period. Psychopharmacology (Berl). 128, 408–412 (1996).

Erb, S., Kayyali, H. & Romero, K. A study of the lasting effects of cocaine pre-exposure on anxiety-like behaviors under baseline conditions and in response to central injections of corticotropin-releasing factor. Pharmacol. Biochem. Behav. 85, 206– 213 (2006).

Eshleman, A. J. et al. Substituted methcathinones differ in transporter and receptor interactions. Biochem. Pharmacol. 85, 1803–1815 (2013).

Estanislau, C. et al. Individual differences in the elevated plus-maze and the forced swim test. Behav. Processes 86, 46–51 (2011).

Everitt, B. J. & Wolf, M. E. Psychomotor stimulant addiction: a neural systems perspective. J. Neurosci. 22, 3312–3320 (2002).

Fantegrossi, W. E. In vivo pharmacology of MDMA and its enantiomers in rhesus monkeys. Exp. Clin. Psychopharmacol. 16, 1–12 (2008).

Fantegrossi, W. E., Ullrich, T., Rice, K. C., Woods, J. H. & Winger, G. 3,4- Methylenedioxymethamphetamine (MDMA, ‘ecstasy’) and its stereoisomers as

102 reinforcers in rhesus monkeys: serotonergic involvement. Psychopharmacology (Berl). 161, 356–364 (2002).

Fantegrossi, W. E. et al. Pharmacological characterization of the effects of 3,4- methylenedioxymethamphetamine (‘ecstasy’) and its enantiomers on lethality, core temperature, and locomotor activity in singly housed and crowded mice. Psychopharmacology (Berl). 166, 202–211 (2003).

Fantegrossi, W. E., Gannon, B. M., Zimmerman, S. M. & Rice, K. C. In vivo effects of abused ‘bath salt’ constituent 3,4-methylenedioxypyrovalerone (MDPV) in mice: drug discrimination, thermoregulation, and locomotor activity. Neuropsychopharmacology 38, 563–73 (2013).

Feltenstein, M. W., Henderson, A. R. & See, R. E. Enhancement of cue-induced reinstatement of cocaine-seeking in rats by : sex differences and the role of the estrous cycle. Psychopharmacology (Berl). 216, 53–62 (2011).

Ferrario, C. R. & Robinson, T. E. Amphetamine pretreatment accelerates the subsequent escalation of cocaine self-administration behavior. Eur. Neuropsychopharmacol. 17, 352–357 (2007).

Fibiger, H. C. Neurobiology of depression: focus on dopamine. Adv. Biochem. Psychopharmacol. 49, 1–17 (1995).

Fibiger, H.C., Phillips AG. Reward, motivation, cognition: Psychobiology of mesotelencephalic dopamine systems. In: Mountcastle VB, Bloom FE, Geiger SR, editors. Handbook of Physiology: The Nervous System. American Physiological Society 4, 647–675 (1986).

Filip, M., Bubar, M. J. & Cunningham, K. A. Contribution of serotonin (5-HT) 5-HT2 receptor subtypes to the discriminative stimulus effects of cocaine in rats. Psychopharmacology (Berl). 183, 482–489 (2006).

Fink, H., Morgenstern, R. & Oelssner, W. Psychotomimetics potentiate locomotor hyperactivity induced by dopaminergic drugs. Pharmacol. Biochem. Behav. 11, 479–482 (1979).

Fletcher, P. J., Grottick, A. J. & Higgins, G. A. Differential effects of the 5-HT(2A) receptor antagonist M100907 and the 5-HT(2C) receptor antagonist SB242084 on cocaine-induced locomotor activity, cocaine self-administration and cocaine- induced reinstatement of responding. Neuropsychopharmacology 27, 576–586 (2002).

103 Fletcher, P. J., Chintoh, A. F., Sinyard, J. & Higgins, G. A. Injection of the 5-HT2C receptor agonist Ro60-0175 into the ventral tegmental area reduces cocaine- induced locomotor activity and cocaine self-administration. Neuropsychopharmacology 29, 308–318 (2004).

Fone, K. C., Shalders, K., Fox, Z. D., Arthur, R. & Marsden, C. A. Increased 5-HT2C receptor responsiveness occurs on rearing rats in social isolation. Psychopharmacology (Berl). 123, 346–352 (1996).

Frazer A, Hensler J Serotonin. In: Basic Neurochemistry: Molecular, Cellular and Medical Aspects (Siegel G, Agranoff B, Albers R, Fisher S, Uhler M, eds), Lippincott Williams & Wilkins. 263-292. (1999)

Froberg, B. A. et al. Acute Methylenedioxypyrovalerone Toxicity. J. Med. Toxicol. 11, 185–194 (2015).

Fuller, R. W., Snoddy, H. D., Krushinski, J. H. & Robertson, D. W. Comparison of norfluoxetine enantiomers as serotonin uptake inhibitors in vivo. Neuropharmacology 31, 997–1000 (1992).

Fuxe, K Evidence for the existence of monoamine neurons in the central nervous system. IV. Distribution of monoamine nerve terminals in the cenral nervous system. Acta Physiol. Scand. Suppl. SUPPL 247:37+ (1965).

Gannon, B. M., Galindo, K. I., Rice, K. C. & Collins, G. T. Individual Differences in the Relative Reinforcing Effects of 3,4-Methylenedioxypyrovalerone under Fixed and Progressive Ratio Schedules of Reinforcement in Rats. J. Pharmacol. Exp. Ther. 361, 181–189 (2017).

Gannon, B. M., Williamson, A., Suzuki, M., Rice, K. C. & Fantegrossi, W. E. Stereoselective Effects of Abused ‘Bath Salt’ Constituent 3,4- Methylenedioxypyrovalerone in Mice: Drug Discrimination, Locomotor Activity, and Thermoregulation. J. Pharmacol. Exp. Ther. 356, 615–623 (2016).

Gatch, M. B., Taylor, C. M. & Forster, M. J. Locomotor stimulant and discriminative stimulus effects of ‘bath salt’ cathinones. Behav. Pharmacol. 24, 437–47 (2013).

Gawin, F. H. Cocaine addiction: psychology and neurophysiology. Science 251, 1580– 1586 (1991).

Gawin, F. H. & Kleber, H. D. Abstinence symptomatology and psychiatric diagnosis in cocaine abusers. Clinical observations. Arch. Gen. Psychiatry 43, 107–113 (1986).

