BEHAVIORAL CHARACTERIZATION OF SUBSTITUTED AMPHETAMINES AND THEIR SYNTHETIC CATHINONE ANALOGUES IN THE RUSTY CRAYFISH (ORCONECTES RUSTICUS)

Sayali Vilas Gore

A Dissertation

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

December 2017

Committee:

Robert Huber, Advisor Arthur Samel, Graduate Faculty Representative

Verner Bingman

Moira Van Staaden

Jon Sprague

Andrea Kalinoski

© 2017

Sayali Vilas Gore

All Rights Reserved iii ABSTRACT

Robert Huber, Advisor

The functional and evolutionary conservation of neural circuits of reward is an essential component of survival. Drugs of abuse are known to “hijack” natural reward systems to produce their euphoric and reinforcing effects. Recently, synthetic cathinones have gained popularity among the drug users due to low cost, potency and widespread availability resulting from unclear legal regulatory status. Sharing a chemical structure with amphetamines, synthetic cathinones are likely to pose a significant public health threat. Even though synthetic cathinones have been in use for over a decade, the neuropharmacology, behavioral and physiological effects still remain obscure. This gap in knowledge needs to be urgently addressed in order to understand the basic pharmacological effects, development of treatment/therapy against synthetic cathinone addiction and to define a consistent legal framework to assure regulatory control. Due to the ease of experimental manipulations, modularly organized nervous system, absence of blood-brain barrier and well-characterized behavioral paradigms for drug addiction-like behaviors, crayfish continues to be an ideal model to study the addictive potential of any drug. With highly stereotyped behaviors, and a modularly organized nervous system, crustaceans offer productive research models to study proximate mechanisms of a wide range of behavioral phenomena. The current project has harnessed the advantages of this model system to investigate the behavioral effects of synthetic cathinone in comparison to known stimulants: 4-methylmethamphetamine

(4-MMA) vs. mephedrone and 3,4-methylenedioxymethamphetamine (MDMA) vs. methylone.

We explored the unconditioned behavioral effects, locomotor activity, sensitization of locomotor response, reward potential and termination effects of the above mentioned drugs at doses of 1, 3, and 10 µg/g. Our results show that all the drugs generate significant locomotor effects in crayfish. When crayfish were exposed to these drugs for the first time, increase in locomotion iv was seen over a brief period of time which faded eventually over the course of trial. Our study indicates that apart from already known exploratory behaviors, unique unconditioned behaviors such as claw waving and circling are observed following drug injections. Typically, these drugs were able to produce psychostimulation at lower doses (1 µg/g) where as depression of locomotion was seen at higher doses (10 µg/g). Initial exposure of these drugs increased locomotion during the infusion itself while repeated drug injections produced psychostimulation that lasted for longer durations. Using the conditioned place preference (CPP) paradigm, we demonstrated that crayfish seek out a particular tactile environment that had previously been paired with the drug. All the drugs at 10 µg/g show preference for the substrate which was paired with the drug. Furthermore, the preference for drug-paired environment persists even after the extinction pairing sessions (when the initially drug-paired compartment was paired with saline).

We also investigated the termination effects of the drug to pinpoint withdrawal like behaviors.

Termination of drug resulted in production of withdrawal-like behaviors and significant differences in locomotion. This study demonstrates that crayfish offer a comparative and complementary approach in addiction research. The current study contributes an evolutionary context to our understanding of a key component in learning and of natural reward as an important life-sustaining process.

Keywords: Addiction; Psychostimulant Sensitization; Invertebrate Reward; locomotor effects;

Conditioned Place Preference (CPP); withdrawal; unconditioned behaviors

v ACKNOWLEDGMENTS

I would like to express my deepest appreciation to my advisor, Dr. Robert Huber, who has always been encouraging and supportive of my career goals. Without his constructive criticism and guidance, this dissertation would not have been possible. I would like to thank my committee members, Dr. Moira van Staaden, Dr. Jon Sprague, Dr. Verner Bingman, and Dr.

Andrea Kalinoski for their constant help and support. I have always enjoyed the brainstorming session during lab meetings with Dr. van Staaden, Dr. Huber, and their lab students.

Collaboration and discussions with Dr. Sprague, Dr. Bingman, and Dr. Kalinoski helped me learn more about various areas of neuroscience and provided me with valuable insights on my research. I would like to thank Biological Sciences, BGSU for providing me with the financial support throughout my doctoral journey.

Thank you to all my friends and colleagues who joined me for crayfishing expeditions.

Many thanks to Steve for his help with the construction of experimental set-up. Thank you

DeeDee, Susan, and Chris for always being supportive and helpful. Special thanks to Ajinkya

Kulkarni for always being a patient listener and for his unending support throughout my doctoral journey. Finally, I would like to thank my family members and friends who provided me with their constant motivation and care. vi

TABLE OF CONTENTS

Page

CHAPTER 1: LOCOMOTOR EFFECTS OF SUBSTITUTED AMPHETAMINES (4-MMA,

MDMA) AND THEIR SYNTHETIC CATHINONE ANALOGUES (METHYLONE,

MEPHEDRONE) IN CRAYFISH ...... 1

Introduction ...... 1

Materials and Methods ...... 7

Animal Maintenance ...... 7

Surgical procedure for implanting cannula in the pericardial space ...... 7

Drugs, doses and injection protocols ...... 7

Experimental Arena and Video Tracking ...... 8

Psychomotor stimulation and Sensitization - Experimental Design ...... 8

Statistical analysis ...... 9

Results ...... 10

Unconditioned effects of locomotion and other behaviors ...... 10

Psychostimulation for Mephedrone, Methylone, 4-MMA and MDMA ...... 11

Sensitization of psychostimulant effects with repeated injections of synthetic

cathinones and their substituted amphetamine analogues……………………. 11

Discussion……………………………………………………………………………... 12

References ...... 16

CHAPTER 2: REWARDING EFFECTS OF SYNTHETIC CATHINONES (METHYLONE,

MEPHEDRONE) AND THEIR SUBSTITUTED AMPHETAMINE ANALOGS (MDMA, 4-

MMA) IN AN INVERTEBRATE MODEL OF DRUG ADDICTION ...... 25 vii

Introduction ...... 25

Materials and Methods ...... 30

Animal Maintenance ...... 30

Surgical procedure for implanting cannula in the pericardial space ...... 31

Drugs, doses and injection protocols ...... 31

Experimental Arena and Video Tracking ...... 31

Conditioned place preference - Experimental Design ...... 32

Statistical analysis ...... 33

Results ...... 33

Crayfish show a slight, initial preference for soft substrate ...... 33

Three days of drug conditioning demonstrate rewarding effects ...... 34

The established rewarding effects are relatively unaffected by the extinction

pairing sessions ...... 34

Discussion…………………………………………………………………………….. 34

References ...... 38

CHAPTER 3: UNCONDITIONED AND TERMINATION EFFECTS OF SYNTHETIC

CATHINONES IN CRAYFISH: COMPARISON TO SUBSTITUTED AMPHETAMINE

ANALOGS ...... 47

Introduction…………………………………………………………………………… 47

Materials and Methods ...... 49

Animal Maintenance ...... 49

Surgical procedure for implanting cannula in the pericardial space ...... 50

Drugs, doses and injection protocols ...... 50 viii

Experimental Arena and Video Tracking ...... 51

Unconditioned and termination effects - Experimental Design ...... 51

Statistical analysis ...... 51

Results ...... 51

Unconditioned effects of the drugs ...... 52

Termination effects of the drugs ...... 53

Discussion ...... 54

References ...... 58

APPENDIX A: FIGURES AND TABLES ...... 67 1

CHAPTER 1: LOCOMOTOR EFFECTS OF SUBSTITUTED AMPHETAMINES (4-

MMA, MDMA) AND THEIR SYNTHETIC CATHINONE ANALOGUES

(METHYLONE, MEPHEDRONE) IN CRAYFISH.

