CHARACTERIZING THE PHARMACOLOGICAL PROFILE OF AND DETERMINING THE ABUSE LIABILITY MECHANISMS

A Dissertation Submitted to the Temple University Graduate Board

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

by Iman A. Saber December 2017

Examining Committee Members:

Ellen Walker, Advisory Chair, Department of Pharmaceutical Sciences Wayne Childers, Department of Pharmaceutical Sciences Ellen Unterwald, Department of , Center for Substance Abuse and Research Sarah Jane Ward, Department of Pharmacology, Center for Substance Abuse and Research Scott Rawls, Department of Pharmacology, Center for Substance Abuse and Research

© Copyright 2017

by

Iman A. Saber

All Rights Reserved

! ii! ABSTRACT

Illicit use has been a growing concern over the past few decades. The rise in use of illegal drove the government and law enforcement to aggressively tackle this problem and crackdown on the illicit use of drugs. However, this sparked a further interest in ‘legal highs.’ Before 2011, among the newly popular ‘legal highs’ was ‘Bath

Salts.’ is a monoamine alkaloid and the active ingredient found in the leaves of the khat plant. The psychoactive form of bath salts may contain a mixture of synthesized , including, 4-methyl-N- (mephedrone), 3,4-methylenedioxy-N- methylcathinone () and methylenedioxypyrovalerone (MDPV). These three are commonly found in bath salts. One of the major psychoactive ingredients in bath salts is mephedrone. Mephedrone grew in popularity due to its low price, accessibility, and the shortage of MDMA, thus making mephedrone the prime drug to sell as a ‘legal high’ up until 2011 when it became banned in the United States.

Before 2012, most of the studies focused on the identification and clinical case reports of mephedrone. During the recent years, other preclinical studies supported the notion that mephedrone might have strong abuse liability and may lead to addiction. The shortage of information about the pharmacological mechanism of the novel drug mephedrone present in preclinical observations inhibits the ability of law enforcement and health care personnel to tackle the problems of its misuse. With this in mind, the overarching intention of this dissertation is to characterize the pharmacological mechanism of mephedrone and further determine the mechanisms that are involved in its abuse liability. Specifically, we want to evaluate the contribution of and

! iii! serotonin to mephedrone’s discriminative and reinforcing effects and drug-seeking behavior in male Sprague Dawley rats.

We first established two doses of mephedrone as discriminative stimuli in drug naïve rats and tested the capacity of various dopaminergic and serotonergic agonists to generalize to 0.5 mg/kg or 3.2 mg/kg mephedrone. We then examined the capacity of dopamine and serotonin receptor antagonists to attenuate the discriminative effects of the training doses 0.5 and 3.2 mg/kg mephedrone. We were able to successfully establish both a low and high dose of mephedrone as discriminative stimuli in male rats. 3,4- methylenedioxymethamphetamine (MDMA) was the only drug to fully substitute for both 0.5 and 3.2 mg/kg mephedrone, while cocaine, , d- and, 2,5-dimethoxy-4-iodoamphetamine (DOI) fully substituted for the low training dose of 0.5 mg/kg mephedrone. The D1 receptor antagonist, SCH23390, was able to significantly attenuate the discriminative stimulus effects of 0.5 mg/kg mephedrone while significantly decreasing the response rates, and the D2/3 receptor antagonist, sulpiride, significantly attenuated the discriminative stimulus effects of 0.5 mg/kg mephedrone while significantly increasing the response rates. The 5-HT2C receptor antagonist,

SB242084, significantly increased the response rates of 0.5 mg/kg mephedrone.

SCH23390 significantly disrupted response rates for 3.2 mg/kg mephedrone and substitution effects could not be measured.

Next, we examined the capacity of mephedrone to reinstate cocaine responding and support self-administration. We established a history of self-administering 0.375 mg/kg/infusion cocaine in rats for 14 days. Rats then went through a period of extinction and were reinstated with mephedrone, methamphetamine, cocaine, or saline. We then

! iv! determined if rats were able to develop a mephedrone self-administering behavior when a history of cocaine self-administration was already established. We also used a D1 receptor antagonist, SCH23390, to inhibit this mephedrone seeking behavior.

Mephedrone and saline did not fully reinstate the extinguished cocaine-seeking behavior; however, methamphetamine and cocaine did. Mephedrone was able to substitute and maintain the cocaine-seeking behavior. A moderate dose of 0.01 mg/kg SCH23390, antagonized the reinforcing effect of cocaine, but not the reinforcing effects of mephedrone, although the responses of individual rats highly suggest a trend in that direction.

Overall, the experiments presented here suggests that mephedrone has a pharmacological mechanism that is more similar to MDMA than cocaine.

! v!

This work is dedicated to my sweet, sweet children. For all

of the happiness they brought to my life, no matter how short-lived it was.

So that my future children may know how hard their mother worked and to know that

absolutely no dream is ever too big to achieve. Also to my husband, who always

challenges me and pushes me to become just a little bit better than I was before.

! vi! ACKNOWLEDGMENTS

The completion of this Ph.D. degree would not be possible without the support and guidance of specific people.

I am and will be forever grateful to have Dr. Ellen Walker as an advisor and a mentor. Throughout this long and tiring journey, she has been very patient with me.

Through her careful guidance, I attained the ability to be independent and the ability to objectively evaluate my research. Watching her over the years, I learned how to effectively and respectively maneuver through the scientific community as a woman. I am also thankful for her endless critique of my scientific writing. Throughout the years, she spent endless hours correcting my writings and consistently allowed anything that I wrote to be my own work. It has been such an honor to be the understudy of someone who is very respected within the field of behavioral pharmacology. Her immense wealth of knowledge will forever serve as a reservoir for me.

I would like to thank my dissertation committee for all of their academic and scientific guidance. My research was highly influenced by the collaborative inputs of

Dr. Ellen Walker, Dr. Wayne Childers, Dr. Sarah Jane Ward, Dr. Ellen Unterwald, and

Dr. Scott Rawls. I always enjoyed the stimulating discussions we had after the presentation of my data and I actually looked forward to them.

I would like to give a special thank you to Dr. Sarah Jane Ward for training me from day one. She was the first person who showed me how to properly handle both mice and rats. She also trained me in drug-discrimination and self-administration, the two behavioral assays that encompassed my graduate research. When I was performing the self-administration studies, she was always eager to answer any of my questions, correct

! vii! my many experimental design issues, and made me really excited about reviewing my results, no matter how confounded they were.

I would like to thank my fellow graduate students and lab mates Dr. Harshini

Neelakantan and Rajesh Sanku for being easy to work around and for being daily, encouraging reminders that graduate students are not robots and still have the ability to socialize. I have to especially thank Dr. Neelakantan for showing me around the lab early on and teaching me the basics of laboratory etiquette. Without knowing it, she taught me what it meant to be a graduate student and showed me how to be an independent and dedicated scientist. I would like to thank the previous animal caretaker of the Pharmacy

School, Beatrice, for taking such good care of my research animals throughout the years.

Also for our daily conversations and the laughs that we shared.

I must acknowledge and thank the Pharmaceutical Department of the Pharmacy

School. I could not have chosen a better department to be apart of and a better environment to flourish in. I would like to thank AlMira Cutler for always taking care of any administrative assistance that I needed and being willing to answer any question that

I had, no matter how irrelevant it was; if she was not able to answer it, she always pointed me in the direction of the person that could. I would like to thank Dr. Daniel Canney of the Pharmacy School and Dr. Zebulon Kendrick of Temple University Graduate

Department for financially supporting me over the years.

I would like to thank my family and especially my dad, for always inquiring about how I am doing in school and for sacrificing so much of himself to ensure my success and happiness. I thank him for instilling in me, from an early age, the importance of a secured education. Most importantly, I would like to thank my husband, future Dr. David

! viii! Burgess, for being by my side through this whole journey. Getting married in the middle of my graduate career was not easy, but it was the best decision I have ever made. He unselfishly endured my long nights studying and me spending every weekend at the lab, without any complaint. He spent countless hours listening to me practice my presentations and explaining my work, even though he had work of his own to focus on.

Over the years of me being a graduate student, he was the stone that made me sharper and the little hint of light than made me shine brighter.

Lastly, I would have to give thanks to God, for without my faith, I would not have made it this far.

! ix! TABLE OF CONTENTS Page ABSTRACT...... iii DEDICATION...... vi ACKNOWLEDGEMENTS...... vii LIST OF TABLES...... xiii LIST OF FIGURES...... xiv CHAPTER 1. INTRODUCTION…………………………………...... 1 Drug Abuse..……...... 1 Bath Salts…….……...………………...... 2 Mephedrone….……...………………...... 3 Neuropharmacology of Addiction….……...……………...... 5 Animal Models of Addiction……...…………...... 6 Drug Discrimination...... 6 Self-Administration………...... 7 Conditioned Place Preference...... 8 Rationale...... 10 Current Aims...... 12 2. EXPERIMENT 1: DRUG DISCRIMINATION...... 14 Drug Discrimination………………...... 14 Rationale……………………………...... 15 Experiment 1A: Low Dose Training Group…………...... 21 Materials and Methods………...... 21 Subjects………………………...... 21 Drugs…………………………...... 22 Apparatus….…………………...... 24 Procedure…………………...... 24 Two-Choice Drug Discrimination Training...... 24 Two-Choice Drug Discrimination Testing...... 26 Data Analysis...………………………...... 26 Results……...... ………………………...... 27 0.5 mg/kg Mephedrone Training...... 27 Low Training Dose-Dopamine Agonist Substitution Tests...... 31 Low Training Dose-Serotonin Agonist Substitution Tests...... 31 Low Training Dose-Dopamine Receptor Antagonist Tests……...33 Low Training Dose-Serotonin Receptor Antagonist Tests……....36 Experiment 1B: High Dose Training Group…………...... 39 Materials and Methods………...... 39

! x! Subjects………………………...... 39 Drugs…………………………...... 39 Apparatus….…………………...... 40 Procedure…………………...... 40 Two-Choice Drug Discrimination Training...... 40 Two-Choice Drug Discrimination Testing...... 41 Data Analysis...………………………...... 42 Results……...... ………………………...... 43 3.2 mg/kg Mephedrone Training...... 43 High Training Dose-Dopamine Agonist Substitution Tests...... 45 High Training Dose-Serotonin Agonist Substitution Tests...... 45 High Training Dose-Dopamine Receptor Antagonist Tests……..47 High Training Dose-Serotonin Receptor Antagonist Tests……...47 Experiment 1 Discussion…………………...... 53 3. EXPERIMENT 2: SELF-ADMINISTRATION...... 61 Self-Administration..………………...... 61 Rationale……………………………...... 62 Experiment 2A: Reinstatement Effects of Mephedrone on Cocaine Trained Rats…..………………………………...……..66 Materials and Methods………...... 66 Subjects………………………...... 66 Drugs…………………………...... 67 Operant Self-Administration Chambers...... 68 Procedure…………………...... 69 Intravenous Catheter Implantation………...... 69 Overall Self-Administration Strategy Procedure...... 70 Cocaine Acquisition Phase………………...... 71 Extinction Phase…………………………...... 72 Reinstatement Phase……………………...... 72 Blood Collection…………………………...... 72 Sample Preparation and UPLC/MS Method………...... 73 Data Analysis...………………………...... 74 Results……...... ………………………...... 75 Experiment 2B: High Dose Training Group…………...... 80 Materials and Methods………...... 80 Subjects………………………...... 80 Drugs…………………………...... 81 Operant Self-Administration Chambers...... 81 Procedure…………………...... 82 Intravenous Catheter Implantation………...... 82 Overall Self-Administration Strategy Procedure...... 82 Self-Administration Cocaine Acquisition Phase…………...... 82 Self-Administration Drug Substitution Phase…...... 83 Self-Administration Antagonist Phase………...... 84 Data Analysis...………………………...... 84

! xi! Results……...... ………………………...... 85 Experiment 2 Discussion…………………...... 87 4. CONCLUSIONS……………………...... 95 Involvement of DA and 5-HT in Reward...... 95 TAAR1 and Regulation of Dopamine and Serotonin...... 98 Summary of Findings………………...... 99 Limitations and Future Directions…...... 104 Final Remarks………………………...... 106 REFERENCES CITED...... 108

! xii! LIST OF TABLES Table Page

1. Number of Rats That ‘Fully’ Reinstated During Reinstatement Phase……………………………………………………………...... 78

2. Concentration of Mephedrone Found in Plasma……………………………..…..….79

! xiii! LIST OF FIGURES Figure Page

1. Mephedrone Potency Comparison………………………………….………………..28

2. Low Training Dose Dopamine Agonist Substitution Tests………………..….....…..30

3. Low Training Dose Serotonin Agonist Substitution Tests.……..……...... 32

4. Low Training Dose DA1 Receptor Antagonist Tests.………………………………..34

5. Low Training Dose DA2/3 Receptor Antagonist Tests ………………………………35

6. Low Training Dose 5-HT2C Receptor Antagonist Tests.…………………………….37

7. Low Training Dose 5-HT2 Receptor Antagonist Tests.……………………………...38

8. High Training Dose Dopamine Agonist Substitution Tests………………..………..44

9. High Training Dose Serotonin Agonist Substitution Tests.……..……...... 46

10. High Training Dose DA1 Receptor Antagonist Tests ……………………………….49

11. High Training Dose DA2/3 Receptor Antagonist Tests ……………………………...50

12. High Training Dose 5-HT2C Receptor Antagonist Tests ……………………………51

13. High Training Dose 5-HT2 Receptor Antagonist Tests.……………………………..52

14. Cocaine Dose Substitution……………………………..…………………………….76

15. Reinstatement of Extinguished Drug-Seeking Behavior………..………………..….78

16. Concentration of Mephedrone Found in Plasma……………..……………………...80

! xiv! 17. Mephedrone Dose Substitution for Cocaine Self-Administration ……………...…...86

18. Dose Antagonism of SCH23390 of Drug Self-Administration……………………...87

! xv! CHAPTER 1

INTRODUCTION

Drug Abuse

Illicit drug use continues to be a growing concern over the past few decades.

Recent reports from the National Survey on Drug Use and Health suggest that, during the past two years, over 27 million people, 12 years of age or older, used illicit drugs

(SAMHSA, 2015; SAMHSA, 2016). This equates to approximately 10.1% of the population, an increase from the 9.4% of the population in 2013 (SAMHSA, 2014). Out of the 27 million people who used illicit drugs, most of the users were between the ages of 18 to 25 and prescription and cocaine were second and third most commonly misused drugs (SAMHSA, 2016).

The increase of illegal drug use has measurable effects on the social and economic structure. The rise in use of illegal drugs drove the government and law enforcement to respond aggressively and sparked new campaigns to tackle this problem, hence, the ‘war on drugs’ (DPA, 2017). This legal crackdown reaction sparked a new alternative to more popular illegal drugs. Drugs that are not illegal, but still have mood- altering properties are commonly referred to as ‘legal highs’. These drugs are commonly used as alternatives and/or replacements for illegal drugs. Before 2011, among the newly popular ‘legal highs’ was ‘Bath Salts.’

! 1! Bath Salts

Cathinone is a monoamine alkaloid and the active ingredient found in the leaves of the khat plant, Catha edulis. Traditionally, the leaves of this plant are chewed and the organic compound cathinone is released. For centuries, chewing khat leaves has been a social custom for those living in the Middle East and Eastern Africa (Abdelwahab et al.,

2015; Bongard et al., 2015). Recently, synthetic cathinones have been marketed for ‘legal highs’ and sold under alias names, such as ‘plant food’, ‘jewelry cleaner’ or ‘bath salts.’

‘Bath Salts’ are a collection of designer drugs that are commonly formulated by clandestine chemists. Although the complete ingredients in bath salts differ from lab to lab, they commonly contain one or more of three main psychoactive synthesized cathinones: 4-methyl-N-methcathinone (mephedrone), 3,4-methylenedioxy-N- methylcathinone (methylone) and methylenedioxypyrovalerone (MDPV) (Murphy et al.,

2013; Prosser and Nelson, 2012).

Users of bath salts report intranasal as the most common route of administration while other less common routes include oral and inhalation (Terry, 2014). Users describe the effects of bath salts to be similar to , in particular, cocaine and a low- dose of 3,4-methylenedioxymethamphetamine (MDMA). They attribute bath salts to be a and slightly euphoric, but after binging behaviors, they begin to experience hallucinations, feelings of psychosis and increased cardiovascular rates (Anonymous,

2011; CanadianBakin, 2012; Johnson and Johnson, 2014; Morrissey, 2012). Bath salts also induces panic attacks, increased sociability and sex drive, suicidal ideals, hyperthermia, vasoconstriction, muscle tremor and spasms, and may lead to overdose and even death (Ross et al., 2011; NIDA, 2016).

! 2! Mephedrone

One of the major psychoactive ingredients in bath salts is mephedrone. Saem de

Burnaga first synthesized mephedrone in 1929 (Karila et al., 2015; Saem de Burnaga

Sanchez, 1929), however, it was not considered a publicly known drug until its reemergence in 2003. On the streets, mephedrone may go by multiple names, such as ‘m- cat,’ ‘meow-meow,’ or ‘drone.’(Carhart-Harris et al., 2011; Weaver et al., 2015).

Mephedrone grew in popularity due to its low price, accessibility, and the shortage of

MDMA, thus making mephedrone the prime drug to sell as a ‘legal high’ up until 2010

(Brunt et al., 2011; Papaseit et al., 2016; Prosser and Nelson, 2012). In 2009, the UK witnessed a spike in mephedrone use, largely due to its cheap price and easy access through the Internet. By March 2011, the International Narcotics Control Board (2011) reported mephedrone to be used recreationally throughout major continents, including

North America, Europe and Australia. Although most of Europe and Asia banned mephedrone and other synthetic cathinones by 2010 (INCB, 2010; INCB, 2011), it was not until 2011 mephedrone became banned in the US. In 2011, the DEA requested information for six cathinone analogs, including mephedrone, methylone, and MDPV as the top three cathinones of concern (DEA, 2013). Before its ban in 2011, mephedrone was primarily sold over the Internet, was one of the highest recreationally used synthetic cathinones, and received a vast amount of media attention (Federal Register, 2011; Green,

2014).

Despite the band on mephedrone, recreational illegal use is still relevant

(Winstock, et al., 2010). An online survey, open to anyone who previously used mephedrone, including those from the US, reported that out of 1,506 responders, over

! 3! 80% were males living in Britain with the average age of 26 years old. Despite being illegal, it was found that 36% of the responder’s still use mephedrone and 53% reported its prohibition to have no impact on its availability (Carhart-Harris et al., 2011).

A UK survey done by Winstock et al. (2011), suggested that intranasal is the preferred route of administration for mephedrone and that about 0.5-1g of mephedrone is used per session. Although intranasal is the preferred method of administration, intravenous administration of mephedrone is a growing choice for chemsex drug use in men who have sex with men (Dolengevich-Segal et al., 2016; Schmidt et al., 2016;

Sewell et al., 2017). Also, most users who reported using both cocaine and mephedrone, consider the high of mephedrone to be the same or greater than the high of cocaine.

Before being temporarily placed into Schedule I in 2011 (Federal Register, 2011), and permanently in 2012 (DEA, 2012; Keim, 2012), most of the studies focused on the identification and clinical case reports of mephedrone (e.g.; Wood et al., 2010;

Gustavsson and Escher, 2009; Ahmed et al., 2010). During the recent years, other preclinical studies suggested that the use of mephedrone might have strong abuse liability and may lead to addiction.

In preclinical studies, such as, planarians, spontaneous discontinuation of mephedrone exposure produced a withdrawal effect (Ramoz et al., 2012). In rhesus monkeys trained to discriminate cocaine from saline, mephedrone served as a discriminative cue and substituted for cocaine (Smith et al., 2016). Furthermore, in both mice and rats, mephedrone supported intravenous self-administration (Aarde et al., 2013;

Creehan et al., 2015; Motbey et al., 2013; Vandewater et al., 2015) and facilitated rewarding intracranial stimulation (Bonano et al., 2015; Robinson et al., 2012). Other

! 4! indices of potential abuse liability, in mice and rats, include studies demonstrating that mephedrone can serve as a discriminative stimulus (Gatch et al., 2013; Harvey and Baker,

2016; Varner et al., 2013;), produce conditioned place preference (Lisek et al., 2012;), and induce locomotor activity (Gatch et al., 2013; Gregg et al., 2013; Huang et al., 2012;

Kehr et al., 2011; López-Arnau et al., 2012; Martínez-Clemente et al., 2013; Marusich et al., 2012; Motbey et al., 2012; Shortall et al., 2013a; Varner et al., 2013; Wright et al.,

2012). The ability of mephedrone to maintain self-administration, serve as a discriminative cue in drug discrimination, and to induce conditioned place preference, suggests that mephedrone may have strong abuse liability.

Neuropharmacology of Addiction

Most addictions begin with minor exposure to a drug. There is an initial exposure to a drug, then the drug becomes regularly or recreationally used, next the drug is used in an abuse-like fashion where binging may occur, followed by addiction. Addiction is characterized as compulsive usage of drug, despite negative consequence. Once a person is addicted to the drug, there is a high probability that there are chemical alterations affecting the central nervous system. These alterations may lead to brain damage, such as cocaine causing apoptotic cell death in the mesencephalon and striatal portion of the brain (Lepsch et al., 2015) or 3,4-methylenedioxypyrovalerone (MDPV) inducing toxicity of the blood-brain barrier (Rosas-Hernandez et al., 2016).

Studies have shown that the reinforcing effects of most stimulant drugs of abuse, is, in part, mediated through the stimulation of the mesolimbic dopamine pathway (e.g.

Büttner, 2011; Cunha-Oliveira et al., 2008; Hyman et al., 2006; Koob and Volkow, 2010;

! 5! Nestler, 2004; Nutt, et al., 2015; Volkow and Morales, 2015; Wise, 2008). The mesolimbic dopamine pathway is often referred to as the reward pathway. The reward pathway consists of dopaminergic neurons that originate in the ventral tegmental area, project into, and release dopamine (DA) in the nucleus accumbens. The release of dopamine into the nucleus accumbens may be responsible for the association of drug- reward and drug stimuli-reward found in drug abuse (Volkow and Morales, 2015; Wise,

2008).

Aside from the neurotransmitter dopamine contributing to the abuse of drugs, adaptation of the serotoninergic system has also been observed when drugs are abused and may have a significant impact on drug relapse (Kirby, et al., 2011; Müller et al.,

2010; Müller and Homberg, 2015). For example, the psychostimulant cocaine causes an increase of both DA and serotonin (5-HT) within the synapse (Andrews and Lucki, 2001).

The reinforcing effects of cocaine have been shown to be modulated through the 5-HT2C receptor (Anastasio et al., 2006; Swinford-Jackson et al., 2016). For example, the non- selective 5-HT2 receptor antagonist ketanserin attenuates the discriminative stimulus effects of cocaine in rats (McMahon and Cunningham, 2001).

Animal Models of Addiction

Drug Discrimination

In behavioral pharmacology, drug discrimination is an assay to model the subjective effects of a drug. Drug discrimination is carried out in an experimental operant chamber in which the subject performs a response in the presence of one stimulus and a different response in the presence of another stimulus in order to obtain or avoid a

! 6! particular consequence. In order for drug discrimination to work, the stimuli presented must be discriminable, meaning, that there must be a component or property between the two stimuli that the subject can differentiate. A drug serves as a discriminative stimulus if, when presented, the drug evokes a response that is followed by a reinforcer. Typically, in a two choice drug discrimination paradigm, subjects are trained to discriminate between two distinctive stimuli, usually a specific drug, at a specific dose, from a vehicle, such as saline, in order to receive a reward. For the drug discrimination analysis, both the chosen training drug and test drugs should have measurable effects on the central nervous system, thus, serving as discriminative stimuli. In general, drug discrimination is a good tool to pharmacologically characterize the neural mechanism in which the training drug, at the training dose, may produce its discriminative stimulus effect (Solinas, 2006; Stolerman,

2014; Young, 2009). Subjects may be tested with various receptor antagonists, ion channel blockers, or even enzyme inhibitors in an attempt to inhibit or increase the discriminative effect of the training drug. Drug discrimination is a sensitive assay such that even partial activation and blockade may be measured (Colpaert, 1999).

Self-Administration

Self-administration is a behavioral assessment that is traditionally used to determine the abuse liability and reinforcing effects of a drug. During the self- administration procedure, the subject is trained to self-administer a drug and various aspects of self-administration behaviors may be studied, such as acquisition, maintenance, extinction, and relapse (Panlilo and Goldberg, 2007). Acquisition is the learning phase of self-administration. During this phase, a new behavior, of self-administering a drug, is

! 7! added to the subject’s behavioral repertoire. Next is the maintenance phase. During this phase, the continuation of the newly acquired behavior of self-administering drug is observed and the behavior does not have to be learned. This phase may only begin after acquisition has occurred. Extinction is the process of discontinuing the presentation of a reinforcer for a previously reinforced behavior. In self-administration, the subject may perform the previous response to self-administer a drug, however, no drug is actually administered. Therefore, the contingency between the operant behavior and the consequence is broken and the learned behavior is significantly reduced. Lastly, relapse or reinstatement, in self-administration, is when the behavior is once again, reinforced or a cue is presented to trigger the previously learned self-administrating behavior. During extinction, the learned behavior is no longer reinforced, and therefore the rate of the behavior is decreased. During relapse, however, the learned behavior is reinforced again and as a consequence, the rate of the behavior increases. In order to initiate the reinstatement phase, the subject is often presented with a cue to indicate a reward is available. This cue may be the drug itself, or a stimulus that was conditioned during the acquisition phase.

Conditioned-Place Preference

Conditioned Place Preference is a behavioral assay that uses predominantly

Pavlovian conditioning to evaluate the associative properties of a drug (Tzschentke,

2007). In the most simplistic model, there are two boxes that are connected by an opening.

Both of the boxes differ in their environments, such as color (white walls vs. black walls) and texture (steel grid floor vs. bedding floor). One box will be associated with a

! 8! particular drug, while the other box will be associated with a vehicle. During conditioning training, the subject is exposed to an unconditioned stimulus (drug or vehicle) and placed in the box that is associated with that stimulus, while having restricted access to the other box. After multiple sessions, the different boxes will then serve as conditioned stimuli to the vehicle or drug and illicit conditioned place preference or avoidance when tested without drugs and given a choice of which chamber to enter.

Conditioned place preference is said to occur if the subject spends more time in the drug- associated box, after conditioning to the drug, and conditioned place aversion is said to occur if the subject spends more time in the vehicle-associated box after conditioning.

This behavioral procedure is used as a model to evaluate rewarding or aversive properties of a drug.