German, C. L., Fleckenstein, A. E. & Hanson, G. R. Bath salts and synthetic cathinones: An emerging designer drug phenomenon. Life Sciences 97, 2–8 (2014).

104 Gerson, S. C. & Baldessarini, R. J. Motor effects of serotonin in the central nervous system. Life Sci. 27, 1435–1451 (1980).

Giorgetti, M. & Tecott, L. H. Contributions of 5-HT(2C) receptors to multiple actions of central serotonin systems. Eur. J. Pharmacol. 488, 1–9 (2004).

Glennon, R. A., Young, R., Martin, B. R. & Dal Cason, T. A. Methcathione (‘cat’): an enantiomeric potency comparison. Pharmacol. Biochem. Behav. 50, 601–606 (1995).

Gobert, A. & Millan, M. J. Serotonin (5-HT)2A receptor activation enhances dialysate levels of dopamine and noradrenaline, but not 5-HT, in the frontal cortex of freely-moving rats. Neuropharmacology 38, 315–317 (1999).

Gonzalez, G. et al. Tiagabine increases cocaine-free urines in cocaine-dependent methadone-treated patients: results of a randomized pilot study. Addiction 98, 1625–1632 (2003).

Grabowska, M., Michaluk, J. & Antkiewicz, L. Possible involvement of brain serotonin in -induced hypothermia. Eur. J. Pharmacol. 23, 82–89 (1973).

Grabowski, J., Rhoades, H., Elk, R., Schmitz, J., Davis, C., Creson, D., Kirby, K. Fluoxetine is ineffective for treatment of cocaine dependence or concurrent opiate and cocaine dependence: two placebo-controlled double-blind trials. J Clin Psychopharmacol. 15, 163-174 (1995).

Gregg, R. A. et al. Stereochemistry of mephedrone neuropharmacology: enantiomer- specific behavioural and neurochemical effects in rats. Br. J. Pharmacol. 172, 883–894 (2015).

Gregg, R. A. et al. Synthetic cathinone MDPV downregulates glutamate transporter subtype I (GLT-1) and produces rewarding and locomotor-activating effects that are reduced by a GLT-1 activator. Neuropharmacology 108, 111–119 (2016).

Gregg, R. A., Tallarida, C. S., Reitz, A., McCurdy, C. & Rawls, S. M. Mephedrone (4- methylmethcathinone), a principal constituent of psychoactive bath salts, produces behavioral sensitization in rats. Drug Alcohol Depend. 133, 746–750 (2013).

Griffin, M. L., Weiss, R. D., Mirin, S. M. & Lange, U. A comparison of male and female cocaine abusers. Arch. Gen. Psychiatry 46, 122–126 (1989).

Grimm, J. W. et al. Time-dependent increases in brain-derived neurotrophic factor protein levels within the mesolimbic dopamine system after withdrawal from cocaine: implications for incubation of cocaine craving. J. Neurosci. 23, 742–747 (2003).

105 Hadlock, G. C. et al. 4-Methylmethcathinone (mephedrone): neuropharmacological effects of a designer stimulant of abuse. J. Pharmacol. Exp. Ther. 339, 530–536 (2011).

Hall, B. J., Slade, S., Wells, C., Rose, J. E. & Levin, E. D. Bupropion-varenicline interactions and nicotine self-administration behavior in rats. Pharmacol. Biochem. Behav. 130, 84–89 (2015).

Haney, M., Gunderson, E. W., Jiang, H., Collins, E. D. & Foltin, R. W. Cocaine-specific antibodies blunt the subjective effects of smoked cocaine in humans. Biol. Psychiatry 67, 59–65 (2010).

Harvey, E. L. & Baker, L. E. Differential effects of 3,4-methylenedioxypyrovalerone (MDPV) and 4-methylmethcathinone (mephedrone) in rats trained to discriminate MDMA or a d-amphetamine + MDMA mixture. Psychopharmacology (Berl). 233, 673–680 (2016).

Heal, D. J., Smith, S. L., Gosden, J. & Nutt, D. J. Amphetamine, past and present--a pharmacological and clinical perspective. J. Psychopharmacol. 27, 479–496 (2013).

Heisler, L. K., Zhou, L., Bajwa, P., Hsu, J. & Tecott, L. H. Serotonin 5-HT(2C) receptors regulate anxiety-like behavior. Genes. Brain. Behav. 6, 491–496 (2007).

Hendershot, C. S., Witkiewitz, K., George, W. H. & Marlatt, G. A. Relapse prevention for addictive behaviors. Subst. Abuse Treat. Prev. Policy 6, 17 (2011).

Hicks, C. et al. Glutamate carboxypeptidase II (GCPII) inhibitor 2-PMPA reduces rewarding effects of the synthetic cathinone MDPV in rats: a role for N- acetylaspartylglutamate (NAAG). Psychopharmacology (Berl). (2017). doi:10.1007/s00213-017-4568-y

Highfield, D. A., Mead, A. N., Grimm, J. W., Rocha, B. A. & Shaham, Y. Reinstatement of cocaine seeking in 129X1/SvJ mice: effects of cocaine priming, cocaine cues and food deprivation. Psychopharmacology (Berl). 161, 417–424 (2002).

Hoyer, D. et al. International Union of Pharmacology classification of receptors for 5- hydroxytryptamine (Serotonin). Pharmacol. Rev. 46, 157–203 (1994).

Hoyer, D., Hannon, J. P. & Martin, G. R. Molecular, pharmacological and functional diversity of 5-HT receptors. Pharmacol. Biochem. Behav. 71, 533–554 (2002).

Hubner, C. B. & Moreton, J. E. Effects of selective D1 and D2 dopamine antagonists on cocaine self-administration in the rat. Psychopharmacology (Berl). 105, 151–156 (1991).

106 Hysek, C. M. et al. Duloxetine inhibits effects of MDMA (‘ecstasy’) in vitro and in humans in a randomized placebo-controlled laboratory study. PLoS One 7, e36476 (2012).

Ikemoto, S. & Bonci, A. Neurocircuitry of drug reward. Neuropharmacology 76 Pt B, 329–341 (2014).

Jesse, C. R., Wilhelm, E. A. & Nogueira, C. W. Depression-like behavior and mechanical allodynia are reduced by bis selenide treatment in mice with chronic constriction injury: a comparison with fluoxetine, , and bupropion. Psychopharmacology (Berl). 212, 513–522 (2010).