Introduction

Stimulant drug abuse continues to be a major health concern all over the world.

Amphetamines such as methamphetamine and 3,4-Methylenedioxymethamphetamine (MDMA) cause severe neurotoxicity, lead to addiction and may induce psychotic disorders or cognitive dysfunctions (Cruickshank & Dyer 2009; Jiao et al., 2015). High abuse potential arises from an activation of reward circuits, enhanced arousal, euphoria, behavioral disinhibition, and transient improvement in cognitive domains for lower doses of amphetamines. Higher doses elicit sympathetic activation and agitation and may lead to violent behavior. Termination of stimulant drugs can induce strong withdrawal effects such as disturbed sleep, depressed mood and anxiety, intense craving and cognitive impairment (Karila et al., 2016). Cathinones are structurally similar to amphetamines and appear to share some of these same behavioral consequences. They are now the second most widely used class of drugs worldwide according to the United Nations

Office On Drugs and Crime (UNODC) 2012 world drug report. Synthetic cathinones are likely posing a public health threat similar to amphetamines, a systematic comparative characterization of their neuropharmacology, behavior and physiological effects in comparison to the stimulant effects of amphetamines still remains to be done.

In 1993, cathinone was classified as a Schedule I controlled substance in the USA, however, the manufacturers and the distributors have managed to evade regulatory scrutiny by marketing synthetic cathinone and its derivatives (Weinstein et al., 2017) as “bath salts” or

“plant food”. A “not for human consumption” label on them has allowed sellers to exploit an ill- 2 defined regulatory framework (Kelly 2011; Marusich et al., 2012). In humans, cathinone increases alertness, euphoria, sensory stimulation, hyperthermia, heart rate, respiration, and blood pressure. Apart from the classic stimulatory effects, cathinones are also known to produce

MDMA-like hallucinogenic effects. Strong craving following termination of synthetic cathinones is reported by a high proportion of synthetic cathinone users (Kelly 2011; Marusich et al., 2012).

Sold in a wide range of specialized outlets (Fass et al., 2012; Rosenbaum et al., 2012), cathinone has acquired popularity due to its low cost, ready availability and powerful psychostimulant effects similar to amphetamines and cocaine. As with the typical amphetamine backbone, the chemical structure of cathinone can be readily altered to generate a wide range of novel chemical derivatives (Watterson & Olive 2014), featuring multiple methyl and di-oxy substitutions. At present, little is known about the pharmacology, physiological effects, behavioral consequences, and abuse potential of this rapidly evolving class of substances.

Acting primarily as psychostimulants, unconditioned responses to cocaine, amphetamine, and cathinones elicit increased locomotor responses during the first exposure (Dougherty &

Ellinwood 1981), an effect which generally intensifies with repeated use. Thought to reflect the onset of craving, enhanced locomotion appears to arise from combined impacts on GABAergic, dopaminergic, noradrenergic, serotonergic, glutamatergic, cholinergic and peptidergic pharmacology. As locomotion increases, associated perceptions of reward are reduced, which is thought to reflect the onset of tolerance (Kalivas & Stewart 1991). Sensitized locomotor response has been attributed to modulation of synaptic dopamine levels in the mesolimbic and medial prefrontal cortex neurons. Dopamine can trigger a cascade of cellular events including its action on GABAergic and glutamatergic systems to produce psychostimulant sensitization

(Cruickshank & Dyer 2009; Jiao et al., 2015; Steketee 2003). Controlling the interplay between 3 sensitization and tolerance, the incentive sensitization theory of addiction provides a theoretical framework for how drugs of abuse may bring about their reinforcing effects. Psychostimulant induced hypersensitivity heightens the incentive salience of drugs and drug-associated stimuli

(Robinson & Berridge 1993, 2003; Robinson et al., 2014) and thereby mediates the potent increase in drug "wanting" of addicted individuals (Vanderschuren et al., 2009). As an exceptionally long-lived effect, rats will display psychostimulant sensitization even a year after termination of amphetamine administrations (Paulson et al., 1991). The presence of strong locomotor sensitization offers a powerful predictor for an individual’s vulnerability to enter an addictive cycle. Intense wanting of a drug is associated with both affective and environmental factors associated with drug use (May et al., 2004). The context dependence and persistence of sensitization explains how the associative learning mechanisms bind specific cues to drug wanting and drug seeking (Robinson & Berridge, 1993; 2003; Kalivas et al., 2004; Vezina

2004). Essential to the initiation, expression and maintenance of drug-seeking behavior, measures of psychostimulant sensitization are a central indicator of drug craving and of inherent vulnerabilities to enter the addictive cycle.

The implementation of mammalian reward resides in mesolimbic dopamine pathways which are also known to play a role in mediating locomotion and stereotyped motor behaviors

(DiChiara & Bassareo 2007; Kelley & Berridge 2002; Robinson & Berridge 1993). The activation of dopamine circuitry triggers systemic changes in synaptic signaling between neurons in reward regions of the brain leading to an enhanced search for drug and drug-associated cues.

In addition to the initial priming the brain’s reward system, neuroadaptations in extended amygdala and prefrontal cortex impair executive processes such as self-regulation, decision making, attribution of salience, etc. Mammals exposed to psychostimulant drugs display changes 4 in behavior comparable to those observed in human drug users, offering a set of validated model systems for the study of addiction. These include arousal, hyperactivity, stereotypic perseverative movements, psychomotor depression, cognitive impairment, hallucinatory-like behaviors, and chronic self-administration of amphetamine, methamphetamine and MDMA (Hall et al., 2008;

Varela et al., 2011). Psychostimulant sensitization and reward tolerance arise from neural substrates (Berman et al., 2009), reflecting the central importance of dopaminergic function

(McNamara et al., 2006; Fuentealba et al., 2007; Fraioli et al., 1999; Salahpour et al., 2008;

Orsini et al., 2004). The parent cathinone as well as synthetic cathinones such as mephedrone and methylone appear to produce effects matching those described for amphetamines (Angoa-

Pérez et al., 2012; Marusich et al., 2012; Budzynska et al., 2017; Motbey et al., 2011; Lisek et al., 2012; Shortall et al., 2013), however, similarities and differences remained vastly underexplored.