Out of these three models of drug abuse liability, we utilized two of these assays to study the pharmacology of mephedrone: drug discrimination and self-administration.

In the drug discrimination procedure, the subjects learned to recognize the stimulus effects of mephedrone. We then compared this novel discriminative stimulus effect with other multiple test drugs with different pharmacological properties. In addition to testing whether other drugs can substitute for the mephedrone discriminative stimulus, we also tested selected antagonists to further refine the pharmacology of mephedrone. In self- administration, we directly studied the reinforcing capacity of different drugs including cocaine, mephedrone or methamphetamine and attempted to block these reinforcing effects with a selected antagonist. This procedure allowed comparisons of the pharmacological mechanisms for the reinforcing effects of cocaine and mephedrone as well as comparison of the potency of mephedrone’s discriminative stimulus and

! 9! reinforcing effects to other drugs. Although self-administration tests reinforcement effects and conditioned place preference is able to test rewarding effects, we used self- administration and not conditioned place preference because we were able to test and compare the effects of multiple drugs within the same subjects and were interested in the direct reinforcing effects of mephedrone.

Rationale

Abusing drugs has both personal and societal consequences. Drug abuse has a significant impact on the economy, social and public health. In 2010, illicit drug use cost the nation over an approximate $193 billion, with the cost of health implication costing approximately $11 billion and with an increase rate of drug use from 9.4% to 10.1% of the population (SAMHSA, 2014). Although scientists presently know more now than they did before about the mechanism and effects of drugs, there has been an increased presence of novel psychoactive substances in recent years, which are being used in place of the more traditional abused drugs (Johnson et al., 2013; Karila et al., 2015; White,

2017).

So far, in vitro studies have shown mephedrone to increase the release of both DA and 5-HT and to inhibit both the dopaminergic transporter and the serotonergic transporter (Kehr et al., 2011; Martínez-Clemente et al., 2012), but the full contribution of either neurotransmitter system is unknown. Mephedrone also displays an affinity for both the 5-HT2 receptor and the D2 receptor, but with a greater affinity for the 5-HT2 receptor than the D2 receptor (Kehr et al., 2011; Martínez-Clemente et al., 2012). In vivo,

Varner et al. (2013) and Gatch et al. (2013) performed a drug discrimination experiment

! 10! in which they concluded that mephedrone might have discriminative mechanisms that are more similar to stimulants than to non-stimulants. Lisek et al. (2012) investigated the locomotor effects of mephedrone and suggested that its locomotor effects are mediated through the D1 and D2 receptor. Creehan et al. (2015) and Aarde et al. (2013) were successful in having rats self-administer mephedrone and suggesting mephedrone to have potential abuse liability, however they did not test the contribution of DA or 5-HT to the reinforcing effects.

Neither the in vitro or in vivo studies mentioned above determined the contributing factors of both DA and 5-HT to the abuse liability of mephedrone. Hence, there is still a void in the knowledge of mephedrone’s mechanism, which limits potential treatment strategies. The shortage of information about the pharmacological mechanism of the novel psychoactive substance mephedrone present in preclinical observations inhibits the ability of health care personnel to tackle the problems of its misuse. To further investigate the pharmacological properties of mephedrone and the relative contributions of 5-HT and DA receptor activation, the following specific aims were established:

1. Establish mephedrone as a discriminative stimulus in male Sprague-

Dawley rats;

2. Determine the effects of mephedrone on cocaine’s abuse liability in rats

with a history of cocaine self-administration.

! 11! Current Aims

The overall objective of this current thesis was to determine the pharmacological mechanisms of action of mephedrone, which may lead to its abuse ability. In order to achieve this objective, two rat models were used: two-choice drug discrimination and self-administration. These current studies were conducted in male Sprague-Dawley rats that were trained to respond on levers in experimental operant chambers.

The first study used drug discrimination to establish mephedrone as a discriminative stimulus in male Sprague-Dawley rats. We tested the hypothesis that, if mephedrone produces a discriminative stimulus, then its mechanism of action will be serotonergic at a low dose and dopaminergic at a high dose. By using the drug discrimination behavioral assay, we were able to determine which other drugs substitute for two specific doses of mephedrone. We determined the discriminative ability and the mechanisms of both a low dose of 0.5 mg/kg mephedrone and a high dose of 3.2 mg/kg mephedrone in groups of 12 rats. This allowed us to attempt to differentiate the mechanism mephedrone uses to illicit its discriminative stimulus effects at different doses.

The chosen mephedrone training doses were 0.5 mg/kg and 3.2 mg/kg and were based upon previous findings of Varner et al. (2013) and preliminary data from our lab. We used a variety of dopaminergic and serotonergic receptor antagonists to further characterize the discriminative mechanism of both a low and high dose of mephedrone.

The second study used self-administration to determine the effects of mephedrone on cocaine’s abuse liability in rats with a history of cocaine self-administration. We tested the hypothesis that, if mephedrone is reinforcing, then its reinforcing effects are mediated through dopaminergic mechanisms. Therefore, a history of self-administrating

! 12! cocaine will establish reinforcement through dopaminergic mechanisms and mephedrone will substitute for the cocaine seeking behavior. Self-administration allowed us to compare the pharmacological effects of cocaine and mephedrone on the self- administering and drug-seeking behavior. This procedure also allowed us to use monoamine receptor antagonists to further determine the mechanistic action of mephedrone’s reward. A DA1 receptor antagonist was used in an attempt to block the drug-seeking behavior produced by mephedrone, and to hone in on the specific receptor and mechanism that is activated when mephedrone is presented as a reward.

The combination of these studies helped to determine the discriminative and reinforcing mechanisms of mephedrone. Analyzing these the discriminative and reinforcing mechanisms of mephedrone helped to determine the pharmacological components that may lead to its abuse and may implicate potential treatment strategies.

! 13! CHAPTER 2

EXPERIMENT ONE: DRUG DISCRIMINATION

Drug Discrimination

Drug discrimination is a model of behavioral pharmacology in which we are able to model the subjective effects of a drug. Subjects are typically trained to discriminate between a specific drug, at a specific dose, from saline or a vehicle. For the drug discrimination analysis, the chosen training drug and test drugs should have measurable effects on the central nervous system, thus, serving as discriminative stimuli. As independent variables, training and test drugs and doses are manipulated and the lever- pressing behavior is directly measured as the dependent variable. In general, drug discrimination is a good tool to further characterize the neural mechanism in which the training drug, at the training dose, may produce its discriminative effect. Subjects may be pretreated with various receptor antagonists, ion channel blockers, or even enzyme inhibitors in an attempt to inhibit the discriminative effects of the drug. Drug discrimination is a sensitive assay such that even partial activation and blockade may be measured (Colpaert, 1999).

For Experiment 1A and 1B, a two-lever drug discrimination procedure was implemented. Rats were injected with either training drug or saline and required to respond on the drug lever or saline lever, respectively. Once the discrimination was learned, the assay was used to test for stimulus generalization and to test the neural mechanism by which the training drug at the particular dose was mediated.

! 14! To test for stimulus generalization, various drugs that emit a primarily dopaminergic or serotonergic cue were used as the test drugs. If stimulus generalization occurred for the training drug at the training dose, then administration of the test drugs would result in responding on the drug lever. This implied that there are similarities in the discriminative stimuli, between the training drug at the training dose and the test drugs.

DA and 5-HT receptor antagonists were also given in combination with mephedrone to further characterize the mechanisms involved in the discriminative effects of mephedrone.

Rationale

As mentioned previously, not many studies focused on the pharmacological mechanism of mephedrone at various doses. In vivo microdialysis studies demonstrate that mephedrone dose dependently increases the DA and 5-HT levels in the nucleus accumbens and striatum. Mephedrone also promotes the release of DA and 5-HT through the dopamine transporter (DAT) and the serotonin transporter (SERT) (Baumann, et al.,

2012; Gołembiowska, et al., 2016; Kehr et al., 2011; Suyama et al., 2016). In vitro, mephedrone not only increases DA and 5-HT levels, but also produces blockade of the

DA and 5-HT transporters and inhibits the synaptosomal uptake of these neurotransmitters (Angoa-Pérez et al., 2012, 2014; Cameron et al., 2013; Hadlock et al.,

2011; López-Arnau et al., 2012; Martínez-Clemente et al., 2012; Suyama et al., 2016).

Only a handful of studies examined the discriminative stimulus properties of mephedrone and even fewer used mephedrone as the training drug. To further understand the pharmacological mechanism of mephedrone and to help determine what other psychostimulants have subjective pharmacologic effects similar to mephedrone, the drug

! 15! discrimination assay was implemented in monkeys, mice, and rats. In rhesus monkeys, a low, moderate, and high dose of mephedrone failed to fully substitute for 0.32 mg/kg cocaine training stimulus (Smith et al., 2016). To the contrary, in mice, 3.0 mg/kg mephedrone fully substituted for the 10 mg/kg cocaine training dose (Gannon and

Fantegrossi, 2016).

In a two choice discrimination assay, mephedrone was able to fully substitute for both 10 mg/kg cocaine and 1.0 mg/kg methamphetamine discriminative stimuli (Gatch et al., 2013). When rats were trained to discriminate between d-amphetamine and saline, doses of 1.0 and 2.0 mg/kg mephedrone fully substituted for 0.5 mg/kg d-amphetamine.

Furthermore, the D1 receptor antagonist, SCH39166, was able to attenuate the discriminative stimulus effects of mephedrone (Harvey et al., 2017). When rats were trained to discriminate between 1.5 mg/kg MDMA and saline, or a combination of 1.5 mg/kg MDMA+0.5 mg/kg d-amphetamine and saline, 2.0 mg/kg mephedrone was able to fully substitute for both 1.5 mg/kg MDMA and for the combination of 1.5 mg/kg

MDMA+0.5 mg/kg d-amphetamine. It is suspected that mephedrone might cross- substitutes for MDMA since MDMA was the common element in both training groups

(Harvey and Baker, 2016).

When rats were trained to discriminate between 3.2 mg/kg mephedrone and saline, cocaine and methamphetamine produced only partial substitution. On the other-hand,

MDMA fully substituted for mephedrone, but also greatly reduced response rates. Other non-stimulants, such as, , morphine, and PCP, did not substitute for mephedrone (Varner et al., 2013). In another study, when rats were trained to discriminate between 3.2 mg/kg mephedrone and saline, full substitution was attained

! 16! with 18 mg/kg cocaine, while d-amphetamine, DOI, PCP, heroin, delta-9-THC, , and MDPV only partially substituted. Furthermore, the selective serotonin reuptake inhibiter, fluoxetine, shifted mephedrone’s dose-response curve dose- dependently, showing signs of both antagonism and potentiation (DeLarge et al., 2017).

Based on the previously mentioned studies, mephedrone appears to produce stimulus effects similar to other stimulant drugs that portray strong DA (methamphetamine, cocaine) and 5-HT (MDMA) underlying mechanisms.

In Experiment 1A, to test the hypothesis, if low-dose mephedrone produces a discriminative stimulus, then its mechanism of action will be more serotonergic than dopaminergic, we trained rats to discriminate between 0.5 mg/kg mephedrone and saline.

Therefore, if the low-dose of 0.5 mg/kg mephedrone produces a discriminative stimulus through 5-HT mechanisms, then both indirect and direct 5-HT agonists should substitute for the low training dose of mephedrone during drug discrimination and 5-HT receptor antagonists should produce blockade of the discriminative stimulus effects of 0.5 mg/kg mephedrone. Similarly, in Experiment 1B, to test the hypothesis, if high-dose mephedrone produces a discriminative stimulus, then its mechanism of action will be more dopaminergic than serotonergic, we trained rats to discriminate between 3.2 mg/kg mephedrone and saline. Therefore, if the high dose of 3.2 mg/kg mephedrone produces a discriminative stimulus through DA mechanisms, then indirect DA agonists should substitute for the high training dose of mephedrone during drug discrimination and DA receptor antagonists should produce blockade of the discriminative stimulus effects of 3.2 mg/kg mephedrone.

! 17! To generate a dose-response curve for mephedrone, a dose range of 0.05-5.0 mg/kg mephedrone was used (Gatch et al., 2013; Harvey and Baker, 2016; Shortall et al.,

2013 a, b; Varner et al., 2013). Since it is suggested that the enantiomers of mephedrone vary in their potencies and efficacies (Gregg et al., 2014; Vouga et al., 2015), we also tested S-mephedrone and R-mephedrone in initial experiments (not shown). We tested a dose of 0.28-5.0 mg/kg morphine as a negative control (not shown; Varner et al., 2013).

To test the dopaminergic discriminative properties of mephedrone, three well- studied drugs of abuse served as DA agonists and were tested for their ability to substitute for the training dose of mephedrone: the monoamine uptake inhibitor cocaine

(0.5-15 mg/kg (Gatch et al., 2013; Varner et al., 2013)), and the monoamine releasers, d-

N-methylamphetamine (methamphetamine) (0.15-3.0 mg/kg (Gatch et al., 2013; Varner et al., 2013)), and d-amphetamine (0.03125-2.0 mg/kg (Varner et al., 2013; Harvey and

Baker, 2016)). Cocaine, d-amphetamine, and methamphetamine were established as discriminative stimuli in other assays in rats with the underlying pharmacological mechanisms predominantly DA as demonstrated by Baker et al., (1993), Dunn and

Killcross, (2006), Kleven and Koek, (1998), Munzar et al., (1999), Munzar and Goldberg,

(2000), and Witkin et al., (1991). In vivo microdialysis shows that cocaine increases both the DA and 5-HT concentrations in the nucleus accumbens, when administered i.p., however, the increase of DA (about 462%) was larger than that of 5-HT (about 281%), when compared to baseline values (Andrews and Lucki, 2001). When administered subcutaneously in rat microdialysis experiments, d-amphetamine induced a greater release of DA in the nucleus accumbens (about 412%), compared to its increase of 5-HT

(about 165%) from baseline levels (Kehr et al., 2011). Similarly, microdialysis shows

! 18! that methamphetamine increased both DA levels in the caudate–putamen and 5-HT levels in the hippocampus when injected into rats i.p., however the increase of DA was again larger (1460%) than that of 5-HT (870%) (Matsumoto, et al., 2014). Therefore, these three drugs possess predominantly dopaminergic stimulation in these microdialysis studies. In the current experiment, to test the first prediction, these drugs were also used as DA cues and tested in their ability to substitute for the training dose of mephedrone.

To further test the dopaminergic properties of mephedrone, the DA1 receptor antagonist

SCH23390 (0.0125-0.06 mg/kg (Brennan et al., 2009; Carati and Schenk, 2011; Hiranita et al., 2010; Lisek et al., 2012; Munzar and Goldberg, 2000)) and the DA2 receptor antagonist sulpiride (2.0 and 4.0 mg/kg (Lisek et al., 2012)) were tested in their ability to antagonize the discriminative cues of mephedrone.

We specifically used D1 and D2 receptor antagonists, to determine if these receptors contribute to the discriminative stimulus effects of mephedrone. These two receptors are thought to have a greater contribution to the rewarding effects of drugs. In rats trained to self-administer methamphetamine, the D1 receptor antagonist SCH23390 was able to attenuate the methamphetamine-seeking behavior (Carati and Schenk, 2011).

In rats trained to self-administer MDMA, the D2 receptor antagonist, eticlopride, attenuated the reinforcing effect of MDMA, thus causing an increase of MDMA self- administration (Brennan et al., 2009). Thus the D1 and D2 receptors may be important in mediating reinforcing effects of drugs like methamphetamine and MDMA.

To test the serotonergic discriminative properties of mephedrone, four well- studied drugs served as 5-HT cues and were tested for their ability to substitute for the training dose of mephedrone: the monoamine transporter substrate 3,4-

! 19! methylenedioxymethamphetamine (MDMA; Ecstasy) (0.5-9.0 mg/kg (Varner et al.,

2013; Fletcher et al., 2006)), the non-selective 5-HT receptor agonist 1-(m-chloro phenyl)piperazine; mCPP) (0.28-1.6 mg/kg (Eriksson et al., 1999)), the 5-HT2 receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) (0.1, 0.5, 1.0 and 2.0 mg/kg (Munzar et al., 2002)), and the 5-HT2C receptor agonist WAY163909 (0.05, 0.15, 0.5, 0.75, and

1.0 mg/kg (Anastasio et al., 2014)). MDMA, mCPP, DOI, and WAY163909 were established as discriminative stimuli in other assays in rats with the underlying pharmacological mechanisms being predominantly 5-HT (Anastasio et al., 2014;

Eriksson et al., 1999; Fletcher et al., 2006; Munzar et al., 2002). MDMA possess predominately serotonergic discriminating cues (Johanson et al., 2006; Steele et al.,

1994) and although MDMA causes the release of both DA and 5-HT neurotransmitters, the release of 5-HT is notably stronger than that of DA (Verrico et al., 2007). In the nucleus accumbens of rats, MDMA increased DA 235% above baseline while increasing

5-HT 911% above baseline (Kehr et al., 2011). To further test the serotonergic properties of mephedrone, the non-selective 5-HT2 receptor antagonist ketanserin (1.0 and 1.5 mg/kg (Asgari et al., 2006; Li et al., 209; McMahon and Cunningham, 2001)) and the selective 5-HT2C receptor antagonist SB242084 (0.5 and 1.0 mg/kg (Fletcher et al.,

2006)) were tested in their ability to antagonize the discriminative cues of mephedrone.

We specifically used 5-HT2 and specifically 5-HT2C receptor antagonists because these receptors were shown to be involved in the modulation of DA release. The 5-HT2C receptor agonist Ro60-0175 significantly reduced the amount of DA release within the ventral tegmental area and the nucleus accumbens. The 5-HT2C receptor antagonist

SB242084 was able to antagonize this effect (Di Matteo et al., 2000). Also, the 5-HT2C

! 20! receptor agonist Ro60-0175 was able to attenuate cocaine-induced hyperactivity and cocaine-self administration (Grottick et al., 2000). Thus the 5-HT2 receptor may be important in mediating reinforcing effects of drugs.

Experiment 1A: Low Training Dose

Materials and Methods

Subjects

A total of 25 drug naïve male Sprague-Dawley rats were purchased from Harlan

Labs (Indianapolis, IN, USA) (n=6) and Taconic Farms (Cranbury, NJ, USA) (n=13) weighing 200-250 g, when introduced into the experiment, and from Sage Labs

(Boyertown, PA, USA) (n=6) weighing 175-200 g each when introduced into the experiment. However, one rat was removed early from the study, before discrimination was learned, due to episodes of repeated seizures. Rats were group-housed to acclimate to the animal facility with food and water available ad libitum. Rats were placed on a reverse 12-hour light/dark cycle with lights off by 10 AM, and experiments were conducted in the morning for 5-7 days a week. A reverse light/dark cycle was also implemented to reduce stress by allowing all experiments to occur during the dark or

‘active’ phase of the rats’ diurnal cycle (Prager, 2011) and because there may be a link between the circadian cycle and the reward system (Hasler, 2012). Therefore, to avoid adding activity and sleep pattern as potential factors, especially for the self-administration studies, the reverse light/dark cycle was adopted. In addition, we adapted the reverse light/dark cycle for drug discrimination, to maintain consistent housing conditions for all experiments involved with characterizing mephedrone.

! 21! One day before the start of the experiment, rats were individually housed and placed on a restricted, maintenance diet in which the amount of food was controlled and adjusted to allow for slow growth. For the remainder of the experiment, rats were maintained at approximately 85% of their free-feeding body weights according to age matched controls provided by the vendors by earning banana-flavored sucrose pellets

(45mg, BioServ, Flemington, NJ) in the operant experimental chambers and receiving approximately 12-15 g of Rodent Chow each day after the session. All animals were maintained in accordance with the guidelines of the Institutional Animal Care and Use

Committee of Temple University (Institution of Laboratory Animal Research, National

Academy Press; Eighth edition, revised 2011).

Drugs

Doses of 0.05, 0.15, 0.5 and 1.6 mg/kg mephedrone were used to generate a dose- response curve for mephedrone. We also tested S-mephedrone (0.05, 0.15, 0.5 mg/kg), R- mephedrone (0.5 and 1.6 mg/kg), and morphine (0.28, 0.5, and 5.0 mg/kg).

To test the dopaminergic discriminative properties of mephedrone, three well- studied drugs of abuse served as DA cues and were tested for their ability to substitute for the training dose of mephedrone: cocaine, methamphetamine, and d-amphetamine. The following doses were tested: cocaine (0.5, 0.9, 5.0, 10 and 15 mg/kg); methamphetamine

(0.15, 0.28, 0.5, 1.0 and 3.0 mg/kg); d-amphetamine (0.03125, 0.0625, 0.125, 0.25, 0.5,

1.0 and 2.0 mg/kg). To further test the dopaminergic properties of mephedrone, the DA1 receptor antagonist SCH23390 (0.0125, 0.025, and 0.06 mg/kg) and the DA2/3 receptor

! 22! antagonist sulpiride (2.0 and 4.0 mg/kg) were tested in their ability to antagonize the discriminative cues of mephedrone.

To test the serotonergic discriminative properties of mephedrone, four well- studied drugs served as 5-HT cues and were tested in their ability to substitute for the training dose of mephedrone: MDMA, mCPP, DOI, and WAY163909. The following doses were tested: MDMA (0.5, 1.6, 5.0, and 9.0 mg/kg); mCPP (0.28, 0.5, and 1.6 mg/kg); DOI (0.1, 0.5, 1.0 and 2.0 mg/kg); WAY163909 (0.05, 0.15, 0.5, 0.75, and 1.0 mg/kg). To further test the serotonergic properties of mephedrone, the non-selective 5-

HT2 receptor antagonist ketanserin (1.0 and 1.5 mg/kg) and the selective 5-HT2C receptor antagonist SB242084 (0.5 and 1.0 mg/kg) were tested for their ability to antagonize the discriminative cues of mephedrone.

Mephedrone, R-mephedrone, and S-mephedrone, were generously donated by the

Fox Chase Chemical Diversity Program (Doylestown, PA). The National Institute on

Drug Abuse Drug Supply Program (Bethesda, MD) provided cocaine, morphine, methamphetamine, and d-amphetamine. MDMA was purchased from Sigma (St. Louis,

MO). DOI, mCPP, and the antagonists, ketanserin, SCH23390, sulpiride, and SB242084 were purchased from Tocris (Ellisville, MO). Wyeth Pharmaceuticals (Princeton, NJ) generously donated WAY163909. Mephedrone, R-mephedrone, S-mephedrone, mCPP, cocaine, morphine, methamphetamine, d-amphetamine, MDMA, DOI and SCH23390 were all dissolved in 0.9% saline. Ketanserin was dissolved in 0.9% saline and sonicated for approximately 1 hour. Sulpiride was first dissolved in and brought up to volume with 0.9% saline. A few drops of 8% lactic acid were used to neutralize the pH.

SB242084 was first dissolved in 5% DSMO and titrated up to final concentration with

! 23! 0.9% saline. All injections were given intraperitoneally (i.p.) in a volume of 0.5 or 1.0 mL/kg of body weight.

Apparatus

Experiments were conducted in twelve aluminum and polycarbonate behavioral operant experimental chambers (30.5 cm x 24.1 cm x 21.0 cm, Model ENV-008CT, Med

Associates, Inc., Georgia, VT, USA) located within ventilated sound attenuating enclosures (ENV-018MD). Each chamber featured, on one wall, two amber stimulus lights (Model ENV-221M) located directly above two retractable levers (Model ENV-

112CM) positioned 2.1 cm above the stainless steel grid floor and 7.62 cm apart from one another, a center receptacle located between the two levers, and a pellet feeder (Model

ENV-200R2M). Located on the opposite wall, a house light, and ventilator fan for white noise was provided. Experimental contingencies were controlled using a software- installed computer and the data were recorded by the computer-driven interface (MED

Associated, St. Albans, VT, USA).

Procedure

Two-Choice Drug Discrimination Training

Rats were initially trained to respond on two levers, on alternate days, on a fixed ratio 1 (FR1) of banana-flavored sucrose pellet (45mg, BioServ, Flemington, NJ) delivery. Rats were weighed then injected i.p. with either saline or a dose of mephedrone and placed in the experimental chamber for a 10 min timeout period. During this time, both levers were retracted and both the house light and stimulus lights were off. After the

! 24! 10 min timeout, the house light and stimulus lights, located above both levers, were illuminated and the levers were inserted into the chamber. Rats were trained to discriminate mephedrone on the left lever and saline on the right lever on a gradually increasing schedule of food delivery from FR1 to FR10. Incorrect responding on the inappropriate lever was not reinforced and reset the ratio requirement. Each trial lasted for 10 reinforcers or 5 min, whichever occurred first.

After stable responding, rats were moved to a two discrete trials training procedure which included a 10 min timeout, followed by a 5 min ratio component, followed by a second 10 min timeout period and 5 min ratio component. The purpose of the two discrete trials training procedure was to expose the rats more frequently to the different training stimuli and to prepare the rats for testing in the antagonism studies.

Throughout the study, sessions were conducted five to seven days per week. The daily injection sequence was randomly selected with the constraint of not administering a particular treatment for more than three consecutive trials over 2 days, for example:

Day 1: S-S

Day 2: S-D

Also, rats were not given two doses of drug within the same day. If a rat received drug for the first trial, saline was given for the second trial, although response on the drug lever was reinforced.

The low training dose group (n=24) was trained to discriminate 0.5 mg/kg mephedrone from saline. Of these 24 rats, 12 of them initially began training at a dose of

3.2 mg/kg mephedrone. However, this dose was gradually decreased to a final dose of 0.5 mg/kg mephedrone due to a disruption of responding rates. The remaining 12 rats

! 25!

immediately began training with the low training dose of 0.5 mg/kg mephedrone. These groups are compared in the Results section.

Two-Choice Drug Discrimination Testing

Rats were trained until seven consecutive individual training trials (approximately

3-4 sessions) were met with the following criteria of: 1) fewer than 10 responses on the inappropriate lever before the first reinforcer; and, 2) greater than 80% of the injection appropriate responding over the entire training trial. Once all criteria were met, testing began. Test sessions were identical to training sessions except the test compound was injected i.p. before the session and a complete FR10 on either lever was reinforced. After each test session, rats had to meet the criteria for two to three consecutive training trials before testing again.

Data Analysis

For this experiment, the percentage of lever accuracy during training, the percentage of lever choice during testing, and the response rate per second were measured as the dependent variables. To compute the percentage of lever accuracy during training, the number of responses made on the appropriate lever was divided by total responses made on both levers for the duration of the trial. Similarly, the percentage of lever choice during testing was determined by dividing the responses made on the mephedrone lever by the total responses made on both levers throughout the trial.