Johnson, M. W., Garcia-Romeu, A. & Griffiths, R. R. Long-term follow-up of psilocybin-facilitated smoking cessation. Am. J. Drug Alcohol Abuse 43, 55–60 (2017).

Kampman, K. M. et al. A pilot trial of topiramate for the treatment of cocaine dependence. Drug Alcohol Depend. 75, 233–240 (2004).

Karch, S. B. Cathinone neurotoxicity (‘The ’3Ms"). Curr. Neuropharmacol. 13, 21–25 (2015).

Karlsson, L., Andersson, M., Kronstrand, R. & Kugelberg, F. C. Mephedrone, methylone and 3,4-methylenedioxypyrovalerone (MDPV) induce conditioned place preference in mice. Basic Clin. Pharmacol. Toxicol. 115, 411–6 (2014)

Khan, Z. U. et al. Dopamine D5 receptors of rat and human brain. Neuroscience 100, 689–699 (2000).

Kehr, J. et al. Mephedrone, compared with MDMA (ecstasy) and amphetamine, rapidly increases both dopamine and 5-HT levels in nucleus accumbens of awake rats. Br. J. Pharmacol. 164, 1949–1958 (2011).

Kerrigan, S., Savage, M., Cavazos, C. & Bella, P. Thermal Degradation of Synthetic Cathinones: Implications for Forensic Toxicology. J. Anal. Toxicol. 40, 1–11 (2016).

Khreit, O. I. G. et al. Elucidation of the Phase I and Phase II metabolic pathways of (+/-)- 4’-methylmethcathinone (4-MMC) and (+/-)-4’-(trifluoromethyl)methcathinone (4-TFMMC) in rat liver hepatocytes using LC-MS and LC-MS(2). J. Pharm. Biomed. Anal. 72, 177–185 (2013).

Khroyan, T. V, Barrett-Larimore, R. L., Rowlett, J. K. & Spealman, R. D. Dopamine D1- and D2-like receptor mechanisms in relapse to cocaine-seeking behavior: effects of selective antagonists and agonists. J. Pharmacol. Exp. Ther. 294, 680–687 (2000).

107 Kimishima, A., Wenthur, C. J., Eubanks, L. M., Sato, S. & Janda, K. D. Cocaine Vaccine Development: Evaluation of Carrier and Adjuvant Combinations That Activate Multiple Toll-Like Receptors. Mol. Pharm. 13, 3884–3890 (2016).

Kimmel, H. L., Manvich, D. F., Blough, B. E., Negus, S. S. & Howell, L. L. Behavioral and neurochemical effects of amphetamine analogs that release monoamines in the squirrel monkey. Pharmacol. Biochem. Behav. 94, 278–284 (2009).

Kimura, A. et al. Overexpression of 5-HT2C receptors in forebrain leads to elevated anxiety and hypoactivity. Eur. J. Neurosci. 30, 299–306 (2009).

King, H. E. et al. Sex Differences in 3,4-methylenedioxypyrovalerone (MDPV)-induced Taste Avoidance and Place Preferences. Pharmacol. Biochem. Behav. 137, 16–22 (2015a).

King, H. E., Wetzell, B., Rice, K. C. & Riley, A. L. An assessment of MDPV-induced place preference in adult Sprague-Dawley rats. Drug Alcohol Depend. 146, 116– 119 (2015b).

Kippin, T. E. et al. Potentiation of cocaine-primed reinstatement of drug seeking in female rats during estrus. Psychopharmacology (Berl). 182, 245–252 (2005).

Kohut, S. J. et al. Differential modulation of cocaine’s discriminative cue by repeated and variable stress exposure: relation to monoamine transporter levels. Neuropharmacology 63, 330–337 (2012).

Kolanos, R. et al. Stereoselective Actions of Methylenedioxypyrovalerone (MDPV) To Inhibit Dopamine and Norepinephrine Transporters and Facilitate Intracranial Self- Stimulation in Rats. ACS Chem. Neurosci. 6, 771–7 (2015).

Koob, G. F. Drugs of abuse: anatomy, pharmacology and function of reward pathways. Trends Pharmacol. Sci. 13, 177–184 (1992).

Koob, G. F. & Le Moal, M. Drug addiction, dysregulation of reward, and allostasis. Neuropsychopharmacology 24, 97–129 (2001).

Kosten, T. A., Gawin, F. H., Kosten, T. R. & Rounsaville, B. J. Gender differences in cocaine use and treatment response. J. Subst. Abuse Treat. 10, 63–66 (1993).

Kosten, T. R. & Domingo, C. B. Can you vaccinate against substance abuse? Expert Opin. Biol. Ther. 13, 1093–1097 (2013).

Kroeze, W. K. et al. PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Nat. Struct. Mol. Biol. 22, 362–369 (2015).

108 Kupferschmidt, D. A., Tribe, E. & Erb, S. Effects of repeated yohimbine on the extinction and reinstatement of cocaine seeking. Pharmacol. Biochem. Behav. 91, 473–480 (2009).

Laurent, K. et al. Synthetic Cathinones: A New Public Health Problem. Curr Neuropharmacol 13, 12–20 (2015).

Laporte, A. M. et al. Quantitative autoradiographic mapping of 5-HT3 receptors in the rat CNS using [125I]iodo- and [3H]zacopride as radioligands. Synapse 10, 271–281 (1992).

Li, C. & Kirby, L. G. Effects of cocaine history on postsynaptic GABA receptors on dorsal raphe serotonin neurons in a stress-induced relapse model in rats. Eur. Neuropsychopharmacol. 26, 45–54 (2016).

Li, L., Everhart, T., Jacob Iii, P., Jones, R. & Mendelson, J. Stereoselectivity in the human metabolism of methamphetamine. Br. J. Clin. Pharmacol. 69, 187–192 (2010).

Li, Y., White, F. J. & Wolf, M. E. Pharmacological reversal of behavioral and cellular indices of cocaine sensitization in the rat. Psychopharmacology (Berl). 151, 175– 183 (2000).

Liechti, M. E. [‘Ecstasy’ (MDMA): pharmacology, toxicology, and treatment of acute intoxication]. Dtsch. Med. Wochenschr. 128, 1361–1366 (2003).

Liechti, M. E., Kunz, I. & Kupferschmidt, H. Acute medical problems due to Ecstasy use. Case-series of emergency department visits. Swiss Med. Wkly. 135, 652–657 (2005).

Lipari, R. N., Hedden, S. L. & Hughes, A. in (2013).