Most addictive alkaloids (such as cocaine, nicotine, heroin, etc.) are part of a plant’s elaborate defenses against insect herbivory (Wink 2015). Basic properties of these substances, such as a bitter taste and severe toxicity, are countered by specialized adaptations for decreased sensitivity in insects through an evolutionary arms race (Mello & Silva-Filho 2002). The ability to enhanced psychostimulant effects from these plant metabolites forms another potent layer of defense as it likely breaks the effectiveness of crypsis that many herbivorous insects depend on.

The same argument may be advanced for mechanisms that encourage the compulsive consumption of the toxins. As these substances interfere with key components of an animal’s interactions with the environment (Alcaro & Panksepp 2011), its predictive cues, and fundamental mechanisms of associative learning, insects may face greater limitations in adapting to the impact. Arguably representing the primary target of these mechanisms, invertebrate 5 models have received increased attention in the study of addiction (Søvik & Barron, 2013). The fact that many of these compounds produce rewarding effects in invertebrates confirms that the common homocentric diagnostic criteria of addiction may benefit from adoption of a broader perspective (Søvik & Barron, 2013).

Recently, the neural consequences of drugs of addiction have received increasing attention in invertebrate models. Various stimulant drugs, including amphetamines, nicotine, and cocaine, produce increased exploration in Caenorhabditis elegans (Engleman et al., 2016), which is mediated via dopaminergic circuits (Carvelli et al., 2010). D-amphetamine, methamphetamine, morphine, and cocaine enhance exploration in crayfish, with increased locomotion and antennal movements (Alcaro et al., 2011; Huber et al., 2011). Exploratory behaviors in crayfish, driven by tactile and olfactory information, are processed mainly in the olfactory lobe of the crayfish brain. The latter also represents a prominent site of action of amphetamine and other drugs. Drosophila displays psychostimulation when exposed to lower doses of alcohol, cocaine and nicotine, which sensitize upon repeated use (Wolf and Heberlein

2002). Locomotor responses sensitize in crayfish with repeated exposure (Nathaniel et al., 2010,

2012). Termination of amphetamines, cocaine, nicotine and cannabinoids are known to produce withdrawal-like behaviors in planaria (Søvik & Barron, 2013). Reinforcing effects of drugs have been investigated using conditioned place preference paradigm in planaria (Hutchinson et al.

2015), crayfish (Huber et al., 2011), and fruit flies (Kaun et al., 2011), while self administration has been reported in Drosophila (Devineni and Heberlein 2009), honey bees (Abramson et al.,

2005), and crayfish (Datta and Huber, unpublished). Flies even cross an electrified grid in order to seek out drug paired stimuli, a paradigm which is thought to mirror drug use despite negative consequences (Kaun et al., 2011). 6

Studies in invertebrates have also provided valuable insights about the molecular targets of mammalian drugs of abuse. For example, similar to rodent models of drug addiction, dopaminergic circuitry is important for producing rewarding effects of mammalian drugs of abuse in Drosophila and C. elegans (Engleman et al., 2016; Carvelli et al., 2010). Also, c-Fos mRNA, a mammalian brain-reward marker, is altered in the accessory lobe of crayfish in response to cocaine-induced reward (Nathaniel et al., 2010). The aforementioned studies suggest that diversity in invertebrate nervous systems can be utilized to explore the basic functional properties of reward circuits and how drugs of abuse alter their function. Featuring highly modular nervous systems with relatively few, large neurons, invertebrate models are amenable to a search for the underlying neural mechanisms of behavior. This approach has already proven its use in important discoveries such as neuronal conduction in giant squid (Hodgkin & Huxley,

1945), molecular pathways of learning in Aplysia (Kandel, 2007), mechanisms underlying circadian rhythms in Drosophila (Konopka, 1987). Despite the distinct advantages invertebrate models may offer for the study of addiction, evaluations of the behavioral effects of synthetic cathinones have remained rare. Acute mephedrone administration produced stereotyped C- shaped movements and displayed shift in preference when subjected to conditioned place preference in Planaria (Hutchinson et al 2015; Ramoz et al., 2012; Vouga et al., 2015).

While crayfish serve as an excellent model to understand various processes involved in psychostimulant sensitization, little is known about the behavioral effects of synthetic cathinones in this system. This study aimed to compare unconditioned and conditioned effects of cathonones to their better known amphetamine analogs - methylone vs. methyl-methamphetamine (4-MMA) and mephedrone vs. methylenedioxymethamphetamine (MDMA) (Figure 1). Towards this goal, we have investigated unconditioned psychostimulant properties during first exposure in order to 7 characterize increased locomotion as a key metric for psychostimulant effects. We then analyzed the potential of these drugs to produce sensitization with repeated exposure. Temporal resolution of drug effects was obtained by parsing changes in locomotion at fine-scale resolution.

Materials and Methods

Animal Maintenance

Crayfish were wild caught in the Portage River near Bowling Green, Ohio, and maintained under controlled environmental conditions in an aerated community tank (water temperature 20°C, pH7, 12:12h light: dark cycle), and fed once a week with rabbit chow. A week prior to the experiment, intermolt males (approximately 5-15 grams of body weight) with complete, intact appendages were chosen and isolated in perforated plastic containers. Maintained in large holding trays, they received a constant flow of filtered and aerated water.

Surgical procedure for implanting cannula in the pericardial space

Animal were anesthetized in crushed ice for 20 minutes. The cannula was implanted into the anterior end of the pericardial cavity, slightly lateral of the midline to avoid damaging the heart, through an incision created with a 26.5-gauge needle. A 10 cm section of deactivated, fine-bore, fused silica (Agilent 160-2655, i.d. = 50 µm, o.d. = 350 µm) was implanted into the pericardial sinus (allowing 2 mm to enter the sinus) and affixed to the carapace using superglue and bonding material. Following the surgery, animals were allowed to recover overnight in their holding containers. Table 1 lists descriptive details for crayfish included in each experimental group.

Drugs, doses and injection protocols

Mephedrone, methylone, MDMA and 4-MMA were obtained from Cayman Chemical, Ann

Arbor, Michigan. Doses of 1, 3 and 10 µg/g of crayfish body weight of each drug was dissolved in 50µl of 125mM NaCl and administered systemically. Control groups received the same 8 amount of isotonic NaCl solution. Tygon microbore tubing (Fisher Scientific ND 100-80, i.d. =

250 µm) was used to connect a 0.5 m section of deactivated, fine-bore, fused silica needle material (Agilent 160-1010, i.d. = 100 µm, o.d. = 190 µm, 0.5 m long) to the implanted animal stub on one end and the blunt-tipped needle of a 1 ml glass syringe (SGE Analytical Sciences,

Model# 008100) on the other. Mounted above the experimental arena, a syringe pump (Razel R-

99E attached with R-ACC-6 Multi Micro Syringe Adapter) held up to eight syringes side by side to allow for concurrent drug application to multiple animals (Figure 2b).