! 26! Response rates were measured as responses per second on both levers throughout the entire trial.

Full substitution was considered to be 80% or greater responding on the mephedrone-appropriate lever, partial substitution was considered to be between 20%-

80% responding on the mephedrone-appropriate lever, and anything below 20% was considered to not substitute for the training dose of mephedrone. These general rules were applied to the interpretation of the discrimination results. Each independent variable

(single dose of a single drug) was expressed as a group mean, along with the standard error of the mean (S.E.M.). The results of substitution for any rat that did not complete one full ratio was not included in the percentage of mephedrone-appropriate lever response analysis, however, the response rate data were included.

To determine if the multiple doses of antagonist pretreatment altered mephedrone responding, data from rats that were included in a dose of a given pretreatment and control dose of mephedrone were analyzed by repeated measures, one-way ANOVA, followed by Dunnett’s multiple comparison test. If only one dose of antagonist pretreatment and control dose of mephedrone were compared, a paired t-test was used where applicable. Significance was set at p<0.05 for all analyses.

Results

0.5 mg/kg Mephedrone Training Dose

For the 0.5 mg/kg mephedrone group (Group 1, n=12; Group 2, n=12), during initial discrimination training, the training dose for mephedrone was 3.2 mg/kg for Group

! 27! Mephedrone Potency Comparison 100

80

Mephedrone_L 60 Mephedrone_H Saline_L 40 Saline_H

20 % Mephedrone Responding Mephedrone % 0

3.0

2.5

2.0

1.5

1.0 Response Rate Response

(responses/second) 0.5

0.0 S 0.015 0.05 0.15 0.5 1.5 5.0 15.0 Mephedrone Dose (mg/kg)

Figure 1. Mephedrone Potency Comparison. Dose-response curves for mephedrone in rats trained to discriminate either 0.5 (closed symbols) or 3.2 (open symbols) mg/kg mephedrone from saline. Abscissae: doses of mephedrone, in mg/kg. Points above S indicate saline. Ordinate: the percentage of the total responses made on the mephedrone- appropriate lever (upper panels) or the response rate measured as total responses made on both levers divided by the total time in seconds (lower panels). The dashed lines in the upper panel represent the range of criteria for no substitution (0-20%), partial substitution (20-80%) and full substitution (80-100%). The dashed lines in the lower panel represent the control response rates during the saline session for the respective high and low training groups. The data from rats that failed to complete one ratio were included in the response rate but not discrimination data. The numbers of rats completing at least one FR 10/number of rats tested for each dose of mephedrone are as follows: High training dose [Saline (10/10); mephedrone (0.05 mg/kg-10/10, 0.15 mg/kg-10/10, 0.5 mg/kg-10/10, 1.6 mg/kg-8/9, 3.2 mg/kg-11/11, 5.0 mg/kg-5/8); morphine (0.28 mg/kg-8/8, 0.5 mg/kg-6/7, 5.0 mg/kg-1/6) (data not shown)]; Low training dose [Saline (22/22); mephedrone (0.05 mg/kg-23/23, 0.15 mg/kg-23/23, 0.5 mg/kg-22/22, 1.6 mg/kg-22/23)]. Vertical lines represent ± S.E.M.

! 28! 1, based on a previous study (Varner et al., 2013). However, this dose dramatically decreased response rates to less than 0.2 responses/s. The mephedrone training dose was reduced to 1.0 mg/kg after 16 days. Thereafter, the response rates remained below 0.9 responses/s, so the training dose was further reduced to 0.5 mg/kg after 6 weeks. Group 2 began initial discrimination training at a dose of 0.5 mg/kg mephedrone. Once all rats were at the final training dose of 0.5 mg/kg mephedrone, it took an average of 101 trials for the twenty-four rats to acquire the discrimination of 0.5 mg/kg mephedrone vs. saline, while one rat failed to discriminate after 253 trials and was transferred to the high training dose group (see Results in Experiment 1B). One rat that failed to learn to discriminate 3.2 mg/kg mephedrone from saline was transferred to the low training dose group and completed training after 59 sessions. When saline was administered during test trials, responses were made on the saline-appropriate lever and the response rates were similar to the response rates obtained during the saline training trials (1.22±0.60 responses/s; see Figure 1). Likewise, when 0.5 mg/kg mephedrone was administered during test trials, responses were directed to the mephedrone-appropriate lever and the response rates were similar to the response rates obtained during the mephedrone training trials (1.05±0.62 responses/s). At doses lower than the training dose, responses were made predominately on the saline-appropriate lever while doses equal to or higher than the training dose resulted in full substitution. At the highest dose tested, 1.6 mg/kg mephedrone, the rates of responding were reduced but not eliminated. Morphine (0.28-

5.0 mg/kg) did not substitute for 0.5 mg/kg mephedrone at any dose although the response rates were decreased to 0.93±0.43 responses/s after 5.0 mg/kg morphine (data not shown).

! 29!

Dopamine Cues 100

80

60

40

20 % Mephedrone %Responding Mephedrone 0

3.0 Saline D-Amphetamine Metham phetam ine 2.5 Cocaine

2.0

1.5

1.0 Response Rate Response

(responses/second) 0.5

0.0 S 0.015 0.05 0.15 0.5 1.5 5.0 15.0 Drug Dose (mg/kg)

Figure 2. Low Training Dose Dopamine Agonist Substitution Tests. Effects of psychostimulants with predominantly DA actions in rats trained to discriminate 0.5 mg/kg mephedrone. Abscissae: doses of mephedrone, in mg/kg. Points above S indicate saline. The data from rats that failed to complete one ratio were included in the response rate but not discrimination data. The numbers of rats completing at least one FR 10/number of rats tested for each dose of mephedrone are as follows: [Cocaine (0.5 mg/kg-12/12, 0.9 mg/kg-12/12, 5.0 mg/kg-12/12, 10 mg/kg-8/8, 15 mg/kg-7/8); d- amphetamine (0.03125 mg/kg-10/10, 0.0625 mg/kg-10/10, 0.125 mg/kg-8/8, 0.25 mg/kg- 9/9, 0.5 mg/kg-17/17, 1.0 mg/kg-6/8, 2.0 mg/kg-6/8); methamphetamine (0.15 mg/kg- 20/20, 0.28 mg/kg-18/18, 0.5 mg/kg-20/20, 1.0 mg/kg-8/8, 3.0 mg/kg-4/8)]. Other details as in Figure 1.

! 30! Low Training Dose-Dopamine Agonist Substitution Tests

To test the dopaminergic properties of mephedrone, three psychostimulants with predominantly dopaminergic properties were tested for their ability to substitute for the low training dose of mephedrone: cocaine, methamphetamine, and d-amphetamine (see

Figure 2). High doses of cocaine (10 and 15 mg/kg), methamphetamine (0.5 and 1.0 mg/kg), and d-amphetamine (1.0 and 2.0 mg/kg) produced full substitution for the 0.5 mg/kg mephedrone training dose. At doses 0.5 mg/kg and lower, cocaine, d-amphetamine, and methamphetamine, increased response rates. At doses higher than 0.5 mg/kg, d- amphetamine and methamphetamine decreased response rates to below 0.03 responses/s.

Low Training Dose-Serotonin Agonist Substitution Tests

To test the serotonergic properties of mephedrone, four drugs with predominantly serotonergic properties, were tested for their ability to substitute for the training doses of mephedrone: MDMA, DOI, WAY163909, and mCPP (see Figure 3). All doses of mCPP partially substituted for the 0.5 mg/kg mephedrone training dose and a higher dose of 1.6 mg/kg mCPP decreased response rates to 0.22±0.19 responses/s. Low doses of

WAY163909 failed to substitute for 0.5 mg/kg mephedrone, however, there was some partial substitution at doses above 0.15 mg/kg WAY163909. The intermediate and higher doses of WAY163909 disrupted responding to the extent that only 3 out of 10 rats responded when tested with 1.0 mg/kg WAY163909. MDMA produced full substitution for 0.5 mg/kg mephedrone at intermediate to high doses (1.6, 5.0 and 9.0 mg/kg), partial substitution at the lowest dose tested (0.5 mg/kg), and dose-dependently decreased the rates of responding. All doses tested of DOI produced partial substitution, except for the

! 31! Serotonin Cues 100

80 Saline 60 DOI mCPP MDMA 40 WAY163909

20 % Mephedrone Responding Mephedrone % 0

3.0

2.5

2.0

1.5

1.0 Response Rate Response

(responses/second) 0.5

0.0 S 0.015 0.05 0.15 0.5 1.5 5.0 15.0 Drug Dose (mg/kg)

Figure 3. Low Training Dose Serotonin Agonist Substitution Tests. Effects of compounds with predominately 5-HT agonist activities in rats trained to discriminate 0.5 mg/kg mephedrone. The data from rats that failed to complete one ratio were included in the response rate but not discrimination data. The numbers of rats completing at least one FR 10/number of rats tested for each dose of mephedrone are as follows: DOI (0.1 mg/kg-9/9, 0.5 mg/kg-5/9, 1.0 mg/kg-2/8, 2.0 mg/kg-1/8); MDMA (0.5 mg/kg- 12/12, 1.6 mg/kg-12/12, 5.0 mg/kg-10/11, 9.0 mg/kg-6/12); WAY163909 (0.05 mg/kg- 8/8, 0.15 mg/kg-7/8, 0.5 mg/kg-7/9, 0.75 mg/kg-5/9, 1.0 mg/kg-3/10); mCPP (0.28 mg/kg-12/12, 0.5 mg/kg-12/12, 1.6 mg/kg-9/12). Other details as in Figure 1.

! 32! high dose of 2.0 mg/kg DOI, which fully substituted for the low training dose of mephedrone, but greatly disrupted response rates. Similar to WAY163909, intermediate and high doses of DOI disrupted responding, only 2 out of 8 rats responded at 1.0 mg/kg

DOI, and only 1 out of 8 rats responded at 2.0 mg/kg DOI.

Low Training Dose-Dopamine Receptor Antagonist Tests

To further determine the dopaminergic properties of mephedrone, two DA receptor antagonists were tested for their ability to inhibit the discriminative stimulus effects of mephedrone: SCH23390 (see Figure 4) and sulpiride (see Figure 5). SCH23390 blocked the discriminative stimulus effects of 0.5 mg/kg mephedrone. One-way ANOVA indicated a significant overall antagonism of discriminative effects (F(2,18)=4.412; p<

0.05) and response rates (F(3,36)=24.79; p< 0.0001). Specifically, Dunnett’s post hoc tests indicated that a dose of 0.025 mg/kg SCH23390 decreased the % mephedrone appropriate responding (p<0.05). Although a high dose of 0.06 mg/kg SCH23390 increased the substitution of 0.15 mg/kg mephedrone from 28% to 68% mephedrone responding, only one rat responded at this combined dose of mephedrone and SCH23390.

Response rates were low after pretreatment with the lower dose of 0.0125 mg/kg, and after pretreatment with a dose of 0.025 mg/kg SCH23390, the response rates were greatly reduced and only 3 out of 10 rats responded. Dunnett’s post hoc tests indicated that a dose of 0.025 and 0.06 mg/kg SCH23390 significantly decreased response rates relative to 0.5 mg/kg mephedrone alone (p<0.05; p<0.05, respectively).

In rats trained to discriminate 0.5 mg/kg mephedrone, one-way ANOVA indicated significant overall sulpiride dose effects for discrimination (F(2,29)=4.153; p< 0.05).

! 33! SCH23390 Antagonist 100

80

60 Mephedrone + SCH23390 0.0125 + SCH23390 0.025 40 + SCH23390 0.06 * 20 % Mephedrone Responding Mephedrone % 0

3.0

2.5

2.0

1.5

1.0 Response Rate Response

(responses/second) 0.5 ** 0.0 A 0.015 0.05 0.15 0.5 1.5 5.0 15.0 Mephedrone Dose (mg/kg)

Figure 4. Low Training Dose DA1 Receptor Antagonist Tests. Effects of DA1 receptor antagonist SCH23390 pretreatment to mephedrone in rats trained to discriminate 0.5 mg/kg mephedrone. Abscissae: doses of mephedrone, in mg/kg. Points above A indicate antagonist dose administered alone. SCH23390 significantly attenuated mephedrone lever responding in rats trained to discriminate 0.5 mg/kg mephedrone (*, p<0.05), and reduced the response rates (**, p<0.0001). See text for details. The data from rats that failed to complete one ratio were included in the response rate but not discrimination data. The numbers of rats completing at least one FR 10/number of rats tested for each dose of mephedrone are as follows: SCH23390 0.0125 mg/kg with mephedrone doses (0.05 mg/kg-4/10, 0.15 mg/kg-3/9, 0.5 mg/kg-9/10, 1.6 mg/kg-7/8); SCH23390 0.025 mg/kg with mephedrone doses (0.05 mg/kg-1/9, 0.15 mg/kg-1/9, 0.5 mg/kg-3/10, 1.6 mg/kg-4/8); SCH23390 0.06 mg/kg with mephedrone doses (0.05 mg/kg-0/7, 0.15 mg/kg-1/6, 0.5 mg/kg-1/11, 1.6 mg/kg-1/7). Other details as in Figure 1.

! 34! Sulpiride Antagonist 100

80 * 60

Mephedrone 40 + Sulpiride 2.0 + Sulpiride 4.0 20 % Mephedrone Responding % Mephedrone 0

3.0

2.5 * 2.0

1.5

1.0 Response Rate Response

(responses/second) 0.5

0.0 A 0.015 0.05 0.15 0.5 1.5 5.0 15.0 Mephedrone Dose (mg/kg)

Figure 5. Low Training Dose DA2/3 Receptor Antagonist Tests. Effects of DA2/3 receptor antagonist sulpiride pretreatment to mephedrone in rats trained to discriminate 0.5 mg/kg mephedrone. Sulpiride significantly attenuated mephedrone lever responding in rats trained to discriminate 0.5 mg/kg mephedrone, while significantly increasing the rate of responses (*, p<0.05). The data from rats that failed to complete one ratio were included in the response rate but not discrimination data. The numbers of rats completing at least one FR 10/number of rats tested for each dose of mephedrone are as follows: Sulpiride 2.0 mg/kg with mephedrone doses (0.05 mg/kg-9/9, 0.15 mg/kg-8/8, 0.5 mg/kg-10/10, 1.6 mg/kg-11/11); sulpiride 4.0 mg/kg with mephedrone doses (0.05 mg/kg-7/7, 0.15 mg/kg-8/8, 0.5 mg/kg-7/7, 1.6 mg/kg-5/8). Other details as in Figure 1.

! 35! Specifically, Dunnett’s post hoc tests indicated that a dose of 2.0 mg/kg sulpiride decreased the % mephedrone-appropriate responding relative to the training dose. This same dose of 2.0 mg/kg sulpiride also significantly increased response rates relative to mephedrone alone (F(2,30)=7.071; p<0.05).

Low Training Dose-Serotonin Receptor Antagonist Tests

To further determine the serotonergic properties of mephedrone, two 5-HT receptor antagonists were tested for their ability to block the discriminative stimulus and rate-decreasing effects of mephedrone: The 5-HT2C antagonist, SB242084 (see Figure 6) and the 5-HT2A/ antagonist, ketanserin (see Figure 7). The higher dose of 1.0 mg/kg

SB242084 produced a slight attenuation of the discriminative stimulus effects of 0.5 mg/kg mephedrone, however, one-way ANOVA indicated a lack of overall antagonist dose effects for discrimination (F(2,27)=3.062; N.S.). However, SB282084 pretreatment did significantly alter response rates (F(2,27)=4.881; p< 0.05) at the training dose of 0.5 mg/kg mephedrone. Specifically, Dunnett’s post hoc test indicated that a dose of 0.5 mg/kg SB242084 significantly increased response rates relative to mephedrone alone

(p<0.05). For ketanserin, one-way ANOVA indicated a lack of overall antagonist dose effects for discrimination (F(2,24)=1.186; N.S.), but not for response rates

(F(2,24)=4.094; p< 0.05), when combined with the training dose of 0.5 mg/kg mephedrone.

! 36! SB242084 Antagonist 100

80

60 Mephedrone 40 + SB242084 0.5 + SB242084 1.0 20 % Responding Mephedrone 0

3.0

2.5 * 2.0

1.5

1.0 Response Rate Response

(responses/second) 0.5

0.0 A 0.015 0.05 0.15 0.5 1.5 5.0 15.0 Mephedrone Dose (mg/kg)

Figure 6. Low Training Dose 5-HT2C Receptor Antagonist Tests. Effects of 5-HT2C receptor antagonist SB242084 pretreatment to mephedrone in rats trained to discriminate 0.5 mg/kg mephedrone. SB242084 did not attenuate mephedrone lever responding in rats trained to discriminate 0.5 mg/kg mephedrone, but did alter the response rates (*, p<0.05). The data from rats that failed to complete one ratio were included in the response rate but not discrimination data. The number of rats completing at least one FR 10/number of rats tested for each dose of mephedrone, are as follows: SB242084 0.5 mg/kg with mephedrone doses (0.05 mg/kg-9/9, 0.15 mg/kg-9/9, 0.5 mg/kg-8/8, 1.6 mg/kg-7/7); SB242084 1.0 mg/kg with mephedrone doses (0.05 mg/kg-10/10, 0.15 mg/kg-8/8, 0.5 mg/kg-10/10, 1.6 mg/kg-11/11). Other details as in Figure 1.

! 37! Ketanserin Antagonist 100

80

Mephedrone 60 + Ketanserin 1.0 + Ketanserin 1.5 40

20 % Responding Mephedrone 0

3.0

2.5

2.0

1.5

1.0 Response Rate Response

(responses/second) 0.5

0.0 A 0.015 0.05 0.15 0.5 1.5 5.0 15.0 Mephedrone Dose (mg/kg)

Figure 7. Low Training Dose 5-HT2 Receptor Antagonist Tests. Effects of 5-HT2 receptor antagonist ketanserin pretreatment to mephedrone in rats trained to discriminate 0.5 mg/kg mephedrone. The data from rats that failed to complete one ratio were included in the response rate but not discrimination data. The numbers of rats completing at least one FR 10/number of rats tested for each dose of mephedrone are as follows: Ketanserin 1.0 mg/kg with mephedrone doses (0.05 mg/kg-8/8, 0.15 mg/kg-8/8, 0.5 mg/kg-8/8, 1.6 mg/kg-7/8); ketanserin 1.5 mg/kg with mephedrone doses (0.05 mg/kg- 9/9, 0.15 mg/kg-8/8, 0.5 mg/kg-9/9, 1.6 mg/kg-4/8). Other details as in Figure 1.

! 38! Experiment 1B: High Training Dose

Materials and Methods

Subjects

A total of 12 drug naïve male Sprague-Dawley rats were purchased from Taconic

Farms (Cranbury, NJ, USA; n=12) weighing 200-250g, at the start of the experiment.

However, one rat was removed early from the study, before discrimination was learned, due to episodes of repeated seizures. As in Experiment 1A, rats were initially group- housed with food and water available ad libitum, and placed on a reverse 12-hour light/dark cycle. This experiment was conducted 5-7 days a week and began approximately in the early afternoon, after the completion of Experiment 1A.

One day before the start of the experiment, rats were individually housed, placed on a restricted diet, and were maintained at approximately 85% of their free feeding body weights. Banana-flavored sucrose pellets were earned in the operant experimental chambers and upon completion of each session, rats received approximately 12-15 mg of

Rodent Chow. All animals were maintained in accordance with the guidelines of the

Institutional Animal Care and Use Committee of Temple University (Institution of

Laboratory Animal Research, National Academy Press; Eighth edition, revised 2011).

Drugs

To generate a dose-response curve for mephedrone, a dose range of 0.05, 0.15,

0.5, 1.6, 3.2, and 5.0 mg/kg mephedrone was used. A dose range of 0.28, 0.5, and 5.0 mg/kg of morphine was tested as a negative control. To test the dopaminergic discriminative properties of mephedrone, cocaine (0.5, 0.9, 5.0, 10 and 15 mg/kg),

! 39! methamphetamine (0.15, 0.28, 0.5, 1.0 and 3.0 mg/kg), and d-amphetamine (0.03125,

0.0625, 0.125, 0.25, 0.5, 1.0 and 2.0 mg/kg) were tested for their ability to substitute for the training dose of mephedrone and the DA1 receptor antagonist SCH23390 (0.0125,

0.025, and 0.06 mg/kg) and the DA2 receptor antagonist sulpiride (2.0 and 4.0 mg/kg) were tested for their ability to antagonize the discriminative cues of mephedrone.

To test the serotonergic discriminative properties of mephedrone, MDMA (0.5,

1.6, 5.0, and 9.0 mg/kg), DOI (0.1, 0.5, 1.0 and 2.0 mg/kg), and WAY163909 (0.05, 0.15,

0.5, 0.75, and 1.0 mg/kg) were tested in their ability to substitute for the training dose of mephedrone and the serotonergic properties of mephedrone, the non-selective 5-HT2 receptor antagonist ketanserin (1.0 and 1.5 mg/kg) and the selective 5-HT2C receptor antagonist SB242084 (0.5 and 1.0 mg/kg) were tested in their ability to antagonize the discriminative cues of mephedrone. All injections were given intraperitoneally (i.p.) in a volume of 0.5 or 1.0 mL/kg of body weight.

Apparatus

Experiments were conducted in the two-choice operant drug discrimination chambers as described above for Experiment 1A.

Procedure

Two-Choice Drug Discrimination Training

Rats were trained in a fashion similar to Experiment 1A. Briefly, rats were trained to respond on both levers, on alternate days, on a FR1 for banana-flavored sucrose pellet delivery. Rats were initially weighed then injected i.p. with either saline or mephedrone

! 40! and placed in the experimental chamber for a 10 min timeout period. After the 10 min timeout, the house light and stimulus lights, located above both levers, were illuminated and the levers were activated. Rats were trained to discriminate mephedrone on the left lever and saline on the right lever on a FR1 and the ratio requirement gradually increased from FR1 to FR10. After stable responding, rats were moved to a two discrete trials training procedure which included a 10 min timeout, followed by a 5 min ratio component, followed by a second 10 min timeout period and 5 min ratio component.

Again, incorrect responding on the inappropriate lever was not reinforced and caused the ratio requirement to reset. Each trial lasted for 10 reinforcers or 5 min, whichever occurred first. Sessions were conducted five to seven days per week and the daily injection sequence was not systematically selected, except with the constraint of not administering a particular treatment for more than three consecutive trials.

The high dose training group (n=11) was trained to discriminate the final training dose of 3.2 mg/kg mephedrone from saline. This group initially started at mephedrone dose of 0.5 mg/kg and the dose was gradually incremented until response rates were above 1.00 responses/s.

Two-Choice Drug Discrimination Testing

The testing criteria were exactly the same as Experiment 1A. Before testing, rats were trained until seven consecutive individual training trials were met with the criteria of: 1) fewer than 10 responses on the inappropriate lever before the first reinforcer; and,

2) greater than 80% of the injection appropriate responding over the entire training trial.

! 41! After each test session, rats had to meet the criteria for two to three consecutive training trials before testing again.

Data Analysis

The data analysis was exactly the same as Experiment 1A. The percentage of lever accuracy during training was computed by dividing the number of responding made on the appropriate lever, by the total responses made on both levers for the duration of the trial. Similarly, the percentage of lever choice during testing was determined by dividing the responses made on the mephedrone lever by the total responses made on both levers throughout the trial. Response rates were measured as responses per second on both levers throughout the entire trial.

Full substitution was considered to be 80% or greater responding on the mephedrone-appropriate lever, partial substitution was considered to be between 20%-

80% responding on the d mephedrone-appropriate lever, and anything below 20% was considered to not substitute for the training dose of mephedrone. Each independent variable was expressed as a group mean, along with the standard error of the mean. The results of substitution for any rat that did not complete one full ratio was not included in the percentage of mephedrone-appropriate lever response analysis, however, the response rate data were included.

To determine if the multiple doses of antagonist pretreatment altered mephedrone responding, data from rats that were included in a dose of a given pretreatment and control dose of mephedrone were analyzed by repeated measures, one-way ANOVA, followed by Dunnett’s multiple comparison test. If only one dose of antagonist

! 42! pretreatment and control dose of mephedrone were compared, a paired t-test was used where applicable. Significance was set at p<0.05 for all analyses.

Results

3.2 mg/kg Mephedrone Training Dose

For the high dose training group (n=11), initial discrimination training began using a dose of 0.5 mg/kg mephedrone and gradually rose in increments for 94±81 trials until a final dose of 3.2 mg/kg was attained. Ten rats acquired the mephedrone vs. saline discrimination after reaching the final training dose within 88±30 trials, while one rat failed to discriminate after 331 trials and was transferred to the low training dose group

(see Results in Experiment 1A). One rat that failed to learn to discriminate 0.5 mg/kg mephedrone from saline was transferred to the high dose group and completed training after 35 sessions. When saline was administered during test trials, responses were directed to the saline-appropriate lever and the response rates were similar to the response rates obtained during the saline training trials (0.73±0.21 responses/s; see Figure 1).

Likewise, when 3.2 mg/kg mephedrone was administered during test trials, responses were made on the mephedrone-appropriate lever and the response rates were similar to the response rates obtained during the mephedrone training trials (0.51±0.33 responses/s).

Higher doses of mephedrone produced full substitution for the training dose, however the response rates were decreased. At doses lower than the training dose, responses shifted predominately onto the saline lever. A low dose of 1.6 mg/kg fully substituted for the training dose of 3.2 mg/kg mephedrone while a lower dose of 0.5 mg/kg partially

! 43! Dopamine Cues 100

80 Saline 60 Cocaine D-Amphetamine Methamphetamine 40

20 % Mephedrone Responding Mephedrone % 0

3.0

2.5

2.0

1.5

1.0 Response Rate Response

(responses/second) 0.5

0.0 S 0.015 0.05 0.15 0.5 1.5 5.0 15.0 Drug Dose (mg/kg)

Figure 8. High Training Dose Dopamine Agonist Substitution Tests. Effects of psychostimulants with predominantly DA actions in rats trained to discriminate 3.2 mg/kg mephedrone Abscissae: doses of mephedrone, in mg/kg. Points above S indicate saline. The data from rats that failed to complete one ratio were included in the response rate but not discrimination data. The numbers of rats completing at least one FR 10/number of rats tested for each dose of mephedrone are as follows: Cocaine (0.5 mg/kg-8/8, 0.9 mg/kg-8/8, 5.0 mg/kg-8/8, 10 mg/kg-8/8, 15 mg/kg-6/6); d- amphetamine (0.03125 mg/kg-6/6, 0.0625 mg/kg-7/7, 0.125 mg/kg-6/6, 0.25 mg/kg-6/6, 0.5 mg/kg-6/6, 1.0 mg/kg-4/6, 2.0 mg/kg-2/7); methamphetamine (0.15 mg/kg-8/8, 0.28 mg/kg-8/8, 0.5 mg/kg-8/8, 1.0 mg/k-6/7, 3.0 mg/kg-3/6). Other details as in Figure 1.