Lisek, R. et al. Mephedrone (’bath salt’) elicits conditioned place preference and dopamine-sensitive motor activation. Drug Alcohol Depend. 126, 257–262 (2012).

Liu, J. et al. Acute administration of leptin produces anxiolytic-like effects: a comparison with fluoxetine. Psychopharmacology (Berl). 207, 535–545 (2010).

Lopez-Arnau, R., Martinez-Clemente, J., Pubill, D., Escubedo, E. & Camarasa, J. Comparative neuropharmacology of three psychostimulant cathinone derivatives: butylone, mephedrone and methylone. Br. J. Pharmacol. 167, 407–420 (2012).

Lynch, W. J. & Taylor, J. R. Sex differences in the behavioral effects of 24-h/day access to cocaine under a discrete trial procedure. Neuropsychopharmacology 29, 943– 951 (2004).

109 Madayag, A. et al. Drug-induced plasticity contributing to heightened relapse susceptibility: neurochemical changes and augmented reinstatement in high-intake rats. J. Neurosci. 30, 210–217 (2010).

Mancino, M. J. et al. Clinical efficacy of sertraline alone and augmented with gabapentin in recently abstinent cocaine-dependent patients with depressive symptoms. J. Clin. Psychopharmacol. 34, 234–239 (2014).

Marinetti, L. J. & Antonides, H. M. Analysis of synthetic cathinones commonly found in bath salts in human performance and postmortem toxicology: Method development, drug distribution and interpretation of results. J. Anal. Toxicol. 37, 135–146 (2013).

Martell, B. A., Mitchell, E., Poling, J., Gonsai, K. & Kosten, T. R. Vaccine pharmacotherapy for the treatment of cocaine dependence. Biol. Psychiatry 58, 158–164 (2005).

Martin, J. R., Ballard, T. M. & Higgins, G. A. Influence of the 5-HT2C receptor antagonist, SB-242084, in tests of anxiety. Pharmacol. Biochem. Behav. 71, 615– 625 (2002).

Martinez-Clemente, J. et al. Mephedrone pharmacokinetics after intravenous and oral administration in rats: relation to pharmacodynamics. Psychopharmacology (Berl). 229, 295–306 (2013).

Marusich, J. A., Grant, K. R., Blough, B. E. & Wiley, J. L. Effects of synthetic cathinones contained in ‘bath salts’ on motor behavior and a functional observational battery in mice. Neurotoxicology 33, 1305–1313 (2012).

Mattingly, B. A., Hart, T. C., Lim, K., & Perkins, C. Selective antagonism of dopamine D1 and D2 receptors does not block the development of behavioral sensitization to cocaine. Psychopharmacology (Berl) 114, 239- 242 (1994).

McClung, J., Fantegrossi, W. & Howell, L. L. Reinstatement of extinguished amphetamine self-administration by 3,4-methylenedioxymethamphetamine (MDMA) and its enantiomers in rhesus monkeys. Psychopharmacology (Berl). 210, 75–83 (2010).

McFarland, K., Lapish, C. C. & Kalivas, P. W. Prefrontal glutamate release into the core of the nucleus accumbens mediates cocaine-induced reinstatement of drug-seeking behavior. J. Neurosci. 23, 3531–3537 (2003).

Merluzzi, A. P. et al. Age-dependent MDPV-induced taste aversions and thermoregulation in adolescent and adult rats. Dev. Psychobiol. 56, 943–954 (2014).

110 Meyer, M. R., Du, P., Schuster, F. & Maurer, H. H. Studies on the metabolism of the -pyrrolidinophenone designer drug methylenedioxy-pyrovalerone (MDPV) in rat and human urine and human liver microsomes using GC-MS and LC-high- resolution MS and its detectability in urine by GC-MS. J. Mass Spectrom. 45, 1426–1442 (2010a).

Meyer, M. R., Wilhelm, J., Peters, F. T. & Maurer, H. H. Beta-keto amphetamines: studies on the metabolism of the designer drug mephedrone and toxicological detection of mephedrone, butylone, and methylone in urine using gas chromatography-mass spectrometry. Anal. Bioanal. Chem. 397, 1225–1233 (2010b).

Meyers, K. et al. The availability and depiction of synthetic cathinones (bath salts) on the Internet: Do online suppliers employ features to maximize purchases? Int. J. Drug Policy 26, 670–674 (2015).

Mierzejewski, P., Koros, E., Goldberg, S. R., Kostowski, W. & Stefanski, R. Intravenous self-administration of and cocaine: a comparative study. Pol. J. Pharmacol. 55, 713–726 (2003).

Millan, M. J., Brocco, M., Gobert, A. & Dekeyne, A. Anxiolytic properties of , an antidepressant with melatoninergic and serotonergic properties: role of 5-HT2C receptor blockade. Psychopharmacology (Berl). 177, 448–458 (2005).

Millan, M. J. Serotonin 5-HT2C receptors as a target for the treatment of depressive and anxious states: focus on novel therapeutic strategies. Therapie 60, 441–460 (2005).

Miotto, K., Striebel, J., Cho, A. K. & Wang, C. Clinical and pharmacological aspects of bath salt use: a review of the literature and case reports. Drug Alcohol Depend. 132, 1–12 (2013).

Monti, J. M. & Jantos, H. The roles of dopamine and serotonin, and of their receptors, in regulating sleep and waking. Prog. Brain Res. 172, 625–646 (2008).

Motbey, C. P. et al. High levels of intravenous mephedrone (4-methylmethcathinone) self-administration in rats: neural consequences and comparison with methamphetamine. J. Psychopharmacol. 27, 823–836 (2013).

Motbey, C. P., Hunt, G. E., Bowen, M. T., Artiss, S. & McGregor, I. S. Mephedrone (4- methylmethcathinone, ‘meow’): acute behavioural effects and distribution of Fos expression in adolescent rats. Addict. Biol. 17, 409–422 (2012).

Moya, P. R. et al. Altered 5-HT2C receptor agonist-induced responses and 5-HT2C receptor RNA editing in the amygdala of serotonin transporter knockout mice. BMC Pharmacol. 11, 3 (2011).

111 Murnane, K. S., Murai, N., Howell, L. L. & Fantegrossi, W. E. Discriminative stimulus effects of psychostimulants and hallucinogens in S(+)-3,4- methylenedioxymethamphetamine (MDMA) and R(-)-MDMA trained mice. J. Pharmacol. Exp. Ther. 331, 717–723 (2009).