Experimental Arena and Video Tracking

Training and testing was performed in a circular arena (Diameter = 0.6 m) with two quadrants of soft- and hard-textured substrates each, arranged diagonally. Removable plexi-glass barriers were inserted into the arena to restrict the movements of crayfish to one particular quadrant. A digital camera (LifeCam Studio, Microsoft, Redmond, WA, USA), mounted above the experimental arena, provided a live video stream for automated, real time video tracking on an

Apple Macintosh (OS X 10.11.4, 3.2 GHz Quad-Core Intel Xeon, 6GB RAM). Spatial coordinates, orientations and object outlines of the test crayfish were extracted and logged to disk using the open-source, public domain JavaGrinders framework (available for free download at iEthology.com). In a subsequent analysis, the saved coordinates were analyzed for spatial descriptives such as distance travelled, mean speed, time stationary, etc. (Figure 2d) for each one minute time interval.

Psychomotor stimulation and Sensitization - Experimental Design

Isolated and surgically prepared crayfish (N=104) were randomly assigned to one of 12 treatment groups (N=8 per group) - mephedrone, methylone, MDMA and 4-MMA at 1, 3, and 10

µg/g dose each, while one additional group (saline-injected) served as control. The timeline of a 9 typical experiment is included (Figure 3). The 5 min drug/saline infusion commenced when the crayfish was placed into the arena.

Statistical analysis

Data were expressed as means ± standard errors. Psychomotor stimulation was determined by analyzing the total distance travelled by treatment groups on Day 3 in comparison the control individuals. Sensitization of psychomotor response was determined by analyzing the total distance travelled on Day 4 and Day 5 in comparison to the locomotion on Day 3. The total distance travelled was parsed into 5 minute intervals and repeated measures ANOVA was used to detect significant effects of each drug at various doses (0,1,3 and 10 µg/g) on the distance travelled with doses as a between-subject variable and day and segment as within-subject

(repeated measures) variables. For each drug, the repeated measures ANOVA detected significance of each of the independent variable (dose, day, segment) along with their interaction

(dose:day, day:segment, dose:segment and dose:day:segment) on the dependent variable

(distance travelled). Statistical significance was followed by Tukey post hoc comparisons.

Differences were considered statistically significant at p<0.05. To understand the temporal characteristics of each drug, distance travelled during every 2 minutes segment was considered in comparison to the saline group.

All statistical analyses were performed in R (version 3.3.3 , ) with

RStudio and additional installed packages 'ez', 'Deducer',’lsr’, 'ggplot2', 'nlme', 'stats', and 'lme4.'

Repeated measures ANOVA was conducted on time series data using functions lme (in library nlme) and aov (base). 10

Results

Unconditioned effects in locomotion and other behaviors

When placed in the arena, treatment as well as saline groups quickly approached the closest arena wall and followed its curve. They explored the edges of the arena with their antennae and with occasional rearing up the side wall. Exploratory behavior decreased in the saline group over the course of the trial. Individuals injected with MDMA and methylone typically showed bouts of repetitive claw waving, grooming, and other stereotyped patterns, followed by prolonged periods of immobility. 4-MMA and mephedrone elicited similar behaviors, including forward walking, claw waving, rearing, grooming, extended posture, and stereotyped movements of walking legs and antennae. At higher concentrations Methylone appeared to trigger aversive behaviors like backward walking, tail flips, tremors, and uncoordinated leg movements. Stereotyped movements increased in frequency over the course of the trial in all treatment groups. These patterns were not observed in saline-treated individuals.

Total distance travelled on days 3, 4, and 5 (Fig 4) are reported for 4-MMA, mephedrone,

MDMA and Methylone at 1, 3, and 10 µg/g respectively (Table 2). Repeated measures ANOVA

(Table 3) on the distance travelled during 5 minute time segment for each drug at 1, 3 and 10

µg/g doses across all days was used to investigate the effects of each drug on locomotion. Significant differences were observed for Day, Segment and Dose: Day for MDMA and methylone. The post hoc Tukey test revealed that 3 µg/g MDMA produced significant differences in locomotion on day 3, 4 and 5 during the initial 25 minute segment. Also, 10 µg/g of MDMA resulted in significant increase in locomotion during initial 5 minute segment on day

3 in comparison to initial 30 minute segments on day 5. Tukey post hoc test for methylone revealed significant difference in locomotion for the 3µg/g dose between the first ten minutes 11 and the later segments of day 4 and 5. Repeated measures ANOVA revealed significant Day and

Dose: Day effect on distance travelled for individuals treated with 4-MMA. Post-hoc tukey test showed significant differences across day 4 in comparison to day 3 and 5 for 3µg/g 4-MMA.

Significant effects were observed between doses of mephedrone, although less powerful post-hoc tests failed to pinpoint these significant differences.

Psychostimulation for Mephedrone, Methylone, 4-MMA and MDMA

Figures (5-8) show distance travelled by test crayfish during 2-min intervals on Day 3. Measures of locomotion confirmed that smaller doses of cathinone and substituted amphetamine analogues are sufficient to produce psychomotor stimulation. Infusion of the lowest dose of each drug produced a distinct spike in distance travelled during the initial 5 minutes on the first day of drug exposure (day 3). The increase in locomotion was transient and rapidly faded again when the drug infusion stopped. All drugs increased locomotion at a 3 µg/g dose. The surge in locomotion for 3 µg/g was maintained throughout the time of the trial suggesting that the drug given during initial 5 minutes is able to produce effects that last at least an hour. 10 µg/g of Methylone and 4-

MMA produced psychomotor depression while mephedrone and MDMA produce increased locomotion at the onset of infusion. The saline group did not exhibit significant effects in distance travelled over time.

Sensitization of psychostimulant effects with repeated injections of synthetic cathinones and their substituted amphetamine analogues

Our data (Figure 5-8) depict sensitized psychomotor stimulation with repeated drug injections.

Increased locomotion was maintained for increasingly longer times. For 1 µg/g dose of all the drugs, sensitization of duration of psychomotor response was apparent between days. 10 µg/g of

4-MMA and Methylone showed depression of psychomotor response upon repeated injections 12 while 10 µg/g of MDMA sensitized the duration of locomotor activity compared to saline individuals. 10 µg/g mephedrone produced sensitization of locomotor response consistent with other doses of mephedrone. In all the drugs, 3 µg/g dose produced the greatest amount of psychostimulation but sensitization of this locomotor response was not consistent across the different drugs. In 3 µg/g of 4-MMA and methylone, repeated drug injections produced depression of locomotor response. Repeated injections of 3 µg/g of mephedrone and MDMA produced locomotor effects but they were not higher in duration or intensity compared to the locomotor effects after first drug injection. In general, only mephedrone showed sensitization of locomotor response across all the doses. 4-MMA, MDMA and methylone showed sensitization of locomotor response at 1 µg/g and depression of locomotion at 10 µg/g and had variable effects at 3 µg/g.