! 44! substituted for the training dose. Further lower doses produced only saline-appropriate responding. Morphine (0.28-5.0 mg/kg) did not substitute for 3.2 mg/kg mephedrone at any dose although the response rates were decreased to 0.09±0.18 responses/s after 5.0 mg/kg morphine.

High Training Dose-Dopamine Agonist Substitution Tests

To test the dopaminergic properties of mephedrone, three psychostimulants with predominant dopaminergic properties were tested for their ability to substitute for the two training doses of mephedrone: cocaine, methamphetamine, and d-amphetamine (see

Figure 8). Intermediate and high doses of methamphetamine (0.28-3.0 mg/kg) and a high dose of cocaine (10 mg/kg) produced partial substitution for the 3.2 mg/kg mephedrone training dose in most rats. At doses 0.5 mg/kg and lower, cocaine, d-amphetamine, and methamphetamine, increased response rates. At doses higher than this, d-amphetamine and methamphetamine decreased response rates. The doses of cocaine tested did not disrupt response rates and higher doses were not tested due to concerns for toxicity.

High Training Dose-Serotonin Agonist Substitution Tests

To test the serotonergic properties of mephedrone, four drugs with predominant serotonergic properties, were tested for their ability to substitute for the training doses of mephedrone: MDMA, DOI, and WAY163909 (see Figure 9). One dose of 0.75 mg/kg

WAY163909 partially substituted for the training dose of 3.2 mg/kg mephedrone; however, a high dose of 1.0 mg/kg WAY163909 extinguished response rates. An

! 45! Serotonin Cues 100

80 Saline 60 DOI MDMA 40 WAY163909

20 % Responding Mephedrone 0

3.0

2.5

2.0

1.5

1.0 Response Rate Response

(responses/second) 0.5

0.0 S 0.015 0.05 0.15 0.5 1.5 5.0 15.0 Drug Dose (mg/kg)

Figure 9. High Training Dose Serotonin Agonist Substitution Tests. Effects of compounds with predominately 5-HT agonist activities in rats trained to discriminate 3.2 mg/kg mephedrone. The data from rats that failed to complete one ratio were included in the response rate but not discrimination data. The numbers of rats completing at least one fixed ratio 10/number of rats tested for each dose of mephedrone are as follows: DOI (0.1 mg/kg-8/8, 0.5 mg/kg-2/6, 1.0 mg/kg-3/6, 2.0 mg/kg-3/6); MDMA (0.5 mg/kg- 7/7, 1.6 mg/kg-8/8, 5.0 mg/kg-8/8, 9.0 mg/kg-5/7); WAY163909 (0.05 mg/kg-6/6, 0.15 mg/kg-6/6, 0.5 mg/kg-5/6, 0.75 mg/kg-5/6, 1.0 mg/kg-2/6). Other details as in Figure 1.

! 46! intermediate dose of 1.6 mg/kg MDMA produced partial substitution and a high dose of

5.0 and 9.0 mg/kg produced full substitution for 3.2 mg/kg mephedrone and the highest dose of 9.0 mg/kg MDMA disrupted response rates. A high dose of 2.0 mg/kg DOI partially substituted for 3.2 mg/kg mephedrone, but did disrupt response rates, while all other doses of DOI did not produce any substitution.

High Training Dose-Dopamine Receptor Antagonist Tests

To further determine the dopaminergic properties of mephedrone, two DA receptor antagonists were tested for their ability to inhibit the discriminative stimulus effects of mephedrone: SCH23390 (see Figure 10) and sulpiride (see Figure 11).

SCH23390 did not block the discriminative stimulus effects of 3.2 mg/kg mephedrone, however, it did significantly decrease the response rates. A two-tailed paired t-test failed to identify significant overall dose effects for discrimination (t (2,3)=1.000; N.S.), but one-way ANOVA found a significant overall decrease for response rates (F(3,21)=19.06; p< 0.0001). Specifically, Dunnett’s post hoc tests indicated that doses of 0.0125, 0.025 and 0.06 mg/kg SCH23390 significantly decreased response rates relative to 3.2 mg/kg mephedrone alone (p<0.0001; p<0.0007; p<0.0001, respectively). Both doses of sulpiride failed to antagonize the discriminative effects and rate decreasing effects (F(2,19)=2.162;

N.S.) of 3.2 mg/kg mephedrone.

High Training Dose-Serotonin Receptor Antagonist Tests

To further determine the serotonergic properties of mephedrone, two 5-HT receptor antagonists were tested for their ability to block the discriminative stimulus and

! 47! rate-decreasing effects of mephedrone: SB242084 (see Figure 12) and ketanserin (see

Figure 13). Pretreatment of SB242084 failed to block the discriminative stimulus effects of 3.2 mg/kg mephedrone, although the response rates were antagonized in an unsystematic fashion. At the 3.2 mg/kg mephedrone training dose, one-way ANOVA failed to indicate a significant overall SB242084 dose effects for response rates

(F(2,17)=0.213; N.S.). Similarly, pretreatments of the higher and lower doses of ketanserin failed to block the discriminative stimulus effects of 3.2 mg/kg mephedrone and the response rates (F(2,18)=2.42; N.S.).

! 48! SCH23390 Antagonist 100

80

Mephedrone 60 + SCH23390 0.0125 + SCH23390 0.025 40 + SCH23390 0.06

20 % Responding Mephedrone 0

3.0

2.5

2.0

1.5

1.0 Response Rate Response

(responses/second) ** 0.5

0.0 A 0.015 0.05 0.15 0.5 1.5 5.0 15.0 Mephedrone Dose (mg/kg)

Figure 10. High Training Dose DA1 Receptor Antagonist Tests. Effects of DA1 receptor antagonist SCH23390 pretreatment to mephedrone in rats trained to discriminate 3.2 mg/kg mephedrone. Abscissae: doses of mephedrone, in mg/kg. Points above A indicate antagonist dose administered alone. SCH23390 significantly disrupted response rates for 3.2 mg/kg mephedrone and therefore substitution effects could not be measured (**, p<0.0001). The data from rats that failed to complete one ratio were included in the response rate but not discrimination data. The numbers of rats completing at least one FR 10/number of rats tested for each dose of mephedrone are as follows: SCH23390 0.0125 mg/kg with mephedrone doses (0.05 mg/kg-0/6, 0.15 mg/kg-0/6, 0.5 mg/kg-0/7, 1.6 mg/kg-3/6, 3.2 mg/kg-0/6); SCH23390 0.025 mg/kg with mephedrone doses (0.05 mg/kg-0/6, 0.15 mg/kg-0/6, 0.5 mg/kg-0/6, 1.6 mg/kg-2/6, 3.2 mg/kg-3/6); SCH23390 0.06 mg/kg with mephedrone doses (0.05 mg/kg-0/6, 0.15 mg/kg-0/6, 0.5 mg/kg-0/6, 1.6 mg/kg-0/6, 3.2 mg/kg-1/6). Other details as in Figure 1.

! 49! Sulpiride Antagonist 100

80

Mephedrone 60 + Sulpiride 2.0 + Sulpiride 4.0 40

20 % Responding Mephedrone 0

3.0

2.5

2.0

1.5

1.0 Response Rate Response

(responses/second) 0.5

0.0 A 0.015 0.05 0.15 0.5 1.5 5.0 15.0 Mephedrone Dose (mg/kg)

Figure 11. High Training Dose DA2/3 Receptor Antagonist Tests. Effects of DA2/3 receptor antagonist sulpiride pretreatment to mephedrone in rats trained to discriminate 3.2 mg/kg mephedrone. The data from rats that failed to complete one ratio were included in the response rate but not discrimination data. The numbers of rats completing at least one fixed ratio 10/number of rats tested for each dose of mephedrone are as follows: Sulpiride 2.0 mg/kg with mephedrone doses (0.05 mg/kg-6/6, 0.15 mg/kg-6/6, 0.5 mg/kg- 7/7, 1.6 mg/kg-6/6, 3.2 mg/kg-6/7); sulpiride 4.0 mg/kg with mephedrone doses (0.05 mg/kg-6/6, 0.15 mg/kg-6/6, 0.5 mg/kg-6/6, 1.6 mg/kg-6/6, 3.2 mg/kg-6/7). Other details as in Figure 1.

! 50! SB242084 Antagonist 100

80

Mephedrone 60 + SB242084 0.5 + SB242084 1.0 40

20 % Responding Mephedrone 0

3.0

2.5

2.0

1.5

1.0 Response Rate Response

(responses/second) 0.5

0.0 A 0.015 0.05 0.15 0.5 1.5 5.0 15.0 Mephedrone Dose (mg/kg)

Figure 12. High Training Dose 5-HT2C Receptor Antagonist Tests. Effects of 5-HT2C receptor antagonist SB242084 pretreatment to mephedrone in rats trained to discriminate 3.2 mg/kg mephedrone. SB242084 did not significantly attenuate mephedrone lever responding in rats trained to discriminate 3.2 mg/kg mephedrone or alter the rate of responses. The data from rats that failed to complete one ratio were included in the response rate but not discrimination data. The number of rats completing at least one FR 10/number of rats tested for each dose of mephedrone are as follows: SB242084 0.5 mg/kg with mephedrone doses (0.05 mg/kg-6/6, 0.15 mg/kg-5/6, 0.5 mg/kg-6/6, 1.6 mg/kg-6/6, 3.2 mg/kg-6/6); SB242084 1.0 mg/kg with mephedrone doses (0.05 mg/kg- 6/6, 0.15 mg/kg-7/7, 0.5 mg/kg-6/6, 1.6 mg/kg-7/8, 3.2 mg/kg-6/6). Other details as in Figure 1.

! 51! Ketanserin Antagonist 100

80 Mephedrone 60 + Ketanserin 1.0 + Ketanserin 1.5 40

20 % Mephedrone Responding Mephedrone % 0

3.0

2.5

2.0

1.5

1.0 Response Rate Response

(responses/second) 0.5

0.0 A 0.015 0.05 0.15 0.5 1.5 5.0 15.0 Mephedrone Dose (mg/kg)

Figure 13. High Training Dose 5-HT2 Receptor Antagonist Tests. Effects of 5-HT2 receptor antagonist ketanserin pretreatment to mephedrone in rats trained to discriminate 3.2 mg/kg mephedrone. The data from rats that failed to complete one ratio were included in the response rate but not discrimination data. The numbers of rats completing at least one FR 10/number of rats tested for each dose of mephedrone are as follows: Ketanserin 1.0 mg/kg with mephedrone doses (0.05 mg/kg-6/6, 0.15 mg/kg-7/7, 0.5 mg/kg-6/6, 1.6 mg/kg-6/6, 3.2 mg/kg-6/7); ketanserin 1.5 mg/kg with mephedrone doses (0.05 mg/kg-6/6, 0.15 mg/kg-7/7, 0.5 mg/kg-6/6, 1.6 mg/kg-6/6, 3.2 mg/kg-6/6). Other details as in Figure 1.

! 52! Experiment 1 Discussion

A high and low training dose of mephedrone were established as discriminative stimuli in two sets of rats although the time to testing criteria and the ease of training these doses were different between the groups. Initial attempts to train rats to discriminate between 3.2 mg/kg mephedrone and saline were difficult at this starting dose due to the disruptive response rates observed. After 70 trials, we continued to reduce the training dose until a final dose of 0.5 mg/kg mephedrone was attained. At this dose, responding was greater than 1.0 responses/s. For the rats discriminating 3.2 mg/kg mephedrone and saline, we implemented a procedure similar to Varner et al. (2013). We began with the initial dose of 0.5 mg/kg mephedrone, and gradually incremented the training dose until a final dose of 3.2 mg/kg mephedrone was attained. On average, rats acquired the 3.2 mg/kg mephedrone discrimination, faster than the 0.5 mg/kg mephedrone. It took an average of 101 trials for rats to discriminate between 0.5 mg/kg mephedrone and saline, compared to an average of 88 trials for rats to discriminate between 3.2 mg/kg mephedrone and saline. Although the acquisition of the higher dose was faster, the response rates were lower. There was additional trouble with maintaining response rates for the high group throughout the experiments. After approximately 550 trials, response rates for 3.2 mg/kg mephedrone decreased to an average of approximately 0.05 responses/s in 9 of the 10 rats. To continue testing this high training dose group, we decreased both the training dose to 0.5 mg/kg mephedrone and the response ratio from

10, to an FR1 and then gradually increased the dose to 3.2 mg/kg mephedrone, but was only able to increase the ratio to an FR5 for 8 of the 10 rats. These differences in response rates between the two training doses groups of rats played a role in determining

! 53! the window available to test the substitution drugs and antagonize discriminative stimulus effects of mephedrone.

In the present experiment, dopamine appears to play a role in mephedrone’s discriminative stimulus effects. Full substitution was achieved in rats trained to discriminate a low dose of 0.5 mg/kg mephedrone with the DA uptake inhibitor cocaine and the DA releasers d-amphetamine and methamphetamine. Partial substitution was obtained in rats trained to discriminate a high dose of 3.2 mg/kg mephedrone with cocaine and methamphetamine. In vivo microdialysis shows that cocaine increases both the DA and 5-HT concentrations; however, the increase of DA is larger than that of 5-HT

(285% and 191% above baseline, respectfully) (Andrews and Lucki, 2001). When administered subcutaneously, d-amphetamine induced a greater release of DA, compared to its increase of 5-HT from baseline levels (412% and 165%, respectfully) (Kehr et al.,

2011). Similarly, methamphetamine increased both DA levels and 5-HT levels, however the increase of DA was again larger than that of 5-HT (1460% and 870%, respectively)

(Matsumoto, et al., 2014). Therefore, these three drugs possess predominantly dopaminergic stimulation in these microdialysis studies. In the current study, the primarily DA1 receptor antagonist, SCH23390, was able to significantly antagonize the discriminative stimulus effects of 0.5 mg/kg mephedrone, suggesting an involvement of the DA receptor in mediating mephedrone’s discriminative stimulus effects at this low training dose. The DA2/3 receptor antagonist, 2.0 mg/kg sulpiride, was also able to significantly attenuate the discriminative stimulus effects of 0.5 mg/kg mephedrone, suggesting further, an involvement of the DA receptors in mediating mephedrone’s discriminative stimulus effects at a low training dose. The DA2/3 receptor antagonist

! 54! sulpiride produced slight antagonism of the discriminative stimulus effects of 3.2 mg/kg mephedrone, although this change was not significant. The DA1 receptor antagonist,

SCH23390 failed to produce any antagonism of mephedrone in the higher training dose group of rats. This may be due to the combined rate-decreasing effects of 3.2 mg/kg mephedrone and SCH23390 and the lack of rats responding at this high dose.

In previous studies, mephedrone fully substituted for training doses of common drugs of abuse that demonstrate dopaminergic discriminative stimulus effects. A dose of

2.0 mg/kg mephedrone was able to fully substitute for 0.5 mg/kg d-amphetamine and the

D1 receptor antagonist, SCH39166, was able to inhibit this substitution (Harvey et al.

2017). Doses of 2.5 and 5.0 mg/kg mephedrone fully substituted for both 10 mg/kg cocaine and 1.0 mg/kg methamphetamine training doses in drug discrimination assays

(Gatch et al., 2013). In rats trained to discriminate mephedrone, both cocaine and methamphetamine partially substituted for 3.2 mg/kg mephedrone, which is in coordination with our findings. In rats trained to discriminate 3.2 mg/kg mephedrone

(Varner et al., 2013), cocaine and methamphetamine produced 75 and 72% substitution, respectively. In the present experiment, cocaine and methamphetamine produced only 25 and 66% mephedrone-appropriate responding, respectively, which is slightly less than what was observed in the previous study. Nevertheless, these studies taken together further support the involvement of dopamine in the discriminative stimulus effects of mephedrone, especially at low doses of mephedrone.

In the present experiment, serotonin also appears to play a role in mephedrone’s discriminative stimulus effects. Full substitution was achieved in rats trained to discriminate a low dose of 0.5 mg/kg mephedrone with the 5-HT2 receptor agonist DOI

! 55! and the monoamine transporter substrate MDMA, and partial substitution was achieved with the 5-HT2C receptor agonist WAY163909 and the 5-HT agonist and releaser mCPP.

In rats trained to discriminate a high dose of 3.2 mg/kg mephedrone, full substitution was achieved with MDMA and partial substitution was achieved with DOI and WAY163909.

Taking into consideration that both the selective 5-HT2C receptor antagonist SB242084 and the nonselective 5-HT2 receptor antagonist ketanserin were able to produce some small antagonism of the discriminative stimulus effects of 0.5 mg/kg mephedrone, suggests a contribution of the serotoninergic activity in mediating mephedrone’s discriminative stimulus effects in the low training dose group. There may also be a contribution of the serotoninergic activity in mediating mephedrone’s discriminative stimulus effects in the high training dose group since ketanserin was able to produce a small antagonism of the discriminative stimulus effects of 3.2 mg/kg mephedrone.

When analyzing the present findings, serotonin also appears to play an active role in mephedrone’s discriminative stimulus effects, since full substitution was observed in both the low and high training dose groups of mephedrone with MDMA. MDMA is known to possess predominantly serotonergic discriminative cues, when it is tested in both human and rats (Johanson et al., 2006; Steele et al., 1994). Although MDMA causes the release of both DA and 5-HT in stably transfected human embryo kidney cells, the release of 5-HT is notably stronger than that of DA (Verrico et al., 2007). When administered subcutaneously, in rats, 3 mg/kg MDMA induced a greater release of increased 5-HT in the nucleus accumbens (about 911%), compared to its increase of DA

(about 235%) in microdialysis experiments from baseline levels (Kankaanpää et al.,

1998; Kehr et al., 2011; Panos and Baker, 2010).

! 56! To further suggest the involvement of 5-HT in the discriminative stimulus effects of mephedrone, previous studies reported on the similarities of mephedrone to MDMA.

For example, not only was MDMA able to partially substitute for 3.2 mg/kg mephedrone

(Varner et al., 2013), similar to the findings in our study, but 2.0 mg/kg mephedrone was also able to fully substitute for 1.5 mg/kg MDMA and a combination of 1.5 mg/kg

MDMA+0.5 mg/kg d-amphetamine (Harvey and Baker, 2016). The ability for mephedrone to cross-substitute for MDMA was suspected, since MDMA was the common element in both training groups. It is important to note that mephedrone fully substituted for MDMA (Varner et al., 2013) and MDMA also fully substituted for mephedrone (Harvey and Baker, 2016; current study) indicating full cross-substitution between these two drugs and therefore likely similar underlying mechanisms.

MDMA is a monoamine transporter substrate for , serotonin, and dopamine. As mentioned earlier, although MDMA may have an affinity for the SERT similar or slightly higher than DAT, it is able to induce a higher release of 5-HT than DA

(Verrico et al., 2007). Nonetheless, MDMA still has an affinity for DAT and is able to induce DA release into the nucleus accumbens. The involvement of both DA and 5-HT in

MDMA’s discriminative stimulus effects is also supported in discrimination studies, in which MDMA was able to fully substitute for d-amphetamine (Harper et al., 2014),

MDMA partially substituted for 10 mg/kg cocaine, and cocaine partially substituted for

1.5 mg/kg MDMA (Kueh and Baker, 2007); furthermore, the 5-HT uptake inhibitor, fenfluramine, fully substituted for 1.5 mg/kg MDMA (Goodwin, et al., 2003). Hence, these results suggest a detectable and measurable dopaminergic and serotonergic component in MDMA’s discriminative stimulus effects.

! 57! Based on our results in which MDMA was the only drug to fully generalize to both training doses of mephedrone, mephedrone appears to be more similar to MDMA than to cocaine and this is further supported by microdialysis. When compared in microdialysis studies, mephedrone’s extracellular concentration of 5-HT in the nucleus accumbens resembled that of MDMA. Like MDMA, in microdialysis experiments, mephedrone induces the release of both DA and 5-HT in the nucleus accumbens of awake rats. Mephedrone increased DA concentrations about twice as much as MDMA.

However, mephedrone increased 5-HT concentrations approximately 1000-fold above baseline in a manner similar to MDMA. When you compare the ratio of released DA:5-

HT of mephedrone’s, it is more similar to that of MDMA, than amphetamine (495:941 vs. 235:911 vs. 412:165, respectively) (Kehr et al., 2011). Hence, both drug discrimination and microdialysis studies indicate mephedrone is more similar to MDMA.

Because mephedrone and MDMA may share similar mechanisms, the presence of both serotonin and dopamine may be needed for the characterization of mephedrone’s discrimination.

Although the similarity to MDMA implies that the mephedrone discrimination may involve predominantly underlying serotonergic activity, all the data taken together indicates that the discriminative stimulus effects of mephedrone may require both dopaminergic and serotonergic activation at both high and low training doses. This notion is supported by the results in this present study, in which the drugs that fully substituted for mephedrone, at both a high and low doses, were those psychostimulants for which both serotonergic and dopaminergic activation is required for discriminative properties.

The only drug that fully substituted for the low training dose of mephedrone, and does

! 58! not have a dopaminergic component was DOI. However, the variability in substitution dose among the rats and the low response rates make strong conclusions about these results difficult at the present time.

Additional support for the requirement for both serotonergic and dopaminergic activation comes from the antagonism studies in that blockade of 3.2 mg/kg mephedrone was not achieved with neither the dopamine nor serotonin antagonists alone. Ketanserin and sulpiride were able to decrease the discriminative stimulus effects of a high dose of

3.2 mg/kg mephedrone, suggesting involvement of both DA and 5-HT, but again, the variability between the subjects was too great, and the response rates were too low to be conclusive. Similarly, support for the requirement of both serotonergic and dopaminergic activation comes from the antagonism studies in that blockade of 0.5 mg/kg mephedrone was not achieved with either the dopamine or serotonin antagonists alone. SCH23390, sulpiride, SB242084, and ketanserin were able to produce a decrease in the discriminative stimulus effects of 0.5 mg/kg mephedrone, further suggesting involvement of both DA and 5-HT. Although SCH23390 is known to be a primarily DA1 receptor antagonist, it also has strong affinity for the 5-HT2C receptor (Millan et al., 2001). Also, SCH23390 has been shown to inhibit the reuptake of [(3)H]serotonin (Zarrindast et al., 2011), indicating that SCH23390 has a serotonergic mechanism, which must be taken into consideration.

Therefore, there is further evidence to support the notion that the discriminative stimulus effects of mephedrone may require both dopaminergic and serotonergic cues, at both a high and low dose, and not one over the other.

Overall, two groups of rats were successfully trained to discriminate a low or a high dose of mephedrone from saline demonstrating that mephedrone possess

! 59! discriminable effects similar to other psychostimulants and cathinone derivatives. In addition, the dose chosen for training determines the capacity for other compounds to substitute for and antagonize the discriminative stimulus properties of mephedrone although the rate-decreasing effects of higher doses of mephedrone did limit the frequency of testing and number of doses that could be examined. However, despite the dose used for training, both serotonin and dopamine activation components appear to be necessary for a full complement of the mephedrone discriminative stimulus. Drugs that possess both dopaminergic and serotonergic cues fully substituted for both training doses of mephedrone. Also, both dopaminergic and serotonergic receptor antagonists were able to antagonize the discriminative stimulus effects of both a high and low dose of mephedrone. Thus, the discriminative stimulus mechanism of mephedrone may be similar to that of MDMA and the involvement of both dopamine and serotonin may play an important role.

! 60! CHAPTER 3

EXPERIMENT 2: SELF-ADMINISTRATION

Self-Administration

Self-administration is a behavioral assessment that is traditionally used to determine the reinforcing effects of a drug and potential abuse liability. The self- administration procedure allows the subject to self-administer a drug with assigned constraints on the amount of intake. Various aspects of self-administration behaviors can then be compared and studied, such as acquisition, maintenance, extinction, and relapse and total amount of infusions, length of learning, pattern of drug intake, and even tolerance (Panlilo and Goldberg, 2007).

For Experiment 2A and 2B, a three phase self-administration procedure was implemented. In the first phase, rats self-administered 0.375 mg/kg/infusion (inf) cocaine until a drug-seeking behavior was acquired. In the second phase, rats that successfully acquired the drug-seeking behavior went through an extinction period. In the third phase, rats that successfully extinguished the drug-seeking behavior were primed with an i.p. injection of a given drug, in order to reinstate the extinguished drug-seeking behavior.

For Experiment 2B, rats also went through an acquisition phase, where they self- administered 0.375mg/kg/inf of cocaine until a drug-seeking behavior was acquired. In the second phase, rats that successfully acquired the drug-seeking behavior were given mephedrone in substitution of cocaine. In the third phase, rats that successfully substituted mephedrone for cocaine, were given a pretreatment of a DA receptor

! 61! antagonist, to characterize the pharmacology of the self-administering behavior of mephedrone.

Rationale

Mephedrone supports self-administration in various strains of male and female

Wistar and Sprague Dawley rats and mephedrone is shown to not only to be intravenously self-administered, but also maintain self-administration behavior over time.

In male Sprague Dawley rats, the self-administration behavior was acquired quicker, with higher breaking points, in rats self-administering mephedrone when compared to rats self-administering methamphetamine. Also, rats increased in their overall intake of mephedrone infusions, compared to methamphetamine (Hadlock et al., 2011; Motbey et al., 2013). In male Wistar and Sprague Dawley rats, 0.5 and 1.0 mg/kg/inf mephedrone maintained self-administration in a dose-dependent manner. Male Wistar rats received more infusions throughout acquisition, than the male Sprague Dawley rats, although both strains averaged around the same number of infusions on the last acquisition day (Aarde et al., 2013). In male Wistar rats, self-administration of 0.5 mg/kg/inf mephedrone was acquired at a higher rate than MDMA or methylone and there was an overall increased intake of mephedrone infusions, than MDMA or methylone (Vandewater et al., 2015). In female Wistar rats, self-administration of 0.5 mg/kg/inf mephedrone was acquired at a higher rate than MDMA or methylone. After the acquisition phase, female rats displayed an equal potency for mephedrone as MDMA and methylone, although the cumulative self-administered dose of mephedrone was the highest (Creehan et al., 2015). Taken altogether, these studies show that mephedrone supports self-administration in various

! 62! strains of rats and the pattern of self-administration may be compared to the pattern of other common drugs of abuse.