Murray, B. L., Murphy, C. M. & Beuhler, M. C. Death following recreational use of designer drug ‘bath salts’ containing 3,4-Methylenedioxypyrovalerone (MDPV). J. Med. Toxicol. 8, 69–75 (2012).

Nair, S. G., Furay, A. R., Liu, Y. & Neumaier, J. F. Differential effect of viral overexpression of nucleus accumbens shell 5-HT1B receptors on stress- and cocaine priming-induced reinstatement of cocaine seeking. Pharmacol. Biochem. Behav. 112, 89–95 (2013).

Navailles, S., Moison, D., Ryczko, D. & Spampinato, U. Region-dependent regulation of mesoaccumbens dopamine neurons in vivo by the constitutive activity of central serotonin2C receptors. J. Neurochem. 99, 1311–1319 (2006).

Navailles, S., Moison, D., Cunningham, K. A. & Spampinato, U. Differential regulation of the mesoaccumbens dopamine circuit by serotonin2C receptors in the ventral tegmental area and the nucleus accumbens: an in vivo microdialysis study with cocaine. Neuropsychopharmacology 33, 237–246 (2008).

Neisewander, J. L. & Acosta, J. I. Stimulation of 5-HT2C receptors attenuates cue and cocaine-primed reinstatement of cocaine-seeking behavior in rats. Behav. Pharmacol. 18, 791–800 (2007).

Nguyen, J. D. et al. Locomotor Stimulant and Rewarding Effects of Inhaling Methamphetamine, MDPV, and Mephedrone via Electronic Cigarette-Type Technology. Neuropsychopharmacology 41, 2759–2771 (2016a).

Nguyen, J. D., Grant, Y., Creehan, K. M., Vandewater, S. A. & Taffe, M. A. Escalation of intravenous self-administration of methylone and mephedrone under extended access conditions. Addict. Biol. (2016b).

Nic Dhonnchadha, B. A., Fox, R. G., Stutz, S. J., Rice, K. C. & Cunningham, K. A. Blockade of the serotonin 5-HT2A receptor suppresses cue-evoked reinstatement of cocaine-seeking behavior in a rat self-administration model. Behav. Neurosci. 123, 382–396 (2009).

Novellas, J. et al. Concentrations of MDPV in rat striatum correlate with the psychostimulant effect. J. Psychopharmacol. 29, 1209–18 (2015).

O’Connor, E. C., Chapman, K., Butler, P. & Mead, A. N. The predictive validity of the rat self-administration model for abuse liability. Neurosci. Biobehav. Rev. 35, 912– 938 (2011).

112 O’Malley, K. L., Harmon, S., Tang, L. & Todd, R. D. The rat dopamine D4 receptor: sequence, gene structure, and demonstration of expression in the cardiovascular system. New Biol. 4, 137–146 (1992).

Oades, R. D. & Halliday, G. M. Ventral tegmental (A10) system: neurobiology. 1. Anatomy and connectivity. Brain Res. 434, 117–165 (1987).

Ogbonmwan, Y. E., Schroeder, J. P., Holmes, P. V & Weinshenker, D. The effects of post-extinction exercise on cocaine-primed and stress-induced reinstatement of cocaine seeking in rats. Psychopharmacology (Berl). 232, 1395–1403 (2015).

Onaivi, E. S., Bishop-Robinson, C., Darmani, N. A. & Sanders-Bush, E. Behavioral effects of (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, (DOI) in the elevated plus-maze test. Life Sci. 57, 2455–2466 (1995).

Opacka-Juffry, J. et al. Stimulant mechanisms of cathinones - effects of mephedrone and other cathinones on basal and electrically evoked dopamine efflux in rat accumbens brain slices. Prog. Neuropsychopharmacol. Biol. Psychiatry 54, 122– 130 (2014).

Pail, P. B., Costa, K. M., Leite, C. E. & Campos, M. M. Comparative pharmacological evaluation of the cathinone derivatives, mephedrone and methedrone, in mice. Neurotoxicology 50, 71–80 (2015).

Paine, T. A., Jackman, S. L. & Olmstead, M. C. Cocaine-induced anxiety: alleviation by diazepam, but not , or . Behav. Pharmacol. 13, 511–523 (2002).

Palvimaki, E. P. et al. Interactions of selective serotonin reuptake inhibitors with the serotonin 5-HT2c receptor. Psychopharmacology (Berl). 126, 234–240 (1996).

Panksepp, J. & Burgdorf, J. 50-kHz chirping (laughter?) in response to conditioned and unconditioned tickle-induced reward in rats: effects of social housing and genetic variables. Behav. Brain Res. 115, 25–38 (2000).

Parsons, L. H., Koob, G. F. & Weiss, F. Extracellular serotonin is decreased in the nucleus accumbens during withdrawal from cocaine self-administration. Behav. Brain Res. 73, 225–228 (1996).

Patel, J. G., Bartoszyk, G. D., Edwards, E. & Ashby, C. R. The Highly Selective 5- Hydroxytryptamine (5-HT)2A Receptor Antagonist, EMD 281014, Significantly Increases Swimming and Decreases Immobility in Male Congenital Learned Helpless Rats in the Forced Swim Test. Synapse 52, 73–75 (2004).

113 Pedersen, A. J., Reitzel, L. A., Johansen, S. S. & Linnet, K. In vitro metabolism studies on mephedrone and analysis of forensic cases. Drug Test. Anal. 5, 430–438 (2013).

Pehek, E. A., McFarlane, H. G., Maguschak, K., Price, B. & Pluto, C. P. M100,907, a selective 5-HT(2A) antagonist, attenuates dopamine release in the rat medial prefrontal cortex. Brain Res. 888, 51–59 (2001).

Pehek, E. A., Nocjar, C., Roth, B. L., Byrd, T. A. & Mabrouk, O. S. Evidence for the preferential involvement of 5-HT2A serotonin receptors in stress- and drug- induced dopamine release in the rat medial prefrontal cortex. Neuropsychopharmacology 31, 265–277 (2006).

Penders, T. M. & Gestring, R. Hallucinatory delirium following use of MDPV: ‘Bath Salts’. Gen. Hosp. Psychiatry 33, 525–526 (2011).

Perrine, S. A., Sheikh, I. S., Nwaneshiudu, C. A., Schroeder, J. A. & Unterwald, E. M. Withdrawal from chronic administration of cocaine decreases delta opioid receptor signaling and increases anxiety- and depression-like behaviors in the rat. Neuropharmacology 54, 355–364 (2008).