Discussion

Psychomotor stimulation is commonly expressed by increased locomotion, an effect that is strongly associated with high addiction potential. Our study demonstrates that synthetic cathinones and substituted amphetamine analogues stimulate locomotion and generally activate behaviors within the lower dose of this study while higher doses were associated with psycho- depressant effects. Effects in which 1 µg/g dose enhanced locomotion while 10 µg/g produced depression, are similar to patterns observed for most of stimulant drugs (Kuczenski and Segal

2001). This is also consistent with the effect observed in humans where lower doses of amphetamines produce enhanced arousal, euphoria, and behavioral disinhibition while higher doses elicit other responses (Kelly 2011). Consistent with our results, higher doses (5 µg/g) of amphetamine produces decreased locomotion and aversive behaviors like tail flip in crayfish

(Alcaro et al., 2011). 13

Initially, the psychostimulant effects were restricted to the initial time period during which the nervous system was exposed to the drug, but these quickly returned to normal when the infusion stopped. While all drugs included here produced psychostimulation, they did differ in their apparent potency, magnitudes of stimulation, and duration of action. Self reports studies in humans suggest that euphoric and stimulant effects of cathinones commence 10-15 minutes , following intravenously administration and that effects last for about 30 minutes (Prosser and

Nelson 2011). These effects are quick in onset and are short-lived in comparison to other known stimulants. Considering the absence of a frank blood-brain barrier in crayfish may explain why these drugs readily act upon the nervous system to produce immediate locomotor effects. Our results are also consistent with the effects of methylone and mephedrone on locomotion in rats where the highest levels of activity are seen right after administration of the drug and diminish over time (Lisek et al., 2012; Marusich et al., 2012).

The duration of psychomotor stimulation strengthened and lasted longer with repeated drug exposure. Our results indicate that repeated drug use resulted in enhanced arousal and exploration compared with a relatively brief spike seen during the first drug infusion. This result is consistent with studies in mice where repeated exposure sensitized the duration of locomotor response (Boehm et al., 2007; Lessov & Phillips, 1998). Sensitized behavioral responses are viewed as indicators for the onset of drug craving and a strong predictor of vulnerabilities to addiction where sensitization is most strongly elicited by intermittent drug dosing (e.g., once daily), compared with constant dosing (Dougherty & Ellinwood 1981).

Crayfish showed increased locomotion when in the whole arena (day 1) as against in quadrant (day 2, 3, 4 and 5) which was expected. On days when crayfish were exposed to quadrant, a pattern which emerged out from all the experimental groups was that crayfish 14 showed increased locomotion on day 2 i.e., when they were walking in drug-free state in a quadrant. A possible explanation for increased locomotion on day 2 is that the quadrant was being perceived as a novel compartment by crayfish. Earlier studies have shown that crayfish explore novel arenas as evident by an increase in distance travelled and exploratory behaviors

(Imeh-Nathaniel et al., 2016; Nathaniel et al., 2010; Nathaniel et al., 2016).

This study demonstrated that crayfish provide insights into complex processes of behavioral sensitization and drug-induced changes in SEEKING drives (Alcaro and Panksepp,

2011) . Very few studies have looked at the effect of mephedrone and methylone on locomotor effects and even fewer studies have investigated sensitization of locomotor responses. Despite the advantages that invertebrate models of addiction offer, the behavioral effects of synthetic cathinones in invertebrates is very sparse. Our data strongly suggests that synthetic cathinones and their substituted amphetamine analogues target the neural pathways in crayfish that serve as essential drivers of behavior, such as those controlling locomotor responses.

An important discussion relevant to our current results and drug addiction in crayfish is monoaminergic circuitry in crustaceans and the targets of synthetic cathinones in the nervous system of crayfish. Our results show distinct effects of MDMA and methylone on locomotion.

Recent findings have revealed monoaminergic systems as the cellular and molecular targets of the synthetic cathinone interacting with plasma membrane transporters for dopamine (i.e., DAT), norepinephrine (i.e., NET) and serotonin (i.e., SERT) (Baumann et al., 2011; Baumann et al.,

2011; Simmler et al., 2012). Results from release assays suggest that mephedrone, methylone, and MDMA are non-selective transporter substrates (i.e., non-selective releasers), while amphetamine is a selective substrate at DAT and NET. Studies using transporter-transfected cell lines have shown that mephedrone, methylone can induce release of 5-HT similar to MDMA. 15

The potency of drugs of abuse to inhibit the NET and DAT or activate NA and DA systems is associated with the psychostimulant effects and enhanced abuse liability (Rothman et al. 2001).

In contrast, increased activation of 5-HT system is linked to reduction in abuse potential and more MDMA-like subjective drug effects. Thus, relative in vitro effects of DAT versus SERT can be used as a predictor of drug characteristics in vivo and vice versa. Crayfish feature a wealth of serotoninergic, dopaminergic, octopaminergic, tyraminergic (Tierney et al., 2003) and peptidergic (Mancillas et al., 1981) systems. The evolutionary conserved D1 and D2-like receptors (Alvarez Alvarado et al. 2005) for dopaminergic and 5-HT1 and 5-HT2 (Tabor and

Cooper 2002) like receptors for serotonergic circuitries are its main features. The findings of our study speculates involvement of serotonergic circuits in producing distinct subjective effects as seen for MDMA and methylone.

Future work will identify specific brain targets and circuits of synthetic cathinones- associated psychostimulation in the brain of crayfish. Identification of the precise neural circuits underlying the effects described here, and the exploration of the overlap between synthetic cathinone and substituted amphetamine analogues will be interesting to study in order to characterize similarity and differences between their mode of action. The pharmacokinetics of drugs in crayfish is also less well known. Knowledge about the metabolites formed following the introduction of these synthetic cathinones along with their half life in crayfish hemolymph will allow us to further understand the temporal component of addiction-related behavioral processes.

In summary, our work supports the notion that crayfish represent a competent model system for studying the primary sites of psychostimulants, to explore the proximate mechanisms, and to identify the primary behavioral causation of drug addiction in this experimentally accessible invertebrate model system. 16

References

Abramson, C. I., Sanderson, C., Painter, J., Barnett, S., & Wells, H. (2005). Development of an ethanol model using social insects: V. Honeybee foraging decisions under the influence of alcohol. Alcohol , 36(3), 187–193.

Alcaro, A., & Panksepp, J. (2011). The SEEKING mind: primal neuro-affective substrates for appetitive incentive states and their pathological dynamics in addictions and depression.

Neuroscience and Biobehavioral Reviews, 35(9), 1805–1820.

Alcaro, A., Panksepp, J., & Huber, R. (2011). d-amphetamine stimulates unconditioned exploration/approach behaviors in crayfish: Towards a conserved evolutionary function of ancestral drug reward. Pharmacology, Biochemistry, and Behavior, 99(1), 75–80.

Alvarez Alvarado, R., Porras Villalobos, M. G., Calderón Rosete, G., Rodríguez Sosa, L., &

Aréchiga, H. (2005). Dopaminergic modulation of neurosecretory cells in the crayfish. Cellular and Molecular Neurobiology, 25(2), 345–370.

Angoa-Pérez, M., Kane, M. J., Francescutti, D. M., Sykes, K. E., Shah, M. M., Mohammed, A.