Surveys show that more and more users with a history of illegal drug use, including cocaine, use mephedrone (Wood et al., 2011) and prefer mephedrone as their choice of drug (McElrath and O’Neill, 2011). Additionally, users say that mephedrone is similar to both cocaine and MDMA (Anonymous, 2011; CanadianBakin, 2012; Carhart-

Harris et al., 2011; DFsGeezaman, 2009; Meph_Test, 2009; Morrissey, 2012).

Furthermore, DA is a major component of drug reward and plays a key component in reinforcement (Di Chiara and Imperato, 1988; Phillips et al., 2003). Although cocaine failed to fully substitute for 3.2 mg/kg mephedrone in Experiment 1, cocaine was able to achieve partial substitution. This agrees with the results of other studies in which cocaine partially substituted for a similar high dose of mephedrone (Berquist II et al., 2017;

Varner et al., 2013). Furthermore, cocaine fully substituted when cocaine was tested in rats trained to discriminate 0.5 mg/kg mephedrone. In another study, mephedrone was able to fully substitute for a training dose of 10 mg/kg cocaine (Gatch et al., 2013) in rats.

Altogether, this indicates that mephedrone may mediate its subjective, discriminative stimulus effects via combined dopamine and serotonin mechanism and, subsequently, share reinforcing effects through similar pharmacological mechanisms as cocaine.

MDMA may also facilitate the drug-seeking behavior in rats. However, when rats were trained to self-administer MDMA, approximately half of the animals failed to acquire MDMA self-administration and for those that did acquire self-administration, the time course was prolonged and the rate of responding was low when compared to other drugs, such as cocaine (e.g. Bradbury et al., 2014; Creehan et al., 2015; Ratzenboeck et

! 63! al., 2001; Schenk et al., 2007). Although MDMA has dopaminergic releasing factors and may also support self-administration in rats in some studies, results from the drug discrimination studies we conducted suggest the pharmacological mechanism of mephedrone may be more similar to MDMA than to cocaine. However, due to DA’s role in supporting drug reinforcement (Di Chiara and Imperato, 1988; Phillips et al., 2003), we wanted to analyze the contribution of DA to mephedrone’s reinforcing effects.

Therefore, we decided to test rats with a history of self-administering cocaine instead of self-administering MDMA due to better reliability and ease of acquisition for cocaine.

In Experiment 2A, to test the hypothesis, if mephedrone shares similar reinforcing mechanisms as cocaine, then mephedrone will reinstate an extinguished cocaine self-administration behavior, we trained rats to self-administer 0.375 mg/kg/inf cocaine, placed them through an extinction period where the established drug-seeking behavior was extinguished. Next, the rats were reinstated them with a primed i.p. injection of 0.5-10 mg/kg mephedrone. Therefore, if mephedrone activates the mesolimbic DA system and produces a rewarding effect through DA mechanisms, then mephedrone should reinstate the extinguished cocaine-seeking behavior and the rats should readily engage in the self-administration behavior after extinction, when presented with the mephedrone induced priming injection. If mephedrone does not reinstate cocaine, it is possible that the history of cocaine may have altered the metabolism of mephedrone.

Therefore, we performed a pilot study to compare the amount of 5.0 mg/kg mephedrone in the plasma of rats with a history of cocaine, to rats without a history of cocaine. This was to ensure that having a history of cocaine does not alter the metabolism of mephedrone and make the reinforcing effects less potent. Similarly, in Experiment 2B, to

! 64! test the hypothesis, if mephedrone shares a similar dopaminergic mechanism to cocaine, then mephedrone will substitute for cocaine and maintain drug-seeking behavior, we trained rats to self-administer 0.375 mg/kg/inf cocaine. Once the behavior was established, we substituted 0.375 mg/kg/inf cocaine with 0.375 mg/kg/inf mephedrone.

Therefore, if mephedrone shares similar mechanisms and possess the similar abuse liability as cocaine, then mephedrone should substitute and maintain the drug-seeking behavior. Also, if the shared mechanisms are primarily dopaminergic, then the DA1 receptor antagonist SCH23390 should antagonize the substituted and maintained drug- seeking behavior of cocaine and mephedrone.

To establish the self-administration behavior, 0.375 mg/kg/inf cocaine was used for all acquisition phases. This dose has been shown to establish a steady cocaine seeking behavior in other studies (Froger-Colléaux and Castagné, 2016). In addition, this dose appeared to be the optimum dose for acquisition based upon preliminary data that were collected for this study (data not shown). For the reinstatement phase, 10 mg/kg cocaine, saline, or 1.0 mg/kg methamphetamine, was used as a drug cue to reinstate the extinguished drug seeking behavior (Jing et al., 2014; Shelton et al., 2013). Cocaine has been shown to reinstate an extinguished cocaine and methamphetamine-seeking behavior

(Pittenger et al., 2017; Shelton et al., 2013; Worley et al., 1994); therefore, cocaine and methamphetamine were used as positive controls. Saline has been shown to not reinstate an extinguished cocaine seeking behavior (Worley et al., 1994) and therefore, was used as a negative control. As a formal study on mephedrone reinstatement of cocaine self- administration has yet to be publicly published, a dose range of 0.5-10 mg/kg mephedrone was used as a prime to test its ability to reinstate the extinguished cocaine

! 65! seeking behavior, based on doses previously shown to be behaviorally active (Gatch et al.,

2013; German et al., 2014; Hadlock et al., 2011; Harvey and Baker, 2016; Shortall et al.,

2013a,b; Varner et al., 2013).

For the substitution phase, rats self-administered a dose range of 0.175-0.575 mg/kg/inf mephedrone or saline. The D1 receptor antagonist 0.0025-0.02 mg/kg

SCH23390 (Awasaki et al., 1997; Brennan et al., 2009; Caine and Koob, 1994; Stairs et al., 2010) was used to antagonize the established drug-seeking behavior. The D1 receptor was examined because this receptor is thought to be an important receptor in mediating the reinforcing effects of cocaine and may also be an important receptor in mediating the reinforcing effects of mephedrone. In D1 receptor knockout mice, cocaine acquisition was not readily attained, when compared to wild type mice. When mice were trained to self- administer the D1-like agonist, SKF82958, the D1 receptor knockout mice failed to self- administer while the wild type mice did not. Additionally, the D1 receptor antagonist

SCH23390 antagonized the reinforcing effects of cocaine in wild type mice during cocaine self-administration (Caine et al., 2007). Thus the D1 receptor may be important in mediating the reinforcing effects of cocaine, specifically, during self-administration.

Experiment 2A: Reinstatement Effects of Mephedrone on Cocaine Trained Rats

Materials and Methods

Subjects

A total of 76 drug naïve male Sprague-Dawley rats were purchased from Taconic

Farms (Cranbury, NJ, USA) weighing 200-250g, at the start of the experiment. For the reinstatement procedure, 70 rats were used. For the blood analysis procedure, 6 rats were

! 66! used. As in Experiment 1, rats were initially group-housed with food and water available ad libitum throughout all phases of the experiment, and placed on a reverse 12-hour light/dark cycle. After 3 days, rats were separated and individually housed. Rats were placed on a food-restricted diet of 15 g of Purina Rodent chow per day, for the first three days of acquisition. This experiment was conducted 7 days a week and began shortly after the beginning of the active dark cycle. All rats were maintained in accordance with the Guidelines of the Institutional Animal Care and Use Committee of Temple University

(Institution of Laboratory Animal Research, National Academy Press; Eighth edition, revised 2011).

Drugs

For the acquisition phase, 0.375 mg/kg/inf cocaine was used. For the reinstatement phase, 10 mg/kg cocaine, 0.5, 1.0, 5.0, and 10 mg/kg mephedrone, saline, and 1.0 mg/kg methamphetamine were used as drug cues for the reinstatement phase.

Mephedrone was generously donated from the Fox Chase Chemical Diversity

Program (Doylestown, PA). The National Institute on Drug Abuse Drug Supply Program

(Bethesda, MD) provided cocaine and methamphetamine. SCH23390 was purchased from Tocris (Ellisville, MO).

Cocaine, methamphetamine, mephedrone and SCH23390 were all dissolved in

0.9% saline. All reinstatement injections were given intraperitoneally (i.p.) in a volume of

1.0 mL/kg of body weight.

! 67! Operant Self-Administration Chambers

Experiments were conducted in six aluminum and polycarbonate behavioral operant experimental chambers, similar to the ones described in Experiment 1. These chambers were located within ventilated, sound-attenuating enclosures and contained removable self-administration attachments. Atop of the chamber was a swivel system

(PHM-110-SAI, Med Associates, Inc., Georgia, VT, USA; Model SIV-20, SAI Infusion

Technologies, Lake Villa, IL, USA) that connected to the rat’s catheter (Model RJVR-10,

SAI Infusion Technologies), via the chew-resistant lead or tether, and allowed for administration of drug or saline over a period of time. This system allowed free movement of the rat while inside the operant experimental chamber. The infusion pump

(Model PHM-100, Med Associates, Inc., Georgia, VT, USA) for the drug administration was located outside of each chamber. Experimental contingencies were controlled using a software-installed computer and the data were recorded by the computer-driven interface

(MED Associates, Inc., St. Albans, VT, USA).

As mephedrone can increase locomotor activity (Aarde et al., 2013), to distinguish between an increased self-administration behavior and an increase in nonspecific motor activity, two levers were extended into the self-administration chamber.

Appropriate responding on the first (right) lever, resulted in presentation of an infusion of drug or saline, at a rate of 1.39 to 2.76 s. Responses on the second (left) lever did not produce any consequences, however, responses were still recorded.

! 68! Procedure

Intravenous Catheter Implantation

To insert the self-administration catheter into the jugular vein, surgery was performed. Before the surgery, rats were pretreated with an analgesic (1.0 mg/kg meloxicam, subcutaneously), 30 min before the first incision. Rats were then anesthetized using a mixture of 5% isoflurane and oxygen. The delivery of isoflurane was reduced and maintained at 2-3% for the remainder of the surgery. Silicone catheters were inserted into the right external jugular vein, as described by Thomsen and Caine (2005). Very briefly, the rat was clipped and incisions were made, in the ventricle portion, directly above the right jugular vein, and on the dorsal portion, directly below the shoulder blades. The catheter was positioned subcutaneously from the back incision to the front incision. Once in place, the jugular vein was located and cleaned. A small incision was made in the jugular vein and the catheter was inserted and guided through until the bead, located on the catheter, was reached. Two silk sutures then held the bead of the catheter in place, the catheter was flushed and locked with a flushing solution (1:1:8 mixture of heparin,

Baytril, and sterile saline), and the front incision was closed using a combination of silk sutures and skin adhesion glue. The purpose of the flushing solution is to administer prophylactic and to prevent clotting. On the dorsal surface of the rat, the catheter button was subcutaneously inserted and placed towards the top of the incision.

The incision was then sutured, starting from the base of the catheter button, cleaned, and stabilized with skin adhesion. Lastly, the rat was immediately placed into a freshly cleaned home cage and on top of a heating pad for approximately 30 min before being placed back into the colony room.

! 69! Patency for all inserted catheters was maintained by flushing approximately 0.1-

0.2 ml of the flushing solution once a day into the catheter. On the day of the surgery, and

2-3 days after, catheters were flushed twice a day. Meloxicam was given for three or more consecutive days, starting the day of the surgery. Once the catheter was implanted, rats recovered and were minimally handled for 3-5 days until normal behavior returned.

Before and after each session, catheters were flushed with the flushing solution, even on days without experiment. Catheters were checked after each session for adequate flow, by pulling back on the syringe to ensure ease of blood flow, right before flushing occurred.

Overall Self-Administration Strategy Procedure

Behavioral studies examining reinstatement properties of mephedrone were conducted in three phases: 1) a cocaine acquisition phase, which lasted for 14 sessions; 2) an extinction phase, which lasted for 7-20 sessions; and, 3) a drug-induced reinstatement phase, which lasted for only one session. At the beginning of each session, rats were weighed and the rate of infusion was calculated to administer the proper dose of drug per infusion. The infusion rate was determined by the equation:

!"#$ ∗ !"#$ℎ! !"#$%&!"!!"#$ = !"#$ ∗ !"#$%

Rats were then placed into the operant experimental chamber, their catheter was connected to the swivel tether, and the session began.

! 70! Cocaine Acquisition Phase

After the recovery period, rats entered the self-administration cocaine acquisition phase. During this period, rats were placed into the chamber for a single 2-hour daily session on a schedule of FR1. Each session immediately started with no timeout pretreatment. At the start of each session, both levers were extended and the house light was illuminated. Each response made on the right lever was accompanied by the illumination of the stimulus light above the right lever, offset of the house light, a tone stimulus, and an infusion of 0.375 mg/kg/inf cocaine over 1.39 to 2.76 s from the infusion pump, as a reinforcer. After delivery, the house light extinguished and any responses made on the right lever did not have any consequences for 20 s. This 20 s post- infusion time-out period allowed the reinforcer to have an effect and helped in prohibiting the rat from overdosing. Throughout the experiment, if a response was made on the left lever, there was no delivery of drug; however, the lever-pressing behavior was still recorded. Before extinction and reinstatement testing occurred, the acquisition period lasted for each rat individually until three criteria were met: 1) completed a minimum of

14 acquisition sessions; 2) self-administered no less than 15 drug infusions for 3 consecutive acquisition sessions; and, 3) performed at least three baseline sessions where responses did not vary more than 10% (Koob et al., 2007). Any rat that did not reach acquisition criteria within 14 sessions was excluded from the study. The last three baseline acquisition sessions for a given rat were averaged together and represented the amount of infusions made during the acquisition phase.

! 71! Extinction Phase

Once criteria were met for the acquisition phase, rats entered the extinction phase.

This phase was similar to the acquisition phase, except the infusion pumps and the sound of the tones were turned off. When a response was made on the right lever, it was still accompanied by the illumination of the stimulus light above the right lever and the offset of the house light for 20 s. Responses made on both levers and the numbers of ‘blank’ infusions were still recorded. Before reinstatement testing occurred, the extinction period lasted for each rat individually until two criteria were met: 1) completed a minimum of 7 extinction sessions; and 2) the rat self-administered no more than 35% of their acquisition baseline session for three consecutive days. Any rat that did not reach criteria within 20 extinction days was removed from the study.

Reinstatement Phase

Cocaine-trained rats underwent a single reinstatement phase with a cocaine, methamphetamine, mephedrone, or saline primed injection. This phase was similar to the acquisition phase, except rats were injected i.p. with 10 mg/kg cocaine, 1.0 mg/kg methamphetamine, 0.5, 1.0, 5.0, or 10 mg/kg mephedrone, or with 0.9% saline. This phase began the day after successful extinction and lasted for one day.

Blood Collection

The concentration of mephedrone in the plasma of three rats who had a history of cocaine was compared to three rats that did not have a history of cocaine. The cocaine group underwent the Intravenous Catheter Implantation, the Cocaine Acquisition Phase,

! 72! and a 10-day Extinction Phase as described above. The control group also underwent the

Intravenous Catheter Implantation, an Acquisition Phase in which rats were placed in the chamber and exposed to the cues for 14 days, and a 10-day Extinction Phase. The day after the Extinction Phase, rats were injected, i.p, with 5.0 mg/kg mephedrone. Blood was collected (maximum of 100 µL) from the catheter at 8 different time points: 0 min, 15 min, 30 min, 60 min, 2 hrs, 4 hrs, 8 hrs, and 24 hrs. The collected blood was immediately stored in Eppendorf tubes that contained 20 µL of dried heparin to prevent clotting, and inverted at least 5 times to evenly distribute the anticoagulant additive. Blood was centrifuged at 10,000 rotations per minute for 10 min at 4°C. The supernatant plasma was extracted and placed into a clean, labeled Eppendorf tube and placed immediately on dry ice. Blood was stored at -80°C for 3 days before UPLC/MS analysis.

Sample Preparation and UPLC/MS Method

Protein was precipitated from samples by adding 2 volumes of acetonitrile containing 10 nM propafenone (internal standard), followed by centrifugation for 10 min at 2300 x g. Supernatants were diluted with one volume of water and then transferred to a

96 well polypropylene plates sealed with a cap mat for UPLC/MS analysis.

Mephedrone concentrations were determined using a Waters Aquity UPLC/Xevo

TQ MS tandem quad mass spectrometer system. 5 µL samples were fractionated on a

Waters Aquity UPLC BEH C18 1.7 µm column (2.1 x 50 mm) equipped with a Vanguard

Aquity UPLC BEH C18 1.7 µM precolumn (2.1 x 5 mm). The column was run at 40°C using a 3 min 5-95% acetonitrile gradient containing 0.1% formic acid (% ACN: 0-0.3 min 5%; 0.3-1.3 min, 5-20%; 1.3-1.9 min, 20-60%; 1.9-2.3 min, 60-95% 2.3-2.4 min, 95-

! 73! 95%; 2.4-3.0 min, 5%) at 0.65 ml/min. The mass spectrometer was operated in electrospray positive mode with tune conditions of capillary voltage 0.60 kV, source temperature 150°C, desolvation temperature 550°C, desolvation gas flow 800 L/hr and collision gas (argon) flow 0.15ml/min. Multiple reaction monitoring (MRM) methods

[compound, precursor ion > product ion; cone voltage (V); collision voltage (V), retention time (min)]: mephedrone (quantitation ion), 178.08>145.03, 20, 20, 1.3; mephedrone (confirmation ion), 178.08>130.04, 20, 28, 1.3; propafenone, 342.30>116.01,

28, 22, 2.1. Analytes were quantified using Waters MassLynx v4.1 software using 11 point standard curves with propafenone as the internal standard.

Data Analysis

For this experiment, the dependent variables that were measured and recorded were the amount of responses made for drug self-administration on the active (right) and inactive (left) lever, and the number of total infusions delivered. Dependent variables are expressed as a group mean, along with the standard error of the mean. The results of any rat that did not complete a given phase were not included in the average of that phase.

‘Full’ reinstatement was considered to be at least a 10% decrease of infusions made during the acquisition phase, or higher. For example, if a rat made 50 infusions during acquisition, full reinstatement would be considered 45 infusions or higher.

To determine if there were significant differences between the cocaine acquisition phases, acquisition data from rats that reinstated with cocaine, methamphetamine, mephedrone, and saline, were analyzed by one-way ANOVA, followed by Dunnett’s multiple comparison test. To determine if there was a significant difference between the

! 74! cocaine acquisition phase and the reinstatement phase of a particular drug, at a particular dose, a paired t-test was performed. Lastly, to compare the reinstatement phase of methamphetamine, mephedrone, and saline to the reinstatement phase of the control, cocaine, a one-way ANOVA, followed by Dunnett’s multiple comparison test, was performed. To determine if there were significant differences between the concentration of mephedrone found in the plasma of rats with a history of cocaine and rats without a history of cocaine, results of both groups were analyzed using a two-way ANOVA.

Significance was set at p<0.05 for all analyses.

Results

Of the 70 rats that began the experiment, 3 did not survive surgery, 15 did not acquire the cocaine self-administration behavior, and 7 did not extinguish the cocaine self-administration behavior. We chose a dose of 0.375 mg/kg/inf cocaine for acquisition based upon preliminary data collected (see Figure 13). A dose of 0.1 and 0.25 mg/kg/inf cocaine produced a number of infusions that were too low to maintain (16.28±13.31 and

14.56±7.77, respectively). A dose of 0.3 mg/kg/inf cocaine produced a number of infusions that were higher than the rest of the doses tested (40.59±5.46), however, this dose was maintained in only 4 of the 5 rats tested. We therefore decided to establish a dose of 0.375 mg/kg/inf cocaine for all acquisition phases. This dose maintained responding in 5 out of 5 rats and produced a number of infusions (24.59±13.84) that was able to fulfill the criterion of making no less than 15 responses (see Procedure) during the cocaine acquisition phase.

! 75! C o c a in e D o s e S u b s titu tio n

8 0 C o c a in e 0 .1 C o c a in e 0 .2 5 s n

o C o c a in e 0 .3 i 6 0 s u

f C o c a in e 0 .3 7 5 N =4 n I

f 4 0 o

N =5 r

e N =5 b N =5

m 2 0 u N

0 0 .1 0 .2 5 0 .3 0 .3 7 5 D o s e m g /k g /in f ! Figure 14. Cocaine Dose Substitution. Maintained responding of 0.1, 0.25, 0.3, and 0.375 mg/kg/inf cocaine responding. Cocaine responding was collected for at least two consecutive days and averaged together. Abscissae: Doses of cocaine administered. Ordinate: Number of total infusions made during a 2-hour session.

After the acquisition dose of cocaine was established, the reinstatement experiments began. A total of 50 rats did reach the reinstatement phase; however, out of the 50 rats that were tested in reinstatement, only 25 reinstated (see Table 1 and Figure

15). A one-way ANOVA failed to detect a significant overall difference between the number of infusions made during the acquisition phases of 10 mg/kg cocaine, 0.5, 1.0,

5.0, and 10 mg/kg mephedrone, saline, and methamphetamine (F(6,41)=1.193; N.S.).

Cocaine and methamphetamine reinstated the extinguished cocaine self-administration behavior. A paired t-test failed to detect a significant difference between the number of infusions made during the acquisition phase and the number of infusions made during the reinstatement phase of 10 mg/kg cocaine (t(7,8)=0.500; p=N.S.). A paired t-test also failed to detect a significant difference between the number of infusions made during the acquisition phase and the reinstatement phase of 1.0 mg/kg methamphetamine

! 76! (t(4,5)=1.021; N.S.). All doses of mephedrone and saline did not reinstate the extinguished cocaine self-administration behavior. A paired t-test detected a significant difference between the number of infusions made during the acquisition phase and the reinstatement phase of 0.5 and 5.0 mg/kg mephedrone (t(5,6)=3.424; p< 0.05; t(6,7)=5.154; p<0.005, respectively) and the acquisition phase and the reinstatement phase of saline (t(6,7)=4.741; p<0.005). A paired t-test failed to detect a significant difference between the number of infusions made during the acquisition phase and the reinstatement phase of 1.0 and 10 mg/kg mephedrone (t(7,8)=1.983; p=0.0878); t(6,7)=2.323; p=0.0588), respectively), although the average number of infusions were not numerically similar (36±114 vs. 25±8.5; 35±6.0 vs. 19±19.1). A one-way ANOVA failed to detect significant overall difference between saline reinstatement and all doses of mephedrone reinstatement (F(4,31)=0.6118; N.S.).

A one-way ANOVA detected a significant overall difference between the 10 mg/kg cocaine reinstatement and the reinstatement of all doses of mephedrone, saline, and methamphetamine (F(6,42)=4.909; p<0.001). Specifically, Dunnett’s post hoc tests indicated that doses of 0.5, 1.0, 5.0, and 10 mg/kg mephedrone, and saline, significantly decreased the number of total infusions rates relative to 10 mg/kg cocaine.

! 77! R e in s ta te m e n t 8 0 A c q u is itio n

N = 8 s * * C o c a in e n

o 6 0 N = 5 i N = 7 M e p h e d ro n e s N = 7 N = 8 N = 7 N = 7 u f S a lin e n I

f 4 0 M e th a m p h e ta m in e o

r

e *

b *

m 2 0 * u N

0 1 0 0 .5 1 .0 5 .0 1 0 0 .0 1 .0 D o s e m g /k g

Figure 15. Reinstatement of Extinguished Drug-Seeking Behavior. Dose effect of 10 mg/kg cocaine, 0.5, 1.0, 5.0, and 10 mg/kg mephedrone, saline, and 1.0 mg/kg methamphetamine on cocaine reinstatement in rats. Infusions during acquisition did not significantly differ between groups. Reinstatement of mephedrone and saline significantly decreased the number of infusions relative to cocaine acquisition (*, p<0.05). The reinstatement of mephedrone and saline were significantly lower than the reinstatement of cocaine (**, p<0.05). Dashed lines separate mephedrone group from other tested groups. Abscissae: Doses of drug administered during reinstatement phase. Ordinate: Number of total infusions made during a 2-hour session. The acquisition of a given group represents the average last three sessions during the respective acquisition phase.

Table 1

Number of Rats That ‘Fully’ Reinstated During Reinstatement Phase Drug Rats ‘Fully’ Reinstated/Total Rats Tested 10 mg/kg Cocaine 6/8 0.5 mg/kg Mephedrone 3/7 1.0 mg/kg Mephedrone 3/8 5.0 mg/kg Mephedrone 2/7 10 mg/kg Mephedrone 2/7 Saline 3/7 1.0 mg/kg Methamphetamine 6/6 Left column: Dose and drug administered i.p. during the reinstatement phase. Right column: Number of total rats that fully reinstated/Number of total rats tested.

! 78! In the metabolism study, mephedrone was detected in the plasma of all rats within

15 min after mephedrone administration and time dependently decreased. Concentrations of mephedrone returned close to baseline after 8 hours (see Table 2 and Figure 16). A matching, stacked two-way ANOVA failed to detect an overall significant difference between the concentration of mephedrone found in rats with a history of cocaine and controls (F(1,4)=0.023; N.S.). However, two-way ANOVA did detect an overall significant difference between the concentration of mephedrone found in rats over a period of time (F(7,28)=16.83; p<0.0001).

Table 2

Concentration of Mephedrone Found in Plasma Time [Mephedrone] ng/mL [Mephedrone] ng/mL (min) With Cocaine History Without Cocaine History Rat 1 Rat 2 Rat 3 Mean± Rat 4 Rat 5 Rat 6 Mean± SEM SEM 0 0.09 0.13 0.07 0.09± 0.10 1.51 0.12 0.58± 0.03 0.81 15 295.34 57.65 789.04 380.68± 560.77 548.41 532.78 547.32± 373.09 14.03 30 368.80 101.57 651.47 373.95± 290.87 316.72 572.20 393.26± 274.99 155.50 60 203.89 206.30 431.72 280.64± 164.26 194.04 253.18 203.83± 130.85 45.26 120 99.14 76.57 81.20 85.64± 34.10 41.03 120.41 65.18± 11.92 47.96 240 17.34 11.32 30.73 19.80± 2.40 4.52 14.67 7.20± 9.94 6.56 480 2.13 0.59 2.05 1.59± 0.31 0.54 0.88 0.58± 0.85 0.29 1440 2.05 0.11 2.71 1.62± 2.10 3.19 0.62 1.97± 1.35 1.29 Concentration of mephedrone is measured in ng/mL

! 79! Mephedrone Blood Analysis 800

600 l m / g n

] Saline-Mephedrone e

n 400

o Cocaine-Mephedrone r d e h p e

M 200 [

0

0 .25 .5 1 2 4 8 Time (hours) Figure 16. Concentration of Mephedrone Found in Plasma. Blood analysis of rats with a history of cocaine self-administration versus blood analysis of rats without a history of cocaine self-administration. Concentration of mephedrone at a given time point, did not differ between groups. Abscissae: Time of blood collected after initial i.p. injection of 5.0 mg/kg mephedrone. Ordinate: Concentration of mephedrone found in the blood.