Pessia, M., Jiang, Z. G., North, R. A. & Johnson, S. W. Actions of 5-hydroxytryptamine on ventral tegmental area neurons of the rat in vitro. Brain Res. 654, 324–330 (1994).

Petit-Demouliere, B., Masse, F., Cogrel, N., Hascoet, M. & Bourin, M. Brain structures implicated in the four-plate test in naive and experienced Swiss mice using injection of diazepam and the 5-HT2A agonist DOI. Behav. Brain Res. 204, 200– 205 (2009).

Pierce, R. C. & Kumaresan, V. The mesolimbic dopamine system: the final common pathway for the reinforcing effect of drugs of abuse? Neurosci. Biobehav. Rev. 30, 215–238 (2006).

Plosker, G. L. Acamprosate: A Review of Its Use in Alcohol Dependence. Drugs 75, 1255–1268 (2015).

Pockros, L. A., Pentkowski, N. S., Swinford, S. E. & Neisewander, J. L. Blockade of 5- HT2A receptors in the medial prefrontal cortex attenuates reinstatement of cue- elicited cocaine-seeking behavior in rats. Psychopharmacology (Berl). 213, 307– 320 (2011).

Pomara, C. et al. Data available on the extent of cocaine use and dependence: biochemistry, pharmacologic effects and global burden of disease of cocaine abusers. Curr. Med. Chem. 19, 5647–5657 (2012).

114 Prosser, J. M. & Nelson, L. S. The Toxicology of Bath Salts: A Review of Synthetic Cathinones. J. Med. Toxicol. 8, 33–42 (2012).

Qu, Y. et al. Pharmacokinetics and pharmacodynamics of norfluoxetine in rats: Increasing extracellular serotonin level in the frontal cortex. Pharmacol. Biochem. Behav. 92, 469–473 (2009).

Quesseveur, G., Nguyen, H. T., Gardier, A. M. & Guiard, B. P. 5-HT2 ligands in the treatment of anxiety and depression. Expert Opin. Investig. Drugs 21, 1701–1725 (2012).

Ramoz, L. et al. Mephedrone (‘bath salt’) pharmacology: insights from invertebrates. Neuroscience 208, 79–84 (2012).

Ranaldi, R., Wang, Z. & Woolverton, W. L. Reinforcing effects of D2 agonists and partial agonists in rhesus monkeys. Drug Alcohol Depend. 64, 209– 217 (2001).

Randall, P. A., Jaramillo, A. A., Frisbee, S. & Besheer, J. The role of varenicline on alcohol-primed self-administration and seeking behavior in rats. Psychopharmacology (Berl). 232, 2443–2454 (2015).

Rankin, M.L., Hazelwood, L.A., Benjamin R, Namkung, F.Y., Rex, E.B., Roof, R.A., Sibley, D.R. Molecular pharmacology of the dopamine receptors, in Dopamine Handbook (Iversen LL, Dunnett SB, Iversen SD, and Bjorklund A ed) Oxford University Press, New York 63–87 (2010).

Ratzenboeck, E., Saria, A., Kriechbaum, N. & Zernig, G. Reinforcing effects of MDMA (‘ecstasy’) in drug-naive and cocaine-trained rats. Pharmacology 62, 138–144 (2001).

Ritz, M. C., Cone, E.J., Kuhar, M.J. Cocaine inhibition of ligand binding at dopamine, norepinephrine and serotonin transporters: A structure-activity study. Life Sci 46, 635–645 (1990).

Ritz, M. C., Lamb, R. J., Goldberg, S. R. & Kuhar, M. J. Cocaine receptors on dopamine transporters are related to self-administration of cocaine. Science 237, 1219–1223 (1987).

Robbins, S. J., Ehrman, R. N., Childress, A. R. & O’Brien, C. P. Comparing levels of cocaine cue reactivity in male and female outpatients. Drug Alcohol Depend. 53, 223–230 (1999).

Roberts, D. C. et al. Self-administration of cocaine analogs by rats. Psychopharmacology (Berl). 144, 389–397 (1999).

115 Robinson, E. S. J. et al. Opposing roles for 5-HT2A and 5-HT2C receptors in the nucleus accumbens on inhibitory response control in the 5-choice serial reaction time task. Neuropsychopharmacology 33, 2398–2406 (2008).

Robinson, J. E., Agoglia, A. E., Fish, E. W., Krouse, M. C. & Malanga, C. J. Mephedrone (4-methylmethcathinone) and intracranial self-stimulation in C57BL/6J mice: comparison to cocaine. Behav. Brain Res. 234, 76–81 (2012).

Rodgers, R. J. et al. Anxiogenic-like effects of and in the mouse elevated plus-maze: profile comparisons with 8-OH-DPAT, CGS 12066B, TFMPP and mCPP. Behav. Pharmacol. 3, 621–634 (1992).

Roni, M. A. & Rahman, S. Effects of lobeline and reboxetine, fluoxetine, or bupropion combination on depression-like behaviors in mice. Pharmacol. Biochem. Behav. 139, 1–6 (2015).

Rosenaum, C. D., Carreiro, S. P. & Babu, K. M. Here today, gone tomorrow...and back again? A review of herbal marijuana alternatives (K2, Spice), synthetic cathinones (bath salts), kratom, , , and . J. Med. Toxicol. 8, 15–32 (2012).

SAMHSA, The NSDUH Report: Substance Use and Mental Health Estimates from the 2013 National Survey on Drug Use and Health: Overview of Findings. (pp. 1–8) (2014).

Sawyer, E. K. et al. Neurobiological changes mediating the effects of chronic fluoxetine on cocaine use. Neuropsychopharmacology 37, 1816–1824 (2012).

Scardochio, T., Trujillo-Pisanty, I., Conover, K., Shizgal, P. & Clarke, P. B. S. The Effects of Electrical and Optical Stimulation of Midbrain Dopaminergic Neurons on Rat 50-kHz Ultrasonic Vocalizations. Front. Behav. Neurosci. 9, 331 (2015).

Schenk, S. & Gittings, D. Effects of SCH 23390 and eticlopride on cocaine-seeking produced by cocaine and WIN 35,428 in rats. Psychopharmacology (Berl). 168, 118–123 (2003).

Schindler, C. W. et al. Reinforcing and neurochemical effects of the ‘bath salts’ constituents 3,4-methylenedioxypyrovalerone (MDPV) and 3,4-methylenedioxy- N-methylcathinone (methylone) in male rats. Psychopharmacology (Berl). 233, 1981–1990 (2016).