M.,Kuhn, D. M. (2012). 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, no–no.

Baumann, M. H., Ayestas, M. A., Partilla, J. S., Sink, J. R., Shulgin, A. T., Daley, P. F., …

Cozzi, N. V. (2011). The Designer Methcathinone Analogs, Mephedrone and Methylone, are

Substrates for Monoamine Transporters in Brain Tissue. Neuropsychopharmacology: Official

Publication of the American College of Neuropsychopharmacology, 37(5), 1192–1203.

Baumann, M. H., Clark, R. D., Woolverton, W. L., Wee, S., Blough, B. E., & Rothman, R. B.

(2011). In Vivo Effects of Amphetamine Analogs Reveal Evidence for Serotonergic Inhibition of 17

Mesolimbic Dopamine Transmission in the Rat. The Journal of Pharmacology and Experimental

Therapeutics, 337(1), 218–225.

Berman, S. M., Kuczenski, R., McCracken, J. T., & London, E. D. (2009). Potential adverse effects of amphetamine treatment on brain and behavior: a review. Molecular Psychiatry, 14(2),

123–142.

Boehm, S. L., Goldfarb, K. J., Serio, K. M., Moore, E. M., & Linsenbardt, D. N. (2007). Does context influence the duration of locomotor sensitization to ethanol in female DBA/2J mice?

Psychopharmacology, 197(2), 191–201.

Budzynska, B., Michalak, A., Frankowska, M., Kaszubska, K., & Biała, G. (2017). Acute behavioral effects of co-administration of mephedrone and MDMA in mice. Pharmacological

Reports: PR, 69(2), 199–205.

Carvelli, L., Matthies, D. S., & Galli, A. (2010). Molecular mechanisms of amphetamine actions in Caenorhabditis elegans. Molecular Pharmacology, 78(1), 151–156.

Cruickshank, C. C., & Dyer, K. R. (2009). A review of the clinical pharmacology of methamphetamine. Addiction , 104(7), 1085–1099.

Devineni, A. V., & Heberlein, U. (2009). Preferential ethanol consumption in Drosophila models features of addiction. Current Biology: CB, 19(24), 2126–2132.

DiChiara, G., & Bassareo, V. (2007). Reward system and addiction: what dopamine does and doesn’t do. Current Opinion in Pharmacology, 7(2), 233–233.

Dougherty, G. G., Jr, & Ellinwood, E. H., Jr. (1981). Chronic D-amphetamine in nucleus accumbens: lack of tolerance or reverse tolerance of locomotor activity. Life Sciences, 28(20),

2295–2298. 18

Engleman, E. A., Katner, S. N., & Neal-Beliveau, B. S. (2016). Caenorhabditis elegans as a

Model to Study the Molecular and Genetic Mechanisms of Drug Addiction. Progress in

Molecular Biology and Translational Science, 137, 229–252.

Fass, J. A., Fass, A. D., & Garcia, A. S. (2012). Synthetic Cathinones (Bath Salts): Legal Status and Patterns of Abuse. The Annals of Pharmacotherapy, 46(3), 436–441.

Fraioli, S., Crombag, H. S., Badiani, A., & Robinson, T. E. (1999). Susceptibility to amphetamine-induced locomotor sensitization is modulated by environmental stimuli.

Neuropsychopharmacology: Official Publication of the American College of

Neuropsychopharmacology, 20(6), 533–541.

Fuentealba, J. A., Gysling, K., & Andrés, M. E. (2007). Increased locomotor response to amphetamine induced by the repeated administration of the selective kappa-opioid receptor agonist U-69593. Synapse , 61(9), 771–777.

Hall, D. A., Stanis, J. J., Marquez Avila, H., & Gulley, J. M. (2008). A comparison of amphetamine- and methamphetamine-induced locomotor activity in rats: evidence for qualitative differences in behavior. Psychopharmacology, 195(4), 469–478.

Hodgkin, A. L., & Huxley, A. F. (1945). Resting and action potentials in single nerve fibers. The

Journal of Physiology, 104(2), 176–195.

Huber, R., Panksepp, J. B., Nathaniel, T., Alcaro, A., & Panksepp, J. (2011). Drug-sensitive reward in crayfish: an invertebrate model system for the study of SEEKING, reward, addiction, and withdrawal. Neuroscience and Biobehavioral Reviews, 35(9), 1847–1853.

Hutchinson, C. V., Prados, J., & Davidson, C. (2015). Persistent conditioned place preference to cocaine and withdrawal hypo-locomotion to mephedrone in the flatworm planaria. Neuroscience

Letters, 593, 19–23. 19

Imeh-Nathaniel, A., Adedeji, A., Huber, R., & Nathaniel, T. I. (2016). The rewarding properties of methamphetamine in an invertebrate model of drug addiction. Physiology & Behavior, 153,

40–46.

Jiao, D., Liu, Y., Li, X., Liu, J., & Zhao, M. (2015). The role of the GABA system in amphetamine-type stimulant use disorders. Frontiers in Cellular Neuroscience, 9, 162.

Kalivas, P. (2004). Glutamate systems in cocaine addiction. Current Opinion in Pharmacology,

4(1), 23–29.

Kalivas, P. W., & Stewart, J. (1991). Dopamine transmission in the initiation and expression of drug- and stress-induced sensitization of motor activity. Brain Research. Brain Research

Reviews, 16(3), 223–244.

Kandel, E. R. (2007). In Search of Memory: The Emergence of a New Science of Mind. W. W.

Norton & Company.

Karila, L., Billieux, J., Benyamina, A., Lançon, C., & Cottencin, O. (2016). The effects and risks associated to mephedrone and methylone in humans: A review of the preliminary evidences.

Brain Research Bulletin, 126(Pt 1), 61–67.

Kaun, K. R., Azanchi, R., Maung, Z., Hirsh, J., & Heberlein, U. (2011). A Drosophila model for alcohol reward. Nature Neuroscience, 14(5), 612–619.

Kelley, A. E., & Berridge, K. C. (2002). The neuroscience of natural rewards: relevance to addictive drugs. The Journal of Neuroscience: The Official Journal of the Society for

Neuroscience, 22(9), 3306–3311.

Kelly, J. P. (2011). Cathinone derivatives: a review of their chemistry, pharmacology and toxicology. Drug Testing and Analysis, 3(7-8), 439–453. 20

Konopka, R. J. (1987). Genetics of Biological Rhythms in Drosophila. Annual Review of

Genetics, 21(1), 227–236.

Kuczenski, R., & Segal, D. S. (2001). Locomotor effects of acute and repeated threshold doses of amphetamine and methylphenidate: relative roles of dopamine and norepinephrine. The Journal of Pharmacology and Experimental Therapeutics, 296(3), 876–883.

Lessov, C. N., & Phillips, T. J. (1998). Duration of sensitization to the locomotor stimulant effects of ethanol in mice. Psychopharmacology, 135(4), 374–382.