Experiment 2B: Maintenance Effects of Mephedrone On Cocaine Trained Rats

Materials and Methods

Subjects

A total of 38 drug naïve male Sprague-Dawley rats were purchased from Taconic

Farms (Cranbury, NJ, USA) weighing 200-250g at the start of the experiment. As in

Experiment 2A, rats were initially group-housed with food and water available ad libitum throughout all phases of the experiment and placed on a reverse 12-hour light/dark cycle.

After 3 days, rats were separated and individually housed. Rats were also placed on a food-restricted diet of 15 g of Purina Rodent chow per day, for only the first three days of acquisition. This experiment was conducted 7 days a week and began shortly after the beginning of the active dark cycle. All animals were maintained in accordance with the guidelines of the Institutional Animal Care and Use Committee of Temple University

! 80! (Institution of Laboratory Animal Research, National Academy Press; Eighth edition, revised 2011).

Drugs

To establish the self-administrating behavior, 0.375 mg/kg/inf cocaine was used for the acquisition phase. For the substitution phase, rats self-administered 0.175, 0.375, or 0.575 mg/kg/inf mephedrone or saline. To antagonize the established drug-seeking behavior, 0.0025, 0.01, and 0.02 mg/kg SCH23390 or 0.9% saline was used as a pretreatment.

Mephedrone was generously donated by the Fox Chase Chemical Diversity

Program (Doylestown, PA). Cocaine was provided by the National Institute on Drug

Abuse (Bethesda, MD) drug supply program. SCH23390 was purchased from Tocris

(Ellisville, MO). Cocaine, mephedrone and SCH23390 were each dissolved in 0.9% saline. All injections were given intraperitoneally (i.p.) in a volume of 1.0 mL/kg of body weight.

Operant Self-Administration Chambers

Experiments were conducted in six aluminum and polycarbonate behavioral operant experimental chambers, exactly as the ones described in Experiment 2A.

Both levers were extended into the self-administration chamber. Appropriate responding on the first (right) lever, or response key, resulted in presentation of an infusion of drug or saline, at a rate of 1.39 to 4.60 s. Responses on the second (left) lever did not produce any consequences, however, responses were still recorded.

! 81!

Procedure

Intravenous Catheter Implantation

To insert the self-administration catheter into the jugular vein, the same surgery was performed as in Experiment 2A.

Overall Self-Administration Strategy Procedure

Behavioral studies examining the self-administration maintenance properties of mephedrone were conducted in three phases: 1) a cocaine acquisition phase, which lasted for 14 sessions; 2) a drug substitution phase, which lasted for 3 sessions at a time; and, 3) an antagonist phase, which lasted for only 1 session. At the beginning of each session, rats were weighed and the rate of infusion was calculated to administer the proper dose of drug per infusion (see Experiment 2A). Rats were then placed into the operant experimental chamber, their catheter was connected to the swivel tether, and the session began.

Self-Administration Cocaine Acquisition Phase

After the recovery period, rats entered the self-administration cocaine acquisition phase. This phase was exactly the same as the cocaine acquisition phase described in

Experiment 2A. During this period, rats were placed into the chamber for a single 2-hour daily session on a schedule of FR1. Each session immediately started with no timeout pretreatment. At the start of each session, both levers were extended and the house light was illuminated. Each response made on the right lever was accompanied by the

! 82! illumination of the stimulus light above the right lever, offset of the house light, a tone stimulus, and an infusion of 0.375 mg/kg cocaine over 1.39 to 2.56 s from the infusion pump, as a reinforcer. After delivery, the house light extinguished and any responses made on the right lever did not have any consequences for 20 s. Throughout the experiment, if a response was made on the left lever, there was no delivery of drug; however, the lever-pressing behavior was still recorded.

Before the substitution phase occurred, the cocaine acquisition period lasted for each rat individually until three criteria were met: 1) completed a minimum of 14 acquisition sessions; 2) self-administered no less than 15 drug infusions for 3 consecutive acquisition sessions; and, 3) performed at least three baseline sessions where responses did not vary more than 10%. Any rat that did not reach acquisition criteria within 14 sessions was excluded from the study. The last three baseline acquisition sessions of a given rat were averaged together and represented the final amount of infusions made during the acquisition phase.

Self-Administration Drug Substitution Phase

Once criteria were met for the cocaine acquisition phase, rats entered the drug substitution phase. This phase was similar to the cocaine acquisition phase, except infusion pumps delivered 0.375 mg/kg/inf cocaine, 0.375 mg/kg/inf mephedrone, or saline. Responses made on both levers and the numbers of infusions were recorded. This phase continued for 3 consecutive sessions. After the third session, rats entered the antagonist phase for one session (see below). Once the antagonist phase was completed, rats re-entered the drug substitution phase, to re-establish the drug substitution baseline.

! 83! This cycle continued until all pretreatment doses were tested or until catheters failed, whichever occurred first.

After the last pretreatment dose, rats re-entered the drug substitution phase and re- established the mephedrone baseline. Thereafter, infusion pumps delivered a dose of

0.175, 0.375, or 0.575 mg/kg/inf mephedrone. This was to establish a dose response curve for mephedrone substitution.

Self-Administration Antagonist Phase

This phase was similar to the drug substitution phase, except rats were injected i.p. with saline or SCH23390 right before the beginning of the session. This phase began the day after the drug substitution phase and lasted for one day. Thereafter, the rats re-entered the drug substitution phase (see above) for another three days and re-entered the antagonist phase, where they were administered a different pretreatment. This cycle continued until all pretreatments were tested or until catheters failed, whichever occurred first.

Data Analysis

Again, for this experiment, the dependent variables that were measured and recorded were the amount of responses made for drug self-administration on the active

(right) and inactive (left) lever, and the number of total infusions delivered. Dependent variables were expressed as a group mean, along with the standard error of the mean.

Any rat that did not re-establish the drug baseline was removed from the study and the responses made for the given phase were not included into the group average. Any rat

! 84! whose catheter failed during a given phase was removed from the study and the responses made for the given phase were not included into the group average.

A one-way ANOVA analysis, followed by a Dunnett’s multiple comparison test, was used to determine if there was a difference between the cocaine acquisition phase and the drug substitution phase. It was also used to determine if the multiple doses of antagonist pretreatment altered responding that was made in the drug substitution phase.

A paired t-test was used to determine if a particular dose of 0.1 mg/kg SCH23390 altered the responding that was made in the drug substitution phase. Significance was set at p<0.05 for all analyses.

Results

In the substitution studies (see Figure 17), one-way ANOVA indicated that there was not a significant difference between 0.375 mg/kg/inf cocaine and the tested doses of mephedrone (F(3,41)=1.105; N.S.). The dose of 0.175 mg/kg/inf mephedrone maintained self-administration in 6 out of 8 rats. The dose of 0.375 mg/kg/inf mephedrone maintained self-administration in 12 out of 13 rats. The dose of 0.575 mg/kg/inf mephedrone maintained self-administration in 8 out of 9 rats.

For the antagonism study (See Figure 18), a one-way ANOVA did identify an overall significant difference between the number of cocaine, mephedrone, and saline infusions, during the drug substitution phase (F(2,20)=23.89; p<0.0001). Specifically,

Dunnett’s post hoc tests indicated that the number of infusions significantly decreased when rats were self-administering saline, relative to cocaine and mephedrone. A one-way

! 85! M e p h e d ro n e D o s e S u b s titu tio n

8 0 C o c a in e 0 .3 7 5 s N =8 n M e p h e d ro n e o

i 6 0 s u

f N = 15 N = 13 N =9 n I

f 4 0 o

r e b

m 2 0 u N

0 0 .3 7 5 0 .1 7 5 0 .3 7 5 0 .5 7 5 D o s e m g /k g /in f

Figure 17. Mephedrone Dose Substitution for Cocaine Self-Administration. The maintenance of 0.375 mg/kg/inf cocaine responding when substituted with 0.175, 0.375, and 0.575 mg/kg/inf mephedrone. Mephedrone substitution was collected for three consecutive days and averaged together. Abscissae: Doses of drug administered during substitution phase. Ordinate: Number of total infusions made during a 2-hour session. Cocaine responding represents the average last three sessions during the acquisition phase. Responses made during the substitution phase were averaged together to represent the respective dose of mephedrone.

ANOVA failed to identify an overall significance of SCH23390 dose effects for number of infusions of 0.375 mg/kg/inf cocaine (F(4,29)=2.045; N.S.); however a paired t-test did find a dose of 0.2 mg/kg SCH23390 to significantly increase the number of infusions of 0.375 mg/kg/inf cocaine (t(6,12)=2.831; p<0.05). Likewise, a one-way ANOVA failed to identify an overall significance of SCH23390 dose effects for number of infusions of

0.375 mg/kg/inf mephedrone (F(4,37)=0.8225; N.S.). A one-way ANOVA did identify an overall significance of SCH23390 dose effects for number of saline infusions

(F(4,25)=4.101; p<0.05). Specifically, Dunnett’s post hoc tests indicated that a dose of

! 86! 0.02 mg/kg SCH23390 decreased the number of saline infusions relative to saline self- administration alone.

S u b s titu tio n A n ta g o n is m C o c a in e 0 .3 7 5 m g /k g /in f 8 0 N =8 N =6 + S C H 2 3 3 9 0

s * N =8

n M e p h e d ro n e 0 .3 7 5 m g /k g /in f N =7 o

i 6 0 N = 10 N =8 N =8

s + S C H 2 3 3 9 0

u N =6 N =7 f N =7 S a lin e n I

f 4 0 + S C H 2 3 3 9 0 o

r

e N =6 N =6 N =6 N =6 N =6 b

m 2 0 ** u N * 0

e e 5 1 2 e e 5 1 2 e e 5 1 2 n n 2 0 0 n n 2 0 0 n n 2 0 0 i li 0 . . li 0 . . li li 0 . . a 0 0 0 o 0 0 0 0 0 0 c a . r a . a a . S 0 + + d S 0 + + S S 0 + + o e C + + h + + + + p e M D o s e m g /k g Figure 18. Dose Antagonism of SCH23390 of Drug Self-Administration. Effects of the D1 receptor antagonist SCH23390 on drug-seeking behavior. The number of total infusions significantly decreased when rats self-administered substituted saline, when compared to self-administering cocaine (**, p<0.0001). SCH23390 significantly attenuated the number of total infusions in rats responding for saline (*, p<0.05). Other details as in Figure 17.

Experiment 2 Discussion

Although mephedrone did not fully reinstate the extinguished cocaine self- administration behavior, mephedrone was able to substitute and maintain cocaine-seeking behavior. In Experiment 2A, over half of the rats tested, failed to reinstate cocaine self- administration behavior when given a prime injection of mephedrone. Out of the 29 rats tested with 0.5-10 mg/kg mephedrone, only 10 rats exhibited full reinstatement. A primed injection of 1.0 mg/kg mephedrone produced the greatest increase of number of infusions during the reinstatement phase; however only 3 out of 8 rats actually fully reinstated.

Although the difference between the cocaine acquisition phase and reinstatement phase

! 87! of 1.0 and 10 mg/kg mephedrone were not significantly different, these doses did not produce full reinstatement. All doses of mephedrone produced reinstatement patterns that were similar to saline, suggesting a difference in the reinforcing effect of cocaine and mephedrone. From this present study, mephedrone appears to only weakly reinstate an extinguished cocaine-seeking behavior and may not be as reinforcing as cocaine or share as much of an underlying mechanism than what was previously suggested by ‘users’ and scientific studies (DeLarge et al., 2017; Varner et al., 2013; Gannon and Fantegrossi,

2016; Gatch et al., 2013; Simmler et al., 2013; Smith et al., 2016; Winstock et al., 2010).

Since 1.0 mg/kg methamphetamine was able to reinstate the extinguished cocaine-seeking behavior and, therefore, able to mimic the cocaine-seeking behavior, methamphetamine appears more similar to cocaine than mephedrone or a stronger reinforcer. Methamphetamine is a monoamine releaser that causes an increase of both

DA and 5-HT. Microdialysis shows that methamphetamine induced a greater increase of

DA levels in the caudate–putamen than 5-HT levels in the hippocampus (1460% and

870%, respectively) (Matsumoto, et al., 2014). Similar to cocaine, methamphetamine causes a greater release of DA than 5-HT neurotransmitters and this similarity is apparent in self-administration studies. In male and female Sprague-Dawley rats, 5.0 and 10 mg/kg cocaine was able to reinstate an extinguished methamphetamine-seeking behavior

(Pittenger et al., 2017).

The blood analysis confirmed that rats with a history of cocaine did not differ in the metabolism of mephedrone, from rats that did have a history of cocaine. Therefore, this small study eliminates the possibility that mephedrone did not reinstate cocaine because previous cocaine exposure changed the metabolism of mephedrone to be less

! 88! potent. A more likely reason for the failure of mephedrone to fully reinstate cocaine may be partially due to the serotonergic component of mephedrone and not the cocaine history of the rats or changes in metabolic processes. Cocaine is a monoamine transporter inhibitor that increases both DA and 5-HT in nucleus accumbens, however the increase of

DA is more prominent than that of 5-HT (285% and 191%, respectfully) (Andrews and

Lucki, 2001). Mephedrone, on the other hand, also binds to monoamine transporters and increases both DA and 5-HT, but the increase of 5-HT is more prominent than the increase of DA (Kher et al., 2011). Mephedrone releases 5-HT in a fashion that is similar to MDMA, therefore, mephedrone should reinstate in a fashion similar to MDMA. When rats were trained to self-administer 0.5 mg/kg/inf cocaine, an initial i.p. injection of cocaine reinstated the extinguished cocaine seeking behavior; however, when rats were trained to self-administer 0.5 mg/kg/inf cocaine, an initial i.p. injection of 10 mg/kg

MDMA did not reinstate the extinguished cocaine seeking behavior. Instead, rats required multiple exposures to i.p. injections of MDMA, before MDMA was able to reinstate the extinguished cocaine seeking behavior (Schenk et al., 2008). Similar to

MDMA, the initial exposure of mephedrone did not reinstate the extinguished cocaine seeking behavior and may require repeated exposure to become an effective drug prime.

It is suggested that cocaine reinforcement is mediated by the inhibition of both

DAT and SERT, not only DAT. When homozygous DAT knockout mice were tested in conditioned place preference and self-administration, cocaine still portrayed prominent reinforcing effects. When double DAT and SERT knockout mice were tested in condition placed preference, the reinforcing effects of cocaine were eliminated. It is further thought that the inhibition of SERT may equally contribute to both the rewarding and aversive

! 89! effects of cocaine and the inhibition of the norepinephrine transporter (NET) may greatly contribute to the aversive effects of cocaine (Uhl et al., 2002). Looking at the inhibition of cocaine, cocaine has a greater potency for inhibiting the DAT and NET than it does for

SERT (IC50 (µM) (95% CI): 0.768, 0.451 and 2.37, respectfully). When comparing this to mephedrone, mephedrone has a greater potency for inhibiting the NET, than it does for

SERT and DAT (0.254, 4.64, and 3.31, respectfully). Also mephedrone has a higher affinity for SERT and NET, than it does for DAT (>30, >25, and 3.4, respectfully)

(Simmler et al., 2013). Hence, mephedrone’s reinforcing effects, of a single exposure, may not be as potent during self-administration and conditioned place preference than cocaine and this may explain why mephedrone did not reinstate the extinguished cocaine seeking behavior.

Since mephedrone did not fully reinstate the extinguished cocaine self- administration behavior, we examined if mephedrone would substitute for cocaine self- administration and maintain responding instead. Similar to MDMA (Schenk et al., 2003), mephedrone was readily self-administered when a history of cocaine self-administration was present and this drug-seeking behavior was maintained. When cocaine was substituted with 0.375 mg/kg/inf mephedrone, the average amount of infusions did not differ from cocaine. Both drugs averaged 38 infusions within a 2-hour period. When the dose of mephedrone decreased to 0.175 mg/kg/inf, the amount of average infusions increased to approximately 48, and when the dose of mephedrone increased to 0.575 mg/kg/inf, the amount of average infusions slightly decreased to an average of 36 infusions within a 2-hour period. Although not dose dependent with this small range of doses, mephedrone substituted for cocaine.

! 90! Mephedrone may have substituted for cocaine, due to the shared dopaminergic effects between both drugs and the occurrence of sensitization. Both drugs cause an increase of both DA and 5-HT. Despite mephedrone causing a greater release of 5-HT than DA, it is possible that mephedrone is still able to increase a reasonable amount of

DA within the nucleus accumbens and this DA release is still able to serve as an interoceptive cue (see results from Experiment 1). Although we did not test this hypothesis, it may be a suggestion as to why mephedrone substituted for cocaine.

Sensitization is characterized as an increase of response when a stimulus is repeatedly presented (Shettleworth, 2010). Drug sensitization occurs when there is an increased drug effect after repeated exposure to a drug. Previous studies show that pretreating rats with the same, or similar stimulant drug sensitizes them to subsequent exposure (see Kalivas and Stewart, 1991; Liu et al., 2007; Schenk et al., 2003). Cross- sensitization occurs with the reinforcing effects of MDMA. In rats, pre-exposure to cocaine resulted in a decreased latency of MDMA acquisition during self-administration, when compared to rats that were pre-exposed to saline (Fletcher et al., 2001; Schenk, et al., 2003). Since mephedrone is similar to MDMA, mephedrone’s reinforcing effects may also be susceptible to sensitization. Since rats were repeatedly exposed to cocaine and had multiple cocaine self-administration sessions, repeated exposure to cocaine may have sensitized rats to the reinforcing effects of mephedrone. In Experiment 2A, desensitization may have occurred during the time of the extinction period for some rats, which may explain why mephedrone did not reinstate the extinguished cocaine-seeking behavior.

! 91! A moderate i.p. dose of the D1 receptor antagonist 0.01 mg/kg SCH23390 appears to antagonize the reinforcing effects of cocaine. Although the overall effects of 0.01 mg/kg SCH23390 did not antagonize the reinforcing effects of mephedrone, the response of individual rats highly suggest a trend in that direction. Out of the 8 rats that were pretreated with 0.01 mg/kg SCH23390, 6 rats had a drastic increase (almost double) of total number of mephedrone infusions when compared to responding with no SCH23390 pretreatment. Thus, the self-administering behavior of both cocaine and mephedrone may be partially mediated through the D1 receptor and may depend on individual animals.

A high dose of 0.02 mg/kg SCH23390 significantly attenuated the drug-seeking behavior when saline was available to self-administer. This is likely due to the rate- decreasing effects of SCH23390 in addition to the lack of reinforcing effects of saline.

When rats self-administering mephedrone were pretreated with 0.02 mg/kg SCH23390, the rate decreasing effects of SCH23390 were evident in individual rats, but not as a whole group. Out of the 8 rats tested, the number of total mephedrone infusions decreased by more than half in 4 rats. Hence, again there is a trend of D1 receptor involvement of the behavioral effects of mephedrone.

It has been shown that methamphetamine, cocaine, and mephedrone cause an increase of DA and the reinforcing effects of cocaine and methamphetamine are modulated through the D1 receptor. For example, Maldonado et al., (1993) and Anderson et al., (2003) found that when SCH23390 was injected directly into the nucleus accumbens, the cocaine-seeking behavior of rats significantly increased, indicating an antagonism of the reinforcing effects of cocaine through the D1 receptor. Similarly, when rats were trained to self-administer 0.1 mg/kg/inf methamphetamine, a dose of 0.2 and

! 92! 0.4 mg/kg SCH23390 significantly attenuated the drug-seeking behavior (Carati and

Schenk, 2011). Additionally, the D1 receptor antagonist SKF-81297 significantly attenuated the drug-seeking behavior in rats trained to self-administer methamphetamine, indicating an involvement of the D1 receptor in methamphetamine-seeking behavior

(Hiranita et al., 2010). Our results suggest that cocaine-seeking behavior is mediated through the D1 receptor and since methamphetamine reinstated for cocaine, they may share similar reinforcing mechanisms. Thus, our results align with what was already found in the literature.

While we only tested the involvement of the D1 receptor in cocaine and mephedrone self-administration, other receptors may be involved in mediating the reinforcing effects of cocaine and mephedrone. The D2 and the D3 receptor have been shown to modulate cocaine self-administration (e.g. Anderson et al., 2006; Caine et al.,

1997; Caine and Koob, 1993; Campiani et al., 2003; Cervo et al., 2003; Peng et al.,

2009). The D3 antagonist S33138 antagonizes the reinforcing effects of 0.5 mg/kg/inf cocaine at moderate doses and caused an increase in the cocaine self-administering behavior, while a high dose of S33138 attenuated the cocaine self-administering behavior

(Peng et al., 2009). A direct injection of the D2 receptor antagonist sulpiride into the nucleus accumbens has been shown to antagonize the drug-seeking behavior in rats trained to self-administer cocaine (Anderson et al., 2006). Besides DA receptors, 5-HT receptors are also shown to modulate cocaine’s behavioral and reinforcing effects (e.g.

Anastasio et al., 2014; Mcmahon and Cunningham, 2001; Nic Dhonnchadha, et al., 2009;

Swinford-Jackson et al., 2016; Walsh, S. and Cunningham, 1997). When the 5-HT2A receptor antagonist M100907 was injected directly into the prefrontal cortex of rats that

! 93! were trained to self-administer 0.75 mg/kg/inf cocaine, there was a significant decrease in the cue-reinstatement of the cocaine-seeking behavior, indicating the involvement of the

5-HT2A receptor in the reinforcing effects of cocaine (Nic Dhonnchadha, et al., 2009;

Pockros et al., 2011). Also, the activation of the 5-HT2C receptor, by MK212, attenuated both cue and drug-induced cocaine-seeking behavior while the 5-HT2C receptor antagonist SB242084 reversed these effects (Neisewander and Acosta, 2007). This further suggests the involvement of the 5-HT receptor in the reinforcing effects of cocaine. Since there are other receptors involved in mediating cocaine’s reinforcing effects, other receptors may also be involved in mediating mephedrone’s reinforcing effects.

The results from this current study suggest that after a history of cocaine self- administration, cocaine and methamphetamine reinstate extinguished cocaine-seeking behavior more fully than mephedrone. This may be due to mephedrone’s high release of the 5-HT neurotransmitter in the nucleus accumbens and its closer resemblance to

MDMA, than to cocaine. Although more work needs to be done, mephedrone appears to be able to substitute for cocaine self-administration and maintain responding on its own and this self-administering maintenance may be mediated through the D1 receptor.

! 94! CHAPTER 4

CONCLUSIONS

Involvement of DA and 5-HT in Reward

Understanding the neurological contributors of reward is very important when evaluating the abuse liability of drugs. When trying to identify the abuse liability of a novel drug that has appeared on the illicit market, it is common to compare it to other known drugs of abuse. The two main neurotransmitters that are involved in the neuro- mechanism of abusing a drug are DA and 5-HT.

In the central nervous system, tyrosine is converted to L-DOPA, which is then converted to DA by the removal of a carboxyl group. Dopamine may also act as a precursor for norepinephrine and epinephrine. Once DA is synthesized, it is transferred from the cytosol into synaptic vesicles, by vesicular monoamine transporter2 (VMAT2), until the neuron is stimulated. When stimulated, the synaptic vesicles fuse to the membrane and release DA into the synaptic cleft. When DA is released from the cytosol of the presynaptic neuron, it may bind to and activate a total of 5 subtypes of dopamine receptors (D1, D2, D3, D4, and D5). These receptors may be located on the dendrite of the postsynaptic neuron or located on the axon terminal of the presynaptic neuron and function as autoreceptors. After DA interacts with the receptor, it dissociates and is then mainly taken up from the synaptic cleft and back into the cytosol by the DAT. Once it is in the cytosol, it is either broken down by the enzymes monoamine oxidase (MAO) or catechol-O-methyl transferase (COMT), or it is transferred and stored back into synaptic vesicles by VMAT2 (Bortolato et al., 2010; Olguín et al., 2016).

! 95! Dopaminergic neurons are mainly found in the ventral tegmental area and in the substantia nigra. The axons originating in the ventral tegmental area are mainly projected into the hippocampus, nucleus accumbens and into the frontal cortex. The dopaminergic axons originating in the substantia nigra are primarily projected into the dorsal striatum region, which consist of the nucleus and the caudate putamen (Olguín et al., 2016; Prasad and Pasterkamp, 2009). Dopamine is largely responsible for the rewarding properties of natural reinforcement, such as food, sex, and shelter (Olguín et al., 2016). It is also largely responsible for the rewarding properties of many drugs that are commonly abused, such as cocaine (Ritz et al., 1987). Although DA is released in response to natural stimuli

(Fiorino et al., 1997), the release of DA is relatively larger when it is in response to drugs of abuse (Hernandez and Hoebel, 1988). Also, the subjective reward, or high, is directly correlated with the amount of DA that is released; a decrease in dopaminergic function is correlated to a decrease in reward (Volkow et al., 2009).

Similar to DA, 5-HT is also synthesized from an amino acid (L-tryptophan) in the central nervous system. Tryptophan is converted into 5-hydroxy-L-tryptophan (5-HTP), which is then converted to 5-HT by the removal of a carboxyl group. Serotonin may also act as a precursor for melatonin. Once 5-HT is synthesized, it is transferred from the cytosol into synaptic vesicles, by VMAT2, and stored near the nerve terminal until the neuron is stimulated by an action potential. When stimulated, the synaptic vesicles fuse to the membrane and release 5-HT into the synaptic cleft. When 5-HT is released from the cytosol of the presynaptic neuron, it may bind to and activate a total of 14 subtypes of serotonin receptors (5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C,

5-HT3, 5-HT4, 5-HT5A, 5-HT5B 5-HT6, and 5-HT7). These receptors may be located on

! 96! the dendrites of the postsynaptic neuron or located on the axon terminal of the presynaptic neuron. After 5-HT interacts with the receptor, it dissociates and is then mainly taken up from the synaptic cleft and back into the cytosol by the SERT. Once it is in the cytosol, it is primarily broken down by the enzymes monoamine oxidase (MAO) or it is transferred and stored back into synaptic vesicles by VMAT2 (Charnay and Leger,

2010; Bortolato et al., 2010).