Schindler, C. W., Thorndike, E. B., Suzuki, M., Rice, K. C. & Baumann, M. H. Pharmacological mechanisms underlying the cardiovascular effects of the ‘bath salt’ constituent 3,4-methylenedioxypyrovalerone (MDPV). Br. J. Pharmacol. 173, 3492–3501 (2016).

116 Schmidt, H. D., Anderson, S. M. & Pierce, R. C. Stimulation of D1-like or D2 dopamine receptors in the shell, but not the core, of the nucleus accumbens reinstates cocaine-seeking behaviour in the rat. Eur. J. Neurosci. 23, 219–228 (2006).

Schmitz, J.M., Averill, P., Stotts, A.L., Moeller, F.G., Rhoades, H.M., Grabowski, J. Fluoxetine treatment of cocaine-dependent patients with major depressive disorder. Drug Alcohol Depend. 63, 207-214 (2001).

Self, D. W., Barnhart, W. J., Lehman, D. A. & Nestler, E. J. Opposite modulation of cocaine-seeking behavior by D1- and D2-like dopamine receptor agonists. Science 271, 1586–1589 (1996).

Shortall, S. E. et al. Behavioural and neurochemical comparison of chronic intermittent cathinone, mephedrone and MDMA administration to the rat. Eur. Neuropsychopharmacol. 23, 1085–1095 (2013).

Shorter, D., Domingo, C. B. & Kosten, T. R. Emerging drugs for the treatment of cocaine use disorder: a review of neurobiological targets and pharmacotherapy. Expert Opin. Emerg. Drugs 20, 15–29 (2015).

Simmler, L. D., Hysek, C. M. & Liechti, M. E. Sex differences in the effects of MDMA (ecstasy) on plasma copeptin in healthy subjects. J. Clin. Endocrinol. Metab. 96, 2844–2850 (2011).

Simmler, L. D. et al. Pharmacological characterization of designer cathinones in vitro. Br. J. Pharmacol. 168, 458–470 (2013).

Simmons, S. J. et al. Comparing rewarding and reinforcing properties between ‘bath salt’ 3,4-methylenedioxypyrovalerone (MDPV) and cocaine using ultrasonic vocalizations in rats. Addict. Biol. (2016).

Sofuoglu, M., DeVito, E. E., Waters, A. J. & Carroll, K. M. Cognitive enhancement as a treatment for drug addictions. Neuropharmacology 64, 452–463 (2013).

Sokoloff, P. et al. The dopamine D3 receptor: a therapeutic target for the treatment of neuropsychiatric disorders. CNS Neurol. Disord. Drug Targets 5, 25–43 (2006).

Sorg, B. A., Li, N., Wu, W. & Bailie, T. M. Activation of dopamine D1 receptors in the medial prefrontal cortex produces bidirectional effects on cocaine-induced locomotor activity in rats: effects of repeated stress. Neuroscience 127, 187–196 (2004).

Sparago, M. et al. Neurotoxic and pharmacologic studies on enantiomers of the N- methylated analog of cathinone (methcathinone): a new drug of abuse. J. Pharmacol. Exp. Ther. 279, 1043–1052 (1996).

117 Spiller, H. A, Ryan, M. L., Weston, R. G. & Jansen, J. Clinical experience with and analytical confirmation of ‘bath salts’ and ‘legal highs’ (synthetic cathinones) in the United States. Clin. Toxicol. (Phila). 49, 499–505 (2011).

Steele, T. D., Nichols, D. E. & Yim, G. K. Stereochemical effects of 3,4- methylenedioxymethamphetamine (MDMA) and related amphetamine derivatives on inhibition of uptake of [3H]monoamines into synaptosomes from different regions of rat brain. Biochem. Pharmacol. 36, 2297–2303 (1987).

Steinbusch, H. W. Distribution of serotonin-immunoreactivity in the central nervous system of the rat-cell bodies and terminals. Neuroscience 6, 557–618 (1981).

Steketee, J. D. & Kalivas, P. W. Drug wanting: behavioral sensitization and relapse to drug-seeking behavior. Pharmacol. Rev. 63, 348–365 (2011).

Stogner, J. M. & Miller, B. L. Investigating the ‘bath salt’ panic: The rarity of synthetic cathinone use among students in the United States. Drug Alcohol Rev. 32, 545–549 (2013).

Strano-Rossi, S., Cadwallader, A. B., de la Torre, X. & Botrè, F. Toxicological determination and in vitro metabolism of the designer drug methylenedioxypyrovalerone (MPDV) by gas chromatography/mass spectrometry and liquid chromatography/quadrupole time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 24, 2706–2714 (2010).

Swanson, L. W. The projections of the ventral tegmental area and adjacent regions: a combined fluorescent retrograde tracer and immunofluorescence study in the rat. Brain Res. Bull. 9, 321–353 (1982).

Tamrazi, B. & Almast, J. Your Brain on Drugs: Imaging of Drug-related Changes in the Central Nervous System. Radiographics 32, 701–719 (2012).

Tanda, G., Ebbs, A., Newman, A. H. & Katz, J. L. Effects of 4’-chloro-3 alpha- (diphenylmethoxy)-tropane on mesostriatal, mesocortical, and mesolimbic dopamine transmission: comparison with effects of cocaine. J. Pharmacol. Exp. Ther. 313, 613–620 (2005).

Thompson, B., Leonard, K. C. & Brudzynski, S. M. Amphetamine-induced 50 kHz calls from rat nucleus accumbens: a quantitative mapping study and acoustic analysis. Behav. Brain Res. 168, 64–73 (2006).

Ujike, H., Akiyama, K. & Otsuki, S. D-2 but not D-1 dopamine agonists produce augmented behavioral response in rats after subchronic treatment with methamphetamine or cocaine. Psychopharmacology (Berl). 102, 459–464 (1990).