Lisek, R., Xu, W., Yuvasheva, E., Chiu, Y.-T., Reitz, A. B., Liu-Chen, L.-Y., & Rawls, S. M.

(2012). Mephedrone (’bath salt') elicits conditioned place preference and dopamine-sensitive motor activation. Drug and Alcohol Dependence, 126(1-2), 257–262.

Mancillas, J. R., McGinty, J. F., Selverston, A. I., Karten, H., & Bloom, F. E. (1981).

Immunocytochemical localization of enkephalin and substance P in retina and eyestalk neurons of lobster. Nature, 293(5833), 576–578.

Marusich, J. A., Grant, K. R., Blough, B. E., & Wiley, J. L. (2012). Effects of synthetic cathinones contained in “bath salts” on motor behavior and a functional observational battery in mice. Neurotoxicology, 33(5), 1305–1313.

May, J., Andrade, J., Panabokke, N., & Kavanagh, D. (2004). Images of desire: Cognitive models of craving. Memory , 12(4), 447–461.

McNamara, R. K., Logue, A., Stanford, K., Xu, M., Zhang, J., & Richtand, N. M. (2006). Dose– response analysis of locomotor activity and stereotypy in dopamine D3 receptor mutant mice following acute amphetamine. Synapse , 60(5), 399–405.

Mello, M. O., & Silva-Filho, M. C. (2002). Plant-insect interactions: an evolutionary arms race between two distinct defense mechanisms. Brazilian Journal of Plant Physiology, 14(2), 71–81. 21

Motbey, C. P., Hunt, G. E., Bowen, M. T., Artiss, S., & McGregor, I. S. (2011). Mephedrone (4- methylmethcathinone, “meow”): acute behavioral effects and distribution of Fos expression in adolescent rats. Addiction Biology, 17(2), 409–422.

Nathaniel, T. I., Panksepp, J., & Huber, R. (2010). Effects of a single and repeated morphine treatment on conditioned and unconditioned behavioral sensitization in Crayfish. Behavioral

Brain Research, 207(2), 310–320.

Nathaniel, T. I., Panksepp, J., & Huber, R. (2012). Alteration of c-Fos mRNA in the accessory lobe of crayfish is associated with a conditioned-cocaine induced reward. Neuroscience

Research, 72(3), 243–256.

Orsini, C., Buchini, F., Piazza, P. V., Puglisi-Allegra, S., & Cabib, S. (2004). Susceptibility to amphetamine-induced place preference is predicted by locomotor response to novelty and amphetamine in the mouse. Psychopharmacology, 172(3), 264–270.

Paulson, P. E., Camp, D. M., & Robinson, T. E. (1991). Time course of transient behavioral depression and persistent behavioral sensitization in relation to regional brain monoamine concentrations during amphetamine withdrawal in rats. Psychopharmacology, 103(4), 480–492.

Prosser, J. M., & Nelson, L. S. (2011). The Toxicology of Bath Salts: A Review of Synthetic

Cathinones. Journal of Medical Toxicology: Official Journal of the American College of Medical

Toxicology, 8(1), 33–42.

Ramoz, L., Lodi, S., Bhatt, P., Reitz, A. B., Tallarida, C., Tallarida, R. J., … Rawls, S. M.

(2012). Mephedrone (“bath salt”) pharmacology: insights from invertebrates. Neuroscience, 208,

79–84.

Robinson, T. E., & Berridge, K. C. (1993). The neural basis of drug craving: an incentive- sensitization theory of addiction. Brain Research. Brain Research Reviews, 18(3), 247–291. 22

Robinson, T. E., & Berridge, K. C. (2003). Addiction. Annual Review of Psychology, 54, 25–53.

Robinson, T. E., Yager, L. M., Cogan, E. S., & Saunders, B. T. (2014). On the motivational properties of reward cues: Individual differences. Neuropharmacology, 76, 450–459.

Rosenbaum, C. D., Carreiro, S. P., & Babu, K. M. (2012). Here today, gone tomorrow…and back again? A review of herbal marijuana alternatives (K2, Spice), synthetic cathinones (bath salts), kratom, Salvia divinorum, methoxetamine, and piperazines. Journal of Medical

Toxicology: Official Journal of the American College of Medical Toxicology, 8(1), 15–32.

Rothman, R. B., Baumann, M. H., Dersch, C. M., Romero, D. V., Rice, K. C., Carroll, F. I., &

Partilla, J. S. (2001). Amphetamine-type central nervous system stimulants release norepinephrine more potently than they release dopamine and serotonin. Synapse , 39(1), 32–41.

Salahpour, A., Ramsey, A. J., Medvedev, I. O., Kile, B., Sotnikova, T. D., Holmstrand, E., …

Caron, M. G. (2008). Increased amphetamine-induced hyperactivity and reward in mice overexpressing the dopamine transporter. Proceedings of the National Academy of Sciences of the United States of America, 105(11), 4405–4410.

Shortall, S. E., Macerola, A. E., Swaby, R. T. R., Jayson, R., Korsah, C., Pillidge, K. E., … King,

M. V. (2013). Behavioral and neurochemical comparison of chronic intermittent cathinone, mephedrone and MDMA administration to the rat. European Neuropsychopharmacology: The

Journal of the European College of Neuropsychopharmacology, 23(9), 1085–1095.

Simmler, L. D., T. A. Buser, M. Donzelli, Y. Schramm, L-H Dieu, J. Huwyler, S. Chaboz, M. C.

Hoener, and M. E. Liechti. (2012) Pharmacological Characterization of Designer Cathinonesin

Vitro. British Journal of Pharmacology 168 (2): 458–70.

Søvik, E., & Barron, A. B. (2013). Invertebrate models in addiction research. Brain, Behavior and Evolution, 82(3), 153–165. 23

Steketee, J. (2003). Neurotransmitter systems of the medial prefrontal cortex: potential role in sensitization to psychostimulants. Brain Research Reviews, 41(2-3), 203–228.

Tabor, J. N., & Cooper, R. L. (2002). Physiologically identified 5-HT2-like receptors at the crayfish neuromuscular junction. Brain Research, 932(1-2), 91–98.

Tierney, A. J., Kim, T., & Abrams, R. (2003). Dopamine in crayfish and other crustaceans: distribution in the central nervous system and physiological functions. Microscopy Research and

Technique, 60(3), 325–335.

Vanderschuren, L. J. M. J., Louk J M, & Christopher Pierce, R. (2009). Sensitization Processes in Drug Addiction. In Current Topics in Behavioral Neurosciences (pp. 179–195).

Varela, M. J., Brea, J., Loza, M. I., Maldonado, R., & Robledo, P. (2011). Sensitization to

MDMA locomotor effects and changes in the functionality of 5-HT2A and D2 receptors in mice.

Behavioral Pharmacology, 22(4), 362–369.

Vezina, P. (2004). Sensitization of midbrain dopamine neuron reactivity and the self- administration of psychomotor stimulant drugs. Neuroscience and Biobehavioral Reviews, 27(8),

827–839.