Serotonergic cell bodies are mainly found in the raphe nucleus. Their axons are projected into many different regions of the brain, including the nucleus accumbens, the striatum, the frontal cortex, and the hippocampus. Serotonin largely modulates mood, cognition, sleep, and memory. Like DA, 5-HT is also involved in drug abuse by modulating drug-reward behavior (Higgins and Fletcher, 2003; Kranz et al., 2010), such as cocaine and methamphetamine. It has been suggested that the reinforcing effects of cocaine may be mediated through the 5-HT1A and 5-HT2C receptor, for antagonists of these receptors were able to attenuate cocaine-seeking behavior (Anastasio et al., 2014;

McMahon and Cunningham, 2001; Swinford-Jackson et al., 2016). The 5-HT2A receptor is involved in the discriminative stimulus effects of cocaine since the 5-HT2A receptor agonist DOI partially substituted for 10 mg/kg cocaine (Munzar et al., 2002). Also, the selective serotonin uptake inhibitor fluoxetine potentiated methamphetamine’s discriminative stimulus effects and caused a leftward shift in the dose-response curve.

This leftward shift was slightly antagonized by the 5-HT1A receptor antagonist

WAY100635 and fully antagonized by the 5-HT2 receptor antagonist ketanserin (Munzar et al., 1999). Serotonin has the ability to modulate the drug-reward behavior for the 5-HT neurons that originate in the raphe nuclei may project onto DA neurons within the ventral

! 97! tegmental area, substantia nigra, in the striatum and in the prefrontal cortex (e.g. Bubar and Cunningham, 2006; Clemett et al., 2000; Navailles et al., 2008). In rats, the 5-HT2A receptor antagonist SR46349B was able to significantly reduce the amount of DA release induced by amphetamine, in both the nucleus accumbens and striatum (Porras et al.,

2002). Hence 5-HT may be a potential target in facilitating the abuse liability of drugs.

TAAR1 and Regulation of Dopamine and Serotonin

Trace amine-associated receptor 1 (TAAR1) is a G-protein coupled receptor that is located in the central nervous system monoaminergic nuclei and may be located within the presynaptic neuron (Borowsky et al., 2001; Lindemann et al., 2008; Xie et al., 2007;

Xie and Miller, 2008; Xie and Miller, 2009). TAAR1 plays an important role in regulating the release of DA and 5-HT into the synaptic cleft (Lindemann et al., 2008;

Wolinsky et al., 2007; Xie et al., 2008). In transfected cells, DA, 5-HT, and norepinephrine activated TAAR1. This activation caused an efflux of DA, 5-HT, and norepinephrine, and inhibited its reuptake through the respective transporters (Xie et al.,

2008).

TAAR1 has a high affinity for amphetamine-like psychostimulants and, along with DA and 5-HT (Xie et al., 2008), amphetamine, methamphetamine, and MDMA are considered potent TAAR1 agonists (Bunzow et al., 2001; Reese et al., 2007; Xie and

Miller, 2009). Amphetamine, methamphetamine, and MDMA, were shown to produce cyclic!adenosine monophosphate production, which is an indication of TAAR1 activation

(Bunzow et al., 2001). In rhesus monkey and mice, methamphetamine was shown to dose dependently interact with TAAR1, which lead to an efflux of DA, inhibited DA uptake,

! 98! and reduced surface DAT levels (Xie and Miller, 2009). Thus, TAAR1 may play an important role in modulating dopaminergic and serotonergic activity, specifically for psychostimulants with an amphetamine structure.

Summary of Findings

In order to determine the pharmacological mechanism of action of mephedrone that may lead to its abuse ability, we used a combination of the drug discrimination and the self-administration assays. Together, these two experimental models are amongst the best to model drug addiction and to predict abuse liability (Solinas, 2006). Drug discrimination is very useful in pinpointing the interoceptive cues of a given drug by determining its discriminative stimulus effects. When testing drugs that are commonly abused, these interoceptive discriminative cues may play a major role in promoting abuse or addiction. Drug self-administration is useful in identifying and pinpointing the reinforcing properties of a drug. It has been proposed that the rewarding properties of a drug are the main contributor to its addictiveness (Nestler, 1992). When you test a novel drug in drug discrimination and self-administration, the results may give a pretty clear indication of the abuse or addiction sustaining properties of that drug (Solinas, 2006).

Hence, screening a drug with drug discrimination, followed by self-administration, is a good predictor for its abuse liability and can be used pre-clinically to study pharmacological mechanisms.

Due to the importance of both DA and 5-HT in drug reward, we focused on the contribution of both DA and 5-HT to the pharmacological profile of mephedrone in hopes of fine-tuning the mechanisms involved in its abuse liability. Although, in

! 99! Experiment 1, we predicted for a lower dose of mephedrone to be more serotonergic than the higher dose and for a higher dose of mephedrone to be more dopaminergic than the lower dose, it appears as if one mechanism is not more important than the other. It appears that both DA and 5-HT must be detectable in a drug for it to fully generalize to mephedrone. For both the high and low dose training dose groups, there is as much of a needed dopaminergic component as there is a serotoninergic component. The only drugs that fully substituted for mephedrone were the drugs that are considered ‘dirty,’ meaning, they interact with both the DAT and SERT or release DA and 5-HT and are not completely selective. In the high dose training group, only DOI, methamphetamine and cocaine partially substituted for mephedrone and in the low dose training group, only

DOI, methamphetamine, cocaine, and d-amphetamine fully substituted for mephedrone, while mCPP and WAY163909 partially substituted for mephedrone. MDMA was the only drug that fully generalized to both the high and low training dose mephedrone, suggesting that out of the drugs that were tested, the discriminative mechanism of these two drugs have the most in common. When rats were trained to discriminate a high and low training dose of mephedrone, those that learned to discriminate a low training dose, were more sensitive to the discriminating effects of mephedrone, while those that learned to discriminate a high training dose of mephedrone, were more sensitive to the rate decreasing effects of mephedrone. The discriminating and rate decreasing effects of a high training dose of mephedrone were not significantly antagonized by any of the DA and 5-HT receptor antagonists tested. However, the discriminating effect of a low training dose of mephedrone was significantly antagonized by SCH23390 and sulpiride and the rate decreasing effect was significantly antagonized by sulpiride and SB242084.

! 100! Thus, the discriminating effects and rate decreasing effects of mephedrone appears to be mediated by both the DA and 5-HT receptor, for both DA and 5-HT receptor antagonists were able to antagonize these effects. This further supports our conclusion that for both the high and low dose training dose groups, there is as much of a needed dopaminergic component as there is a serotoninergic component and the discriminative stimulus mechanism of mephedrone may be similar to that of MDMA and the involvement of both

DA and 5-HT may play an important role.

In Experiment 2, we predicted for mephedrone to be reinforcing and for the rewarding effects to likely be mediated through dopaminergic mechanisms. In our study, mephedrone was reinforcing when substituted and maintained after a history of cocaine self-administration behavior. However, mephedrone did not reinstate the cocaine-seeking behavior once it was extinguished in our reinstatement studies. Although there was not antagonism amongst the entire group, our results suggest that the reinforcing effects of mephedrone may have a trend similar to cocaine that is mediated through the DA1 receptor, since the DA1 receptor antagonist SCH23390 was able to both antagonize the reinforcing properties of mephedrone and attenuate the drug-seeking behavior in more than half of the tested rats. When analyzing the group of rats that were self-administering mephedrone, the reinforcing effects of mephedrone appears to be weakly blocked by 0.01 mg/kg SCH23390. However, when looking at the rats individually, most of the rats did have an increase of mephedrone infusions, which implies antagonism. Since there was no obvious difference between the two rats and the other six rats in the behavior during the cocaine acquisition phase and the mephedrone substitution phase, we suspect that the low responding of the two rats after pretreatment of 0.01 mg/kg SCH23390 represent the

! 101! normal behavioral variability of self-administration results and perhaps the two rats may have a greater sensitivity to the antagonism or rate decreasing effects of SCH23390. It is possible that testing higher or lower doses of mephedrone in combination with the fixed dose of SCH23390 would reveal a shift to the right of either the ascending or descending limb of the mephedrone dose effect curve. However, since patency was confirmed in the catheters of both rats and the number of self-administered mephedrone infusions returned to baseline the very next day in both rats, we did not exclude the results from the group analysis. To account for individual variability, it may have been better to test multiple doses of SCH23390 without the presence of mephedrone, in the same rats. This may have supported our explanation for why we did not see significant antagonism of mephedrone with 0.01 mg/kg SCH23390, but we did see it with cocaine.

When observing the results form Experiment 1 and Experiment 2, we noticed that

SCH23390 displayed a similar pattern in both assays. In drug discrimination, only one dose of SCH23390 was able to significantly attenuate the discriminative effects of mephedrone, also, in self-administration, only one dose of SCH23390 was able to attenuate the reinforcing effects of mephedrone in some rats. The ability of SCH23390 to inhibit the effects of mephedrone was not dose dependent and did not antagonize mephedrone in every single rat. This suggests, while the D1 receptor may partially mediate the behavioral effects of mephedrone, it is not its only mechanism and other receptors may be involved. In both experiments, cocaine and mephedrone displayed inconsistent results when we tested for the substitution of cocaine for mephedrone and the substitution of mephedrone for cocaine. In Experiment 1, high doses of cocaine were able to fully substitute for a low training dose of mephedrone, but only partially substitute

! 102! for a high training dose of mephedrone. Although we did not test the ability of cocaine to substitute for a mephedrone-seeking behavior, we did test the ability of mephedrone to substitute for a cocaine-seeking behavior. We observed that high and low doses of mephedrone did not fully reinstate an extinguished cocaine-seeking behavior. On the other hand, we did observe for mephedrone to directly substitute for a cocaine-seeking behavior when self-administering similar doses. Also, like cocaine, the reinforcing effect of mephedrone appears to be mediated through the D1 receptor, but only in some rats.

This inconsistency between the mechanisms of cocaine and mephedrone in the two experiments suggest that cocaine and mephedrone may have a common pharmacological mechanism for their reinforcing effects; however, they may differ in their mechanism of discriminative stimulus effects. Hence, cocaine and mephedrone may be similar in some cases, but different in others.

Based upon the conclusions of this body of work, we presume for mephedrone to be, primarily, a monoamine releaser, similar to MDMA. Mephedrone may also activate the DA and 5-HT receptor, however, its main mechanism may be to bind to the DAT and

SERT as a reverse transporter and increase the concentration of DA and 5-HT within the synaptic cleft. This conclusion agrees with the results of previous studies. Our conclusion is parallel to what was found in in vitro studies. It has been shown that, in rat brains, mephedrone was able to dose dependently block the reuptake of both DA and 5-HT, because mephedrone has an affinity for both the DAT and for the SERT as an inhibitor.

Furthermore, mephedrone was able to competitively displace both the 5-HT2 receptor tag

[³H]ketanserin and the DA2 receptor tag [³H]raclopride, indicating an affinity for the DA and 5-HT receptor, although in these radioligand binding experiments it was not

! 103! determined if mephedrone acts as a DA and 5-HT receptor agonist or antagonist

(Martinez-Clemente et al., 2012). Nonetheless, in vivo, mephedrone is shown to increase both DA and 5-HT neurotransmitter in rats (Kehr et al., 2011), which agrees with our behavioral results.

Limitations and Future Direction

When combining the results from this study and the results of other studies, the characterization and mechanism of mephedrone is better understood; however, there were a few limitations in the study, which prompts for more work to be done. In Experiment 1, we tested the ability of various drugs to generalize to different training doses of mephedrone. We categorized these drugs as either dopaminergic or serotonergic, even though most of these drugs involved the stimulation of both the DA and 5-HT neurotransmitter. We did test the generalization of a few selective 5-HT receptor agonists, such as DOI and WAY163909, but we did not test any selective DA receptor agonists.

We believe that testing drugs that are both dopaminergic and serotonergic selective agonists would further contribute to understanding the pharmacological profile of mephedrone. We predict that if rats were tested for the generalization of more selective drugs, we would see a better shift or separation in responding. We expected for there to be a more dominant contribution of serotonin at the low training dose and a shift to a more dominant contribution of dopamine at the high training dose, similar to MDMA

(Harper et al., 2014). Although mephedrone’s pharmacological mechanism did appear most similar to MDMA in the drug discrimination assays, clearly 5-HT and DA increases

! 104! were necessary for substitution of any drug tested across both groups (see Experiment 1

Discussion).

In Experiment 2, we attempted to reinstate an extinguished cocaine-seeking behavior with mephedrone, but failed. To try and find out why, we analyzed the blood of the rats to ensure the history of cocaine did not interfere with the metabolism of the prime injection of mephedrone, and it did not. We believe that one may perform microdialysis experiments to compare rats with a history of cocaine self-administration to rats without a history of cocaine self-administration. Microdialysis is a procedure that may be performed in vivo on awake rats to measure the amount of neurotransmitters in the neuronal extracellular fluid of specific brain areas (Chefer et al., 2009; Darvesh, et al.,

2011). Measuring the concentrations of DA released into the nucleus accumbens during the reinstatement phase would be a better indicator of determining if the history of cocaine has any effect on the reinforcing properties of mephedrone, which we presume it does (see Experiment 2 Discussion). Additionally, the D1 receptor may not be the only receptor involved in the mediation of mephedrone’s reinforcing mechanism. To determine other factors, a variety of DA and 5-HT receptor antagonists should be tested in their ability to attenuate the drug-seeking behavior of mephedrone. We would predict if rats were pretreated with a D3, D2, or 5-HT2C receptor antagonist, we would be able to attenuate the reinforcing effects of mephedrone to some degree since these receptors were shown to also mediate the reinforcing effects of cocaine (see Experiment 2 Discussion). It is possible too that we may need both a DA and 5-HT receptor antagonist in combination to see a full shift to the right in the mephedrone dose-response curve. We presume that if more dopaminergic and serotonergic agonists and antagonists were tested, in both

! 105! experiments, we would see a wide range of receptors that are involved in the discriminative and reinforcing effects of mephedrone. In addition, we had the option of establishing a history of cocaine or MDMA in rats. We decided to establish a history of cocaine, instead of a history of MDMA. One could establish a history of MDMA and test if mephedrone will reinstate an extinguished MDMA-seeking behavior.

The data from this thesis will aid in the translation of results found in animals, to allow health officials understand how this drug works. This will help to diagnose and treat conditions and diseases such as overdose and addiction, because unfortunately, mephedrone is still very prevalent as a recreational drug on the club scene. Lately, many epidemiological reports seem to focus on the emergence of chemsex involving mephedrone, largely within the community of men who have sex with men. Chemsex is a term used to describe the act of recreationally taking a drug to enhance the experience of sex (Ahmed et al., 2016; Elliot et al., 2017; Giorgetti et al., 2017; Ottaway et al., 2017;

Sewell et al., 2017; Tomkins et al., 2017; Weatherburn et al., 2017). This may imply that mephedrone may be an important component in the process of exposing oneself to sexual infections and viruses.

Final Remarks

All together, we were able to establish both a low and high dose of mephedrone as discriminative stimuli in male rats. We found that MDMA was the only drug to fully substitute for both a low and high dose of mephedrone, without interrupting response rates, suggesting that both dopamine and serotonin must be detectable as interoceptive cues in order to discriminate mephedrone and that MDMA and mephedrone have similar

! 106! mechanisms. Although mephedrone did not reinstate the extinguished cocaine-seeking behavior, it was able to substitute for the cocaine self-administration behavior.

Additionally, this drug-seeking behavior seems to follow a trend of mediation through the

D1 receptor. When pretreated with SCH23390, most rats increased the number of mephedrone infusions from baseline, which suggest that some of the reinforcing effects of mephedrone may be mediated through the D1 receptor. Taking the results from the two experiments, we were able to obtain our overall objective of determining the pharmacological mechanism of action of mephedrone, which likely leads to its abuse ability. We conclude that mephedrone is another ‘dirty drug’ whose mechanism involves both dopamine and serotonin. When trying to determine the mechanism that leads to the abusive behaviors associated with mephedrone, dopamine is not more important than serotonin and serotonin is not more important than dopamine. Our findings agree with what was found in vitro. We conjecture that mephedrone may bind to both the DAT and to the SERT as an inhibitor, enter into the presynaptic neuron and bind to TAAR1 or enters into synaptic vesicles by binding to VMAT, to release DA and 5-HT. When it binds to DAT and SERT, it may bind as a TAAR1 agonist and reverse the transporter, prohibiting DA and 5-HT reuptake and causing an efflux of the neurotransmitter into the synaptic cleft, respectively, in a fashion that is more similar to MDMA, than it is to cocaine and methamphetamine.

! 107! REFERENCES CITED

Aarde, et al., “Mephedrone (4-methylmethcathinone) supports intravenous self- administration in Sprague-Dawley and Wistar rats,” Addiction Biology 2013 (18): 786- 799.

Abdelwahab et al., “Khat (Catha edulis Forsk.) Dependence Potential and Pattern of Use in Saudi Arabia,” Biomedical Research International 2015 doi: 10.1155/2015/604526.

Ahmed et al., “Methaemoglobinaemia due to mephedrone ('snow'),” BMJ Case Reports 2010 (22): doi: 10.1136/bcr.04.2010.2879.

Ahmed et al., “Social norms related to combining drugs and sex ("chemsex") among gay men in South London,” International Journal of Drug Policy 2016 (38): 29- 35.

Anastasio et al., “Variation within the serotonin (5-HT) 5-HT₂C receptor system aligns with vulnerability to cocaine cue reactivity,” Translational Psychiatry 2014 (369): 1-9.

Anderson et al., “Administration of the D1-like dopamine receptor antagonist SCH-23390 into the medial nucleus accumbens shell attenuates cocaine priming-induced reinstatement of drug-seeking behavior in rats,” Psychopharmacology (Berl) 2003 (168): 132-138.

Anderson et al., “Administration of the D2 dopamine receptor antagonist sulpiride into the shell, but not the core, of the nucleus accumbens attenuates cocaine priming- induced reinstatement of drug seeking,” 2006 (31): 1452- 1461.

Andrews and Lucki, “Effects of cocaine on extracellular dopamine and serotonin levels in the nucleus accumbens,” Psychopharmacology 2001 (155): 221-229.

Angoa-Pérez et al., “Mephedrone, an abused psychoactive component of ‘Bath Salts’ and methamphetamine congener, does not cause neurotoxicity to dopamine nerve endings of the striatum,” Journal of Neurochemistry 2012 (120): 1097-1107.

Angoa-Pérez et al., “Effects of combined treatment with mephedrone and methamphetamine or 3,4-methylenedioxymethamphetamine on serotonin nerve endings of the hippocampus,” Life Sciences 2014 (97): 31-36.

Anonymous, "Most Devilish Powdered eVil: An Experience with MDPV (ID 90252)," Erowid.org Apr 30, 2011. Accessed December 2013.

! 108!

Asgari et al., “Effects of 5-HT2A receptor stimulation on the discrimination of durations by rats,” Behavioural Pharmacology 2006 (17): 51-59.

Awasaki et al., “Dopamine D(1) antagonist SCH23390 attenuates self- administration of both cocaine and fentanyl in rats,” Environmental Toxicology and Pharmacology 1997 (3): 115-122.

Baker et al., “The role of monoamine uptake in the discriminative stimulus effects of cocaine and related compounds,” Behavioural Pharmacology 1993 (4): 69-79.

Baumann, et al., “The designer methcathinone analogs, mephedrone and methylone, are substrates for monoamine transporters in brain tissue,” Neuropsychopharmacology 2012 (37): 1192-203.

Berquist II et al., “Evaluation of training dose in male Sprague-Dawley rats trained to discriminate 4-methylmethcathinone,” Psychopharmacology 2017 doi: 10.1007/s00213-017-4716-4.

Bonano et al., “Abuse-related neurochemical and behavioral effects of para- substituted methcathinone analogs in rats. I. Quantitative structure-activity relationship (QSAR) analysis of the pharmacology of para-substituted methcathinone analogues,” British Journal of Pharmacology 2015 (172): 2433-2444.

Bongard et al., “Khat chewing and acculturation in East-African migrants living in Frankfurt am Main/Germany,” Journal of Ethnopharmacology 2015 (164): 223-228.

Borowsky et al., “Trace amines: Identification of a family of mammalian G protein-coupled receptors,” Proceedings of the National Academy of Sciences of the USA 2001 (98): 8966-8971.

Bortolato et al., “CHAPTER 2.4 - The Degradation of Serotonin: Role of MAO,” Handbook of Behavioral Neuroscience 2010 (21): 203-218.

Bradbury et al., “Acquisition of MDMA self-administration: pharmacokinetic factors and MDMA-induced serotonin release,” Addiction Biology 2014 (19): 874-884.

Brennan et al., “Effect of D1-like and D2-like receptor antagonists on methamphetamine and 3,4-methylenedioxymethamphetamine self-administration in rats,” Behavioural Pharmacology 2009 (20): 688-694.

Brunt et al., “Instability of the ecstasy market and a new kid on the block: mephedrone,” Journal of Psychopharmacology 2011 (25): 1543-1547.

! 109! Bubar M. and Cunningham K., “Serotonin 5-HT2A and 5-HT2C receptors as potential targets for modulation of psychostimulant use and dependence,” Current Topics in Medicinal Chemistry 2006 (6): 1971-1985.

Bunzow et al., “Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabolites of the catecholamine neurotransmitters are agonists of a rat trace amine receptor,” Molecular Pharmacology 2001 (60): 1181-1188.

Büttner, A., “Review: The neuropathology of drug abuse,” Neuropathology and Applied Neurobiology 2011 (37): 118-134.

Caine et al., “D3 receptor test in vitro predicts decreased cocaine self- administration in rats,” Neuroreport 1997 (8): 2373-2377.

Caine et al., “Lack of Self-Administration of Cocaine in Dopamine D1 Receptor Knock-Out Mice,” Journal of Neuroscience 2007 (27): 13140-13150.

Caine S. and Koob G., “Modulation of cocaine self-administration in the rat through D-3 dopamine receptors,” Science 1993 (260): 1814-1816.

Caine, S. and Koob, G., “Effects of dopamine D-1 and D-2 antagonists on cocaine self-administration under different schedules of reinforcement in the rat,” Journal of Pharmacology and Experimental Therapeutics 1994 (270): 209-218.

Cameron et al., “Mephedrone and methylenedioxypyrovalerone (MDPV), major constituents of "bath salts," produce opposite effects at the human dopamine transporter,” Psychopharmacology 2013 (227): 493-499.

Campiani et al., “Synthesis and pharmacological evaluation of potent and highly selective D3 receptor ligands: inhibition of cocaine-seeking behavior and the role of dopamine D3/D2 receptors,” Journal of Medicinal Chemistry 2003 (46): 3822-3839.

CanadianBakin, “What drugs effects are comparable to mephedrone? And other questions...,” drugs-forum.com October 6, 2012 Accessed December 2013.

Carhart-Harris et al., “A web-based survey on mephedrone,” Drug and Alcohol Dependence 2011 (118): 19-22.

Carati, C. and Schenk, S., “Role of dopamine D1- and D2-like receptor mechanisms in drug-seeking following methamphetamine self-administration in rats,” Pharmacology Biochemistry and Behavior 2011 (98): 49-454.

Chefer et al., “Overview of Brain Microdialysis,” Current Protocols in Neuroscience 2009 doi: 10.1002/0471142301.ns0701s47.

! 110! Clemett et al., “Immunohistochemical localization of the 5-HT2C receptor protein in the rat CNS,” Neuropharmacology 2000 (39): 123-132.

Charnay, Y. and Leger, L., “Brain serotonergic circuitries,” Dialogues in clinical neuroscience 2010 (12): 471-487.

Cervo et al., “Cocaine-seeking behavior in response to drug-associated stimuli in rats: involvement of D3 and D2 dopamine receptors,” Neuropsychopharmacology 2003 (28): 1150-1159.

Colpert, F. C., “Drug Discrimination in Neurobiology,” Pharmacology Biochemistry and Behavior 1999 (64): 337-345.

Creehan et al., “Intravenous self-administration of mephedrone, methylone and MDMA in female rats,” Neuropharmacology 2015 (92): 90-97.

Cunha-Oliveira et al., “Cellular and molecular mechanisms involved in the neurotoxicity of and psychostimulant drugs,” Brain Research Reviews 2008 (58): 192–208.

Darvesh, et al., “In vivo brain microdialysis: advances in neuropsychopharmacology and drug discovery,” Expert Opinion on Drug Discovery 2011 (2): 109-127.

[DEA] Drug Enforcement Administration, “4-Methylmethcathinone (Mephedrone),” Office of Diversion Control: Drug & Chemical Evaluation Section May 2013 Retrieved from: https://www.deadiversion.usdoj.gov/drug_chem_info/mephedrone.pdf.

DeLarge et al., “Atypical binding at dopamine and serotonin transporters contribute to the discriminative stimulus effects of mephedrone,” Neuropharmacology 2017 (119): 62-75.

DFsGeezaman, "Surprisingly Like E: An Experience with 4- Methylmethcathinone (exp77952)," Erowid.org. (2009): erowid.org/exp/77952

Di Chiara, G. and Imperato, A., “Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats,” Proceedings of the National Academy of Sciences of the USA 1988 (85): 5274-5278.

Di Matteo et al., “Biochemical and electrophysiological evidence that RO 60- 0175 inhibits mesolimbic dopaminergic function through serotonin(2C) receptors,” Brain Research 2000 (865): 85-90.

! 111! Dolengevich-Segal et al., “Severe Psychosis, Drug Dependence, and Hepatitis C Related to Slamming Mephedrone,” Case Reports in Psychiatry 2016 doi: 10.1155/2016/8379562.

[DPA] Drug Policy Alliance, “A Brief History of the Drug War,” 2017 Retrieved from: http://www.drugpolicy.org/facts/new-solutions-drug-policy/brief-history-drug-war- 0.

Dunn, M. and Killcross, S., “Differential attenuation of d-amphetamine-induced disruption of conditional discrimination performance by dopamine and serotonin antagonists,” Psychopharmacology 2006 (188): 183-192.

Elliot et al., “Recreational drug use and chemsex among HIV-infected in-patients: a unique screening opportunity,” HIV Medicine 2017 (18): 525-531.

Eriksson et al., “Effects of mCPP on the extracellular concentrations of serotonin and dopamine in rat brain,” Neuropsychopharmacology 1999 (20): 287–296.

Federal Register “Schedules of Controlled Substances: Temporary Placement of Three Synthetic Cathinones into Schedule I” Federal Register Proposed Rules 2011 (76): 55616-55619.

Fletcher et al., “Pre-exposure to (±)3,4-methylenedioxymethamphetamine (MDMA) facilitates acquisition of intravenous cocaine self-administration in rats,” Neuropsychopharmacology 2001 (25): 195-203.

Fletcher et al., “The effects of the 5-HT(2C) receptor antagonist SB242084 on locomotor activity induced by selective, or mixed, indirect serotonergic and dopaminergic agonists,” Psychopharmacology 2006 (187): 515-525.