118 United Nations, Office on Drugs and Crime (2013). World Drug Report (pp. 1–113). Vienna. Retrieved from http://www.unodc.org/unodc/secured/wdr/wdr2013/ World_Drug_Report_2013.pdf

United Nations, Office on Drugs and Crime (2016). World Drug Report (pp. 1–174). Vienna. Retrieved from https://www.unodc.org/doc/wdr2016/WORLD_DRUG_ REPORT_2016_web.pdf

United States Department of Justice, D. E. A. (2011a). Federal Register /Vol. 76, No. 204/Friday, October 21, 2011/Rules and Regulations (Vol. 76, pp. 65371-65375). Retrieved from https://www.deadiversion.usdoj.gov/fed_regs/rules/2011/fr1021_3.htm

United States Department of Justice, D. E. A. (2011b). Synthetic Cathinones - DEA Request for Information. System (pp. 1–2). Retrieved from http://www.dea.gov/pr/micrograms/2011/mg0411.pdf

United States Department of Justice, D. E. A. (2017). Lists of: Scheduling Actions Controlled Substances Regulated Chemicals (pp. 1–78). Retrieved from https://www.deadiversion.usdoj.gov/schedules/orangebook/orangebook.pdf

Valente, M. J., Guedes de Pinho, P., de Lourdes Bastos, M., Carvalho, F. & Carvalho, M. Khat and synthetic cathinones: a review. Arch. Toxicol. 88, 15–45 (2014).

Vandewater, S. A., Creehan, K. M. & Taffe, M. A. Intravenous self-administration of entactogen-class stimulants in male rats. Neuropharmacology 99, 538–545 (2015).

Varner, K. J. et al. Comparison of the behavioral and cardiovascular effects of mephedrone with other drugs of abuse in rats. Psychopharmacology (Berl). 225, 675–685 (2013).

Vazquez-Gomez, E. et al. Bupropion-induced inhibition of alpha7 nicotinic acetylcholine receptors expressed in heterologous cells and neurons from dorsal raphe nucleus and hippocampus. Eur. J. Pharmacol. 740, 103–111 (2014).

Verrico, C. D., Miller, G. M. & Madras, B. K. MDMA (Ecstasy) and human dopamine, norepinephrine, and serotonin transporters: implications for MDMA-induced neurotoxicity and treatment. Psychopharmacology (Berl). 189, 489–503 (2007).

Volkow, N. D. et al. Relationship between subjective effects of cocaine and dopamine transporter occupancy. Nature 386, 827–830 (1997).

Vouga, A. et al. Stereochemistry and neuropharmacology of a ‘bath salt’ cathinone: S- enantiomer of mephedrone reduces cocaine-induced reward and withdrawal in invertebrates. Neuropharmacology 91, 109–116 (2015).

119 Waldmeier, P. C. & Delini-Stula, A. A. Serotonin--dopamine interactions in the nigrostriatal system. Eur. J. Pharmacol. 55, 363–373 (1979).

Walf, A. A. & Frye, C. A. The use of the elevated plus maze as an assay of anxiety- related behavior in rodents. Nat. Protoc. 2, 322–328 (2007).

Wang, Z. & Woolverton, W. L. Estimating the relative reinforcing strength of (+/-)-3,4- methylenedioxymethamphetamine (MDMA) and its isomers in rhesus monkeys: comparison to (+)-methamphetamine. Psychopharmacology (Berl). 189, 483–488 (2007).

Warner, E. A. Cocaine abuse. Ann. Intern. Med. 119, 226–235 (1993).

Watterson, L. R. et al. Potent rewarding and reinforcing effects of the synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV). Addict. Biol. 19, 165–174 (2014).

Wee, S. et al. Relationship between the serotonergic activity and reinforcing effects of a series of amphetamine analogs. J. Pharmacol. Exp. Ther. 313, 848–854 (2005).

Weiner, R. I. & Ganong, W. F. Role of brain monoamines and histamine in regulation of anterior pituitary secretion. Physiol. Rev. 58, 905–976 (1978).

Winstanley, E.L., Bigelow, G.E., Silverman, K., Johnson, R.E., Strain, E.C. A randomized controlled trial of fluoxetine in the treatment of cocaine dependence among methadone-maintained patients. Journal of substance abuse treatment. 40, 255-264 (2001).

Winstock, A. et al. Mephedrone: use, subjective effects and health risks. Addiction 106, 1991–1996 (2011).

Wintink, A. J. & Brudzynski, S. M. The related roles of dopamine and glutamate in the initiation of 50-kHz ultrasonic calls in adult rats. Pharmacol. Biochem. Behav. 70, 317–323 (2001).

Wong, W. C. & Marinelli, M. Adolescent-onset of cocaine use is associated with heightened stress-induced reinstatement of cocaine seeking. Addict. Biol. 21, 634– 645 (2016).

Wood, S., Sage, J. R., Shuman, T. & Anagnostaras, S. G. Psychostimulants and cognition: a continuum of behavioral and cognitive activation. Pharmacol. Rev. 66, 193–221 (2014).

Woolverton, W. L. Effects of a D1 and a D2 on the self- administration of cocaine and by rhesus monkeys. Pharmacol. Biochem. Behav. 24, 531–535 (1986).

120 Wright, J. M., Gourdon, J. C. & Clarke, P. B. S. Identification of multiple call categories within the rich repertoire of adult rat 50-kHz ultrasonic vocalizations: effects of amphetamine and social context. Psychopharmacology (Berl). 211, 1–13 (2010).

Wright, M. J. J. et al. Effect of ambient temperature on the thermoregulatory and locomotor stimulant effects of 4-methylmethcathinone in Wistar and Sprague- Dawley rats. PLoS One 7, e44652 (2012).

Yamawaki, S., Lai, H. & Horita, A. Dopaminergic and serotonergic mechanisms of thermoregulation: mediation of thermal effects of apomorphine and dopamine. J. Pharmacol. Exp. Ther. 227, 383–388 (1983).

Young, R. & Glennon, R. A. Discriminative stimulus effects of S(-)-methcathinone (CAT): a potent stimulant drug of abuse. Psychopharmacology (Berl). 140, 250– 256 (1998).

Zetterstrom, T., Sharp, T., Marsden, C. A. & Ungerstedt, U. In vivo measurement of dopamine and its metabolites by intracerebral dialysis: changes after d- amphetamine. J. Neurochem. 41, 1769–1773 (1983).

Zhao, W. & Becker, J. B. Sensitization enhances acquisition of cocaine self- administration in female rats: estradiol further enhances cocaine intake after acquisition. Horm. Behav. 58, 8–12 (2010).

Zhou, L. et al. Orexin-1 receptor mediation of cocaine seeking in male and female rats. J. Pharmacol. Exp. Ther. 340, 801–809 (2012).

Zimmerman, J. L. Cocaine intoxication. Crit. Care Clin. 28, 517–526 (2012).

121