Fiorino et al., “Dynamic Changes in Nucleus Accumbens Dopamine Efflux During the Coolidge Effect in Male Rats,” The Journal of Neuroscience 1997 (17): 4849- 4855.

Froger-Colléaux, C., and Castagné, V., “Effects of baclofen and raclopride on reinstatement of cocaine self-administration in the rat,” European Journal of Pharmacology 2016 (777):147-155.

Gannon, B. and Fantegrossi, W., “Cocaine-Like Discriminative Stimulus Effects of Mephedrone and in Mice,” Journal of Drug and Alcohol Research 2016: doi: 10.4303/jdar/236009.

Gatch et al., “Locomotor stimulant and discriminative stimulus effects of 'bath salt' cathinones,” Behavioural Pharmacology 24 (2013) 437-447.

! 112! German et al., “Mephedrone alters basal ganglia and limbic neurotensin systems,” Journal of Neurochemistry 2014 (130): 402-407.

Giorgetti et al., “When "Chems" Meet Sex: A Rising Phenomenon Called "ChemSex",” Current Neuropharmacology 2017 (15): 762-770.

Gołembiowska, et al., “Effect of Some Psychoactive Drugs Used as 'Legal Highs' on Brain Neurotransmitters,” Neurotoxicity Society 2016 (29): 394-407.

Goodwin, et al., “Serotonergic-dopaminergic mediation of MDMA's discriminative stimulus effects in a three-choice discrimination,” Pharmacology Biochemistry and Behavior 2003 (74): 987-995.

Green et al., “The preclinical pharmacology of mephedrone; not just MDMA by another name,” British Journal of Pharmacology 2014 (171): 2251-2268.

Gregg et al., “Mephedrone interactions with cocaine: prior exposure to the 'bath salt' constituent enhances cocaine-induced locomotor activation in rats,” Behavioral Pharmacology 2013 (24): 684-688.

Gregg, et al., “Stereochemistry of mephedrone neuropharmacology: enantiomer- specific behavioural and neurochemical effects in rats,” British Journal of Pharmacology 2014 (172): 883-894.

Grottick et al., “Studies to investigate the role of 5-HT(2C) receptors on cocaine- and food-maintained behavior,” The Journal of Pharmacology and Experimental Therapeutics 2000 (295): 1183-1191.

Gustavsson, D. and Escher C., “Mephedrone--Internet drug which seems to have come and stay. Fatal cases in Sweden have drawn attention to previously unknown substance,” Lakartidningen 2009 (106): 2769-2771.

Hernandez, L. and Hoebel, B., “Food reward and cocaine increase extracellular dopamine in the nucleus accumbens as measured by microdialysis,” Life Sciences 1988 (42): 1705-1712.

Hadlock et al., “4-Methylmethcathinone (mephedrone): neuropharmacological effects of a designer stimulant of abuse,” Journal of Pharmacology and Experimental Therapeutics 2011 (339): 530-536.

Harper et al., “A 3-lever discrimination procedure reveals differences in the subjective effects of low and high doses of MDMA,” Pharmacology, Biochemistry and Behavior 2014 (116): 9-15.

! 113! Harvey et al. “Effects of D1 and D2 receptor antagonists on the discriminative stimulus effects of methylendioxypyrovalerone and mephedrone in male Sprague-Dawley rats trained to discriminate D-amphetamine,” Behavioural Pharmacology 2017 (28): 586- 589.

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

Hasler et al., “Circadian Rhythms, Sleep, and Substance Abuse,” Sleep Medicine Reviews 2012 (16): 67-81.

Higgins, G. and Fletcher, P., “Serotonin and drug reward: focus on 5-HT2C receptors,” European Journal of Pharmacology 2003 (480): 151-162.

Hiranita et al., “A tryptamine-derived catecholaminergic enhancer, (-)-1- (benzofuran-2-yl)-2-propylaminopentane [(-)-BPAP], attenuates reinstatement of methamphetamine-seeking behavior in rats,” Neuroscience 2010 (165): 300-312.

Huang et al., “Contrasting effects of d-methamphetamine, 3,4- methylenedioxymethamphetamine, 3,4-methylenedioxypyrovalerone, and 4- methylmethcathinone on wheel activity in rats,” Drug and Alcohol Dependence 2012 (126): 168-175.

Hutchinson et al., “Persistent conditioned place preference to cocaine and withdrawal hypo-locomotion to mephedrone in the flatworm planaria,” Neuroscience Letters (593): 19-23.

Hyman et al., “Neural mechanisms of addiction: the role of reward-related learning and memory,” Annual Reviews of Neuroscience 2006 (29): 565–598.

[INCB] International Narcotics Control Board, “Report of the International Narcotics Control Board for 2011,” 2011 Retrieved from: https://www.incb.org/documents/Publications/AnnualReports/AR2011/AR_2011_Englis h.pdf.

[INCB] International Narcotics Control Board “Report of the International Narcotics Control Board for 2010,” 2010 Retrieved from: https://www.incb.org/documents/Publications/AnnualReports/AR2010/AR_2010_Englis h.pdf.

Jing et al., “Effects of the cannabinoid CB₁ receptor allosteric modulator ORG 27569 on reinstatement of cocaine- and methamphetamine-seeking behavior in rats,” Drug and Alcohol Dependence 2014 (143): 251-256.

! 114! Johanson et al., “Discriminative stimulus effects of 3,4- methylenedioxymethamphetamine (MDMA) in humans trained to discriminate among d- amphetamine, meta-chlorophenylpiperazine and placebo,” Drug and Alcohol Dependence 2006 (81): 27-36.

Johnson et al., “Current "legal highs",” Journal of Emergency Medicine 2013 (44): 1108-1115.

Johnson, P. and Johnson, M., “Investigation of "bath salts" use patterns within an online sample of users in the United States,” Journal of Psychoactive Drugs 2014 (46): 369-78.

Kankaanpää et al., “The acute effects of amphetamine derivatives on extracellular serotonin and dopamine levels in rat nucleus accumbens,” Pharmacology, Biochemistry, and Behavior 1998 (59): 1003-1009.

Kalivas P. and Stewart J., “Dopamine transmission in the initiation and expression of drug- and stress-induced sensitization to motor activity,” Brain Res Rev 1991 (16): 223-244.

Karila et al., “Synthetic Cathinones: A New Public Health Problem,” Current Neuropharmacology 2015 (13): 12-20.

Kehr et al., “Mephedrone, compared with MDMA (Ecstasy) and amphetamine, rapidly increases both dopamine and 5-HT levels in nucleus accumbens of awake rats,” British Journal of Pharmacology 2011 (164): 1949-1958.

Keim, Brandon (2012-07-12). "New Federal Ban on Synthetic Drugs Already Obsolete". Wired. Retrieved 2014-03-18.

Kirby, et al., “Contributions of serotonin in addiction vulnerability,” Neuropharmacology 2011 (61): 421-432.

Kleven M. and Koek W., “Discriminative stimulus properties of cocaine: enhancement by monoamine reuptake blockers,” The Journal of pharmacology and experimental therapeutics 1998 (284): 1015-1025.

Koob et al., “Drug self-administration and microdialysis in rodents,” In: Short Course I: What’s Wrong with My Mouse? Strategies for Rodent Behavior Phenotyping, (J.N. Crawley, Ed.) (2007) pp. 35-51, Society for Neuroscience, Washington D.C.

Koob, G.F. and Volkow, N.D., “Neurocircuitry of addiction,” Neuropsychopharmacology 2010 (35): 217–38.

Kranz et al., “Reward and the serotonergic system,” Neuroscience 2010 (166): 1023-1035.

! 115!

Kueh and Baker, “Reinforcement schedule effects in rats trained to discriminate 3,4-methylenedioxymethamphetamine (MDMA) or cocaine,” Psychopharmacology 2007 (189): 447-457.

Lepsch et al., “Cocaine Causes Apoptotic Death in Rat Mesencephalon and Striatum Primary Cultures,” Biomedical Research International 2015 doi: 10.1155/2015/750752.

Li et al., “Discriminative stimulus effects of 1-(2,5-dimethoxy-4-methylphenyl)- 2-aminopropane (DOM), ketanserin, and (R)-(+)-{alpha}-(2,3-dimethoxyphenyl)-1-[2- (4-fluorophenyl)ethyl]-4-pipidinemethanol (MDL100907) in rats,” The Journal of Pharmacology and Experimental Therapeutics 2009 (331): 671-679.

Lindemann et al., “Trace Amine-Associated Receptor 1 Modulates Dopaminergic Activity,” Journal of Pharmacology and Experimental Therapeutics 2008 (324): 948-956.

Lisek, et al., “Mephedrone ('bath salt') elicits conditioned place preference and dopamine-sensitive motor activation,” Drug and Alcohol Dependence 2012 (126): 257- 262.

Liu et al., “ Cross-sensitization of the reinforcing effects of cocaine and amphetamine in rats,” Psychopharmacology 2007 (195): 369-375.

López-Arnau et al., “Comparative neuropharmacology of three psychostimulant cathinone derivatives: , mephedrone and methylone,” British Journal of Pharmacology 2012 (167): 407-420.

Maldonado et al., “D1 dopamine receptors in the nucleus accumbens modulate cocaine self-administration in the rat,” Pharmacology Biochemistry and Behavior 1993 (45): 239-242.

Martínez-Clemente et al., “Interaction of mephedrone with dopamine and serotonin targets in rats,” European Neuropsychopharmacology 2012 (22): 231-236.

Martínez-Clemente et al., “Mephedrone pharmacokinetics after intravenous and oral administration in rats: relation to pharmacodynamics,” Psychopharmacology (Berl) 2013 (229): 295-306.

Marusich et al., “Effects of synthetic cathinones contained in "bath salts" on motor behavior and a functional observational battery in mice,” Neurotoxicology 2012 (33): 1305-1313.

Matsumoto, et al., “5-hydroxytryptamine- and dopamine-releasing effects of ring- substituted amphetamines on rat brain: a comparative study using in vivo microdialysis,” European Neuropsychopharmacology 2014 (24): 1362-1370.

! 116!

McElrath, K. and O'Neill, C., "Experiences with mephedrone pre- and post- legislative controls: perceptions of safety and sources of supply," International Journal of Drug Policy 2011 (22): 120-127.

McMahon, L. and Cunningham K., “Antagonism of 5-hydroxytryptamine(2a) receptors attenuates the behavioral effects of cocaine in rats,” The Journal of Pharmacology and Experimental Therapeutics 2001 (297): 357-363.

Meph_Test, "Good Alternative to MDMA: An Experience with 4- Methylmethcathinone (Mephedrone) & Alcohol (exp82321)," Erowid.org (2009): erowid.org/exp/82321

Millan et al., “The "selective" dopamine D1 receptor antagonist, SCH23390, is a potent and high efficacy agonist at cloned human serotonin2C receptors,” Psychopharmacology 2001 (158): 58-62.

Morrissey, T., “This Is Your Brain on Bath Salts: What It's Like to Do the Scary Drug du Jour,” Jezebel.com June 1, 2012. http://jezebel.com/5914694/this-is-your-brain- on-bath-salts-what-its-like-to-do-the-scary-drug-du-jour Accessed December 2013.

Motbey et al., “Mephedrone (4-methylmethcathinone, 'meow'): acute behavioural effects and distribution of Fos expression in adolescent rats,” Addiction Biology 2012 (17): 409-22.

Motbey, et al., “High levels of intravenous mephedrone (4-methylmethcathinone) self-administration in rats: Neural consequences and comparison with methamphetamine,” Journal of Psychopharmacology 2013 (27): 823-836.

Müller et al., “The Role of Serotonin in Drug Addiction,” Handbook of Behavioral Neuroscience (2010) 21: 507-545.

Müller, C., and Homberg, J., “The role of serotonin in drug use and addiction” Behavioural Brain Research (2015) 277: 146–192.

Munzar et al., “Effects of various serotonin agonists, antagonists, and uptake inhibitors on the discriminative stimulus effects of methamphetamine in rats,” Journal of Pharmacology and Experimental Therapeutics 1999 (291): 239-50.

Munzar et al., “Differential involvement of 5-HT(2A) receptors in the discriminative-stimulus effects of cocaine and methamphetamine,” European Journal Of Pharmacology 2002 (436):75-82

Munzar, P. and Goldberg, S., “Dopaminergic involvement in the discriminative- stimulus effects of methamphetamine in rats,” Psychopharmacology 2000 (148): 209- 216.

! 117! Murphy et al., ““Bath Salts” and “Plant Food” Products: the Experience of One Regional US Poison Center,” Journal of Medical Toxicology 2013 (9): 42-48.

Navailles et al., “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 2008 (33): 237-246.

Nestler, E., “Molecular mechanisms of drug addiction,” Journal of Neuroscience 1992 (12): 2439-2450.

Neisewander, J. and Acosta J., “Stimulation of 5-HT2C receptors attenuates cue and cocaine-primed reinstatement of cocaine-seeking behavior in rats,” Behavioural Pharmacology 2007 (18): 791-800.

Nestler, E.J., “Molecular mechanisms of drug addiction,” Neuropharmacology 2004 (47): 24-32.

[NIDA] National Institute on Drug Abuse. Synthetic Cathinones (“Bath Salts”) November 2016 Retrieved from: http://www.drugabuse.gov/publications/drugfacts/synthetic-cathinones-bath-salts.

Nic Dhonnchadha, et al., “Blockade of the serotonin 5-HT2A receptor suppresses cue-evoked reinstatement of cocaine-seeking behavior in a rat self-administration model,” Behavioural Neuroscience 2009 (123): 382-396.

Nutt, et al., “The dopamine theory of addiction: 40 years of highs and lows,” Nature Reviews Neuroscience 2015 (16): 305-312.

Olguín et al., “The role of dopamine and its dysfunction as a consequence of oxidative stress,” Oxidative Medicine and Cellular Longevity 2016 (2016): doi:10.1155/2016/9730467.

Ottaway et al., “Men who have sex with men diagnosed with a sexually transmitted infection are significantly more likely to engage in sexualised drug use,” International Journal of STD & AIDS 2017 (28): 91-93.

Panlilio, L. V., and Goldberg, S. R., “Self-administration of drugs in animals and humans as a model and an investigative tool,” Addiction 2007 (102): 1863-1870.

Panos and Baker, “An in vivo microdialysis assessment of concurrent MDMA and cocaine administration in Sprague-Dawley rats,” Psychopharmacology 2010 (209): 95-102.

Papaseit et al., “Human Pharmacology of Mephedrone in Comparison with MDMA,” Neuropsychopharmacology 2016 (41): 2704-2713.

! 118!

Peng et al., “The preferential dopamine D3 receptor antagonist S33138 inhibits cocaine reward and cocaine-triggered relapse to drug-seeking behavior in rats,” Neuropharmacology 2009 (56): 752-760.

Phillips et al., “Subsecond dopamine release promotes cocaine seeking,” Nature 2003 (422): 614-618.

Pittenger et al., “- and cocaine-triggered methamphetamine reinstatement in female and male Sprague-Dawley rats,” Pharmacology Biochemistry and Behavior 2017 (159): 69-75.

Prager et al., “The importance of reporting housing and husbandry in rat research,” Frontiers in Behavioral Neuroscience 2011 (5): 1-4.

Prasad, A. and Pasterkamp, J., “Axon guidance in the dopamine system,” Advances in Experimental Medicine and Biology 2009 (651): 91-100.

Pockros et al., “Blockade of 5-HT2A receptors in the medial prefrontal cortex attenuates reinstatement of cue-elicited cocaine-seeking behavior in rats,” Psychopharmacology (Berl) 2011 (213): 307-320.

Porras et al., “5-HT2A and 5-HT2C/2B Receptor Subtypes Modulate Dopamine Release Induced in Vivo by Amphetamine and Morphine in Both the Rat Nucleus Accumbens and Striatum” Neuropsychopharmacology 2002 (26): 311-324.

Prosser J. and Nelson L., “The toxicology of bath salts: a review of synthetic cathinones,” Journal of Medical Toxicology 2012 (8): 33-42.

Ramoz et al., “Mephedrone ("bath salt") pharmacology: insights from invertebrates,” Neuroscience 2012 (208): 79-84.

Ratzenboeck et al., “Reinforcing Effects of MDMA (‘Ecstasy’) in Drug-Naive and Cocaine-Trained Rats,” Pharmacology 2001 (62): 138-144.

Ritz et al., “Cocaine receptors on dopamine transporters are related to self- administration of cocaine,” Science 1987 (238): 1219-1223.

Robinson et al., “Mephedrone (4-methylmethcathinone) and intracranial self- stimulation in C57BL/6J mice: comparison to cocaine,” Behavioural Brain Research 2012 (234): 76-81.

Rosas-Hernandez et al., “Methamphetamine, 3,4- methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxypyrovalerone (MDPV) induce differential cytotoxic effects in bovine brain microvessel endothelial cells,” Neuroscience Letters 2016 (629): 125-130.

! 119!

Ross et al., “"Bath salts" intoxication,” The New England Journal Medicine 2011 (365): 967-968.

[SAMHSA] Substance Abuse and Mental Health Services Administration “Results from the 2013 National Survey on Drug Use and Health: Summary of National Findings,” 2014 Retrieved from: https://www.samhsa.gov/data/sites/default/files/NSDUHresultsPDFWHTML2013/Web/ NSDUHresults2013.pdf.

[SAMHSA] Substance Abuse and Mental Health Services Administration “Behavioral Health Trends in the United States: Results from the 2014 National Survey on Drug Use and Health,” 2015 Retrieved from: https://www.samhsa.gov/data/sites/default/files/NSDUH-FRR1-2014/NSDUH-FRR1- 2014.pdf.

[SAMHSA] Substance Abuse and Mental Health Services Administration “Results From The 2015 National Survey On Drug Use and Health: Detailed Tables,” 2016 Retrieved from: https://www.samhsa.gov/data/sites/default/files/NSDUH-DetTabs- 2015/NSDUH-DetTabs-2015/NSDUH-DetTabs-2015.pdf.

Saem de Burnaga Sanchez, J. "Sur un homologue de l'éphédrine" [On an analogue of ]. Bulletin de la Societé Chimique de France 1929 (45): 284-286.

Schenk et al., “Development, maintenance and temporal pattern of self- administration maintained by ecstasy (MDMA) in rats,” Psychopharmacology 2003 (169): 21-27.

Schenk et al., “MDMA self-administration in rats: acquisition, progressive ratio responding and serotonin transporter binding,” European Journal of Neuroscience 2007 (26): 3229-3236.

Schenk et al., “Effects of priming injections of MDMA and cocaine on reinstatement of MDMA- and cocaine-seeking in rats,” Drug and Alcohol Dependence 2008 (96): 249-255.

Schmidt et al., “Illicit drug use among gay and bisexual men in 44 cities: Findings from the European MSM Internet Survey (EMIS),” International Journal of Drug Policy 2016 (38): 4-12.

Sewell et al., “Poly drug use, chemsex drug use, and associations with sexual risk behaviour in HIV-negative men who have sex with men attending sexual health clinics,” International Journal of Drug Policy 2017 (43): 33-43.

! 120! Shelton et al., “Efficacy of buspirone for attenuating cocaine and methamphetamine reinstatement in rats,” Drug and Alcohol Dependence 2013 (129): 210-216.

Shettleworth, S. J. (2010). Cognition, Evolution and Behavior (2nd ed.). New York: Oxford.

Shortall et al., “Behavioural and neurochemical comparison of chronic intermittent cathinone, mephedrone and MDMA administration to the rat,” European Neuropsychopharmacology 2013a (23): 1085-1095.

Shortall et al., “Differential effects of cathinone compounds and MDMA on body temperature in the rat, and pharmacological characterization of mephedrone-induced hypothermia,” British Journal of Pharmacology 2013b (168): 966-977.

Simmler, et al., “Pharmacological characterization of designer cathinones in vitro,” British Journal of Pharmacology 2013 (168): 458-470.

Smith et al., “Cocaine-like discriminative stimulus effects of alpha- pyrrolidinovalerophenone, methcathinone and their 3,4-methylenedioxy or 4-methyl analogs in rhesus monkeys,” Addiction Biology 2016 DOI:10.1111/adb.12399.

Solinas et al., “Using drug-discrimination techniques to study the abuse-related effects of psychoactive drugs in rats,” Nature Protocols 2006 (1): 1194–1206.

Stairs et al., “Nicotine and cocaine self-administration using a multiple schedule of intravenous drug and sucrose reinforcement in rats,” Behavioural Pharmacology 2010 (21): 182-193.

Steele et al., “3,4-Methylenedioxymethamphetamine (MDMA, “Ecstasy”): pharmacology and toxicology in animals and humans,” Addiction 1994 (89): 539-551.

Stolerman, I., “Drug Discrimination,” Encyclopedia of Psychopharmacology 2014: 1-7.

Swinford-Jackson et al., “Incubation of cocaine cue reactivity associates with neuroadaptations in the cortical serotonin (5-HT) 5-HT2C receptor (5-HT2CR) system,” Neuroscience 2016 (324): 50-61.

Suyama et al., “Abuse-Related Neurochemical Effects of Para-Substituted Methcathinone Analogs in Rats: Microdialysis Studies of Nucleus Accumbens Dopamine and Serotonin,” The Journal of Pharmacology and Experimental Therapeutics 2016 (36): 182-190.

Terry, S.M., “Bath Salt Abuse: More Than Just Hot Water,” Journal of Emergency Nursing 2014 (40): 88-91.

! 121!

Thomsen, M., and Caine, S., “Chronic Intravenous Drug Self-Administration in Rats and Mice,” Current Protocols in Neuroscience 2005:!doi: 10.1002/0471142301.ns0920s32.

Tomkins et al., “Prevalence of recreational drug use reported by men who have sex with men attending sexual health clinics in Manchester, UK,” International Journal of STD & AIDS 2017: doi: 10.1177/0956462417725638.

Tzschentke, T., “Measuring reward with the conditioned place preference (CPP) paradigm: update of the last decade,” Addiction Biology 2007 (12): 227-462.

Uhl et al., “Cocaine, reward, movement and monoamine transporters,” Molecular Psychiatry 2002 (7): 21-6.

Vandewater et al., “Intravenous self-administration of entactogen-class stimulants in male rats,” Neuropharmacology 2015 (99): 538-545.

Varner et al., “Comparison of the behavioral and cardiovascular effects of mephedrone with other drugs of abuse in rats,” Psychopharmacology 2013 (225): 675- 685.

Verrico et al., “MDMA (Ecstasy) and human dopamine, norepinephrine, and serotonin transporters: implications for MDMA-induced neurotoxicity and treatment,” Psychopharmacology 2007 (189): 489-503.

Volkow et al., “Imaging dopamine's role in drug abuse and addiction,” Neuropharmacology 2009 (56): 3-8.

Volkow, N. and Morales, M., “The Brain on Drugs: From Reward to Addiction,” Cell 2015 (162): 712-725.

Vouga et al., “Stereochemistry and neuropharmacology of a 'bath salt' cathinone: S-enantiomer of mephedrone reduces cocaine-induced reward and withdrawal in invertebrates,” Neuropharmacology 2015 (91): 109-116.

Young, R., “Drug Discrimination” In: Buccafusco JJ, editor. Methods of Behavior Analysis in Neuroscience. 2nd edition. Boca Raton (FL): CRC Press/Taylor & Francis; 2009. Chapter 3. Available from: https://www.ncbi.nlm.nih.gov/books/NBK5225/

Walsh, S. and Cunningham K., “Serotonergic mechanisms involved in the discriminative stimulus, reinforcing and subjective effects of cocaine,” Psychopharmacology (Berl) 1997 (130): 41-58.

! 122! Weatherburn et al., “Motivations and values associated with combining sex and illicit drugs ('chemsex') among gay men in South London: findings from a qualitative study,” Sexually Transmitted Infections 2017 (93): 203-206.

Weaver et al., “Designer drugs 2015: assessment and management,” Addiction Science & Clinical Practice 2015 (10): 1-9.

Winstock et al., “Mephedrone: still available and twice the price,” The Lancet 2010 (376): 1537.

Winstock et al., “Mephedrone, new kid for the chop? Addiction 2011 (106): 154- 161.

Wise, R.A., “Dopamine and reward: the anhedonia hypothesis 30 years On,” Neurotoxicity Research 2008 (14): 69-183.

Witkin et al., “Behavioral effects of selective dopaminergic compounds in rats discriminating cocaine injections,” The Journal of Pharmacology and Experimental Therapeutics 1991 (257): 706-713.

White, T., “Illicit drugs are beginning to replace prescription as source of national drug epidemic,” October 2017 Stanford The Freeman Spogli Institute For International Studies Retrieved from: https://fsi.stanford.edu/news/hospital-discharges- prescription-opioids-down-heroin-discharges-surge

Wright et al., “Effect of ambient temperature on the thermoregulatory and locomotor stimulant effects of 4-methylmethcathinone in Wistar and Sprague-Dawley rats,” Public Library of Science one 2012 (7): e44652.

Wolinsky et al., “The Trace Amine 1 receptor knockout mouse: an animal model with relevance to schizophrenia” Genes, Brain, and Behavior 2007 (6): 628-639.

Wood et al., “Case series of individuals with analytically confirmed acute mephedrone toxicity,” Clinical Toxicology (Philadelphia, PA) 2010 (48): 924-927.

Wood et al., “Limited use of novel psychoactive substances in South London nightclubs,” Association of Physicians of Great Britain and Ireland 2011 (22): 120-127.

Worley et al., “Reinstatement of extinguished cocaine-taking behavior by cocaine and caffeine,” Pharmacology Biochemistry and Behavior 1994 (48): 217-221.

Xie et al., “Rhesus monkey trace amine-associated receptor 1 signaling: enhancement by monoamine transporters and attenuation by the D2 autoreceptor in vitro,” Journal of Pharmacology and Experimental Therapeutics 2007 (321): 116-127.

! 123! Xie et al., “Modulation of monoamine transporters by common biogenic amines via trace amine-associated receptor 1 and monoamine autoreceptors in human embryonic kidney 293 cells and brain synaptosomes,” Journal of Pharmacology and Experimental Therapeutics 2008 (325): 629-640.

Xie, Z. and Miller, G., “β-Phenylethylamine alters monoamine transporter function via trace amine-associated receptor 1: implication for modulatory roles of trace amines in brain,” Journal of Pharmacology and Experimental Therapeutics 2008 (325): 617-628.

Xie, Z and Miller, G., “A receptor mechanism for methamphetamine action in dopamine transporter regulation in brain,” Journal of Pharmacology and Experimental Therapeutics 2009 (330): 316-325.

Zarrindast et al., “SKF 38393 and SCH 23390 inhibit reuptake of serotonin by rat hypothalamic synaptosomes,” Pharmacology 2011 (87): 85-89.

! 124!