Vouga, A., Gregg, R. A., Haidery, M., Ramnath, A., Al-Hassani, H. K., Tallarida, C. S., …

Rawls, S. M. (2015). Stereochemistry and neuropharmacology of a “bath salt” cathinone: S- enantiomer of mephedrone reduces cocaine-induced reward and withdrawal in invertebrates.

Neuropharmacology, 91, 109–116.

Watterson, L. R., & Olive, M. F. (2014). Synthetic cathinones and their rewarding and reinforcing effects in rodents. Advances in Neuroscience (Hindawi), 2014, 209875. 24

Weinstein, A. M., Rosca, P., Fattore, L., & London, E. D. (2017). Synthetic Cathinone and

Cannabinoid Designer Drugs Pose a Major Risk for Public Health. Frontiers in Psychiatry /

Frontiers Research Foundation, 8, 156.

Wink, M. (2015). Modes of Action of Herbal Medicines and Plant Secondary Metabolites.

Medicines (Basel, Switzerland), 2(3), 251–286.

Wolf, F. W., & Heberlein, U. (2002). Invertebrate models of drug abuse. Journal of

Neurobiology, 54(1), 161–178.

25

CHAPTER 2: REWARDING EFFECTS OF SYNTHETIC CATHINONES

(METHYLONE, MEPHEDRONE) AND THEIR SUBSTUTUTED AMPHETAMINE

ANALOGS (MDMA, 4-MMA) IN AN INVERTEBRATE MODEL OF DRUG

ADDICTION.

Introduction

Drugs of abuse are known to “hijack” natural reward systems to produce their pleasurable and rewarding effects. Stimulant drug abuse continues to be a major health concern all over the world. Amphetamines, such as methamphetamine and MDMA, cause severe neurotoxicity, lead to addiction and may induce psychotic disorders or cognitive dysfunctions (Cruickshank and

Dyer 2009; Jiao et al., 2015). High abuse potential for amphetamines arise from a transient activation of neural substrates for arousal, reward, euphoria, behavioral disinhibition, and cognitive domains. Higher doses elicit sympathetic activation and agitation, may lead to violent behavior, and their termination induces strong withdrawal effects such as disturbed sleep, depressed mood and anxiety, intense craving and cognitive impairment.

Cathinone is a naturally occurring plant alkaloid, producing effects similar to amphetamines with a matching backbone but featuring a beta ketone moiety. Cathinone is found in the khat shrub (Catha edulis) native to Arabian peninsula and the horn of Africa where the leaves have been chewed for its psychostimulant effects for hundreds of years (Kelly 2011;

Marusich et al., 2012). The chemical structure of cathinone can be readily altered to generate a number of novel derivatives (Watterson & Olive, 2014) with methyl and di-oxy substitutions. In

1993, cathinone was classified as a Schedule I controlled substance in the USA, however, the manufacturers and the distributors have managed to evade regulatory scrutiny by marketing synthetic cathinone and its derivatives (Weinstein et al., 2017) as “bath salts” or “plant food”. A 26

“not for human consumption” label has allowed sellers to exploit a unclear regulatory framework

(Kelly 2011; Marusich et al., 2012). Amphetamine analogues, including synthetic cathinones, are the second most widely abused class of drugs worldwide (United Nations Office On Drugs and

Crime world drug report, UNODC 2012). Being structurally similar to amphetamines, cathinones appear to bring about some of the same behavioral consequences. Apart from the classic sympatho-mimetic stimulatory effects, some cathinones are also known to produce MDMA-like hallucinogenic effects. Strong craving following termination of synthetic cathinones is reported by a high proportion of synthetic cathinone users (Kelly 2011; Marusich et al., 2012). Despite widespread abuse, little is known about the pharmacology, physiological effects, behavioral consequences, and abuse potential of this rapidly evolving class of substances.

Drugs of abuse are known to modulate the neural reward circuitry to produce their effects ranging from attentional, euphoric and reinforcing effects. As environmental cues are repeatedly associated with the drug, they become conditionally linked. Compulsive promotion of drug seeking/craving, and of the cures that provide predictable access, are primary drivers of relapse.

Enhanced learning and memory consolidation for paired cues exceed responses from cues associated with natural rewards (Torregrossa et al., 2011; Hyman et al., 2006). These conditioned responses become deeply ingrained and can trigger strong cravings for a drug long after initial use had been discontinued. Manipulations that inhibit cue memory reconsolidation, or that enhance consolidation of cue extinction, offer potential therapeutic targets for the treatment of drug addiction and the prevention of relapse (Taylor et al., 2009; Sorg 2012).

Neural circuits in the nucleus accumbens, amygdala, and prefrontal cortex are implicated in the development of drug-dependence and for the extinction and reconsolidation of drug associated memories (Jentsch and Taylor 1999; Taylor et al., 2009). The implementation of mammalian 27 reward resides in mesolimbic dopamine pathways which are also known to play a role in mediating locomotion and stereotyped motor behaviors (Dichiara and Bassareo 2007; Kelley and

Berridge 2002; Robinson and Berridge 1993; Alcaro et al., 2007). The drug induced activation of dopamine circuitry triggers changes in signaling between neurons in various reward regions of the brain and brings about persistent rewiring of brain circuitry within reward and motivational systems (Schultz 2002; Koob and Le Moal 2005). In addition to co-opting and resetting the brain’s reward system, neuroadaptations of repeated drug use generate negative affect via activation of the amygdala. Addicts frequently express frustration about why they continue to pursue the drug even when it no longer seems pleasurable. Accompanying changes in the prefrontal cortex impair executive processes for self-regulation, decision making, and the attribution of salience.

Psychostimulant drugs when administered in mammalian models display comparable changes in behavior to those observed in human drug users. This work explores arousal, hyperactivity, stereotypic movements, psychomotor depression, cognitive impairment, hallucinatory-like behaviors, and chronic self- administration (Hall et al., 2008; Varela et al.,

2011). Psychostimulant sensitization and reward tolerance (Berman et al., 2009) reflect the central importance of dopaminergic function (McNamara et al., 2006; Fuentealba et al., 2007;

Fraioli et al., 1999; Salahpour et al., 2008; Orsini et al., 2004). Synthetic cathinone derivatives, e.g., mephedrone and methylone, produce effects matching those described for their amphetamine equivalents (Angoa-Pérez et al., 2012; Marusich et al., 2012; Budzynska et al.,

2017; Motbey et al., 2011; Shortall et al., 2013; Lisek et al., 2012), however, comparative work exploring the similarities and differences remains vastly underexplored. Specifically, rodents readily self administer synthetic cathinones such as mephedrone, MDPV and methylone. Studies 28 involving intracranial self stimulation paradigm demonstrate that reward circuitry is the source of euphoric and reinforcing effects of mephedrone (Robinson et al., 2012), MDPV, and methylone

(Watterson and Olive 2014; Lisek et al., 2012).

Research into the biological basis of addiction has increasingly recognized the value of invertebrate preparations (Søvik & Barron, 2013). Addictive alkaloids (cocaine, nicotine, morphine, etc.) are integral to a plant’s multi-level defenses against herbivory (Wink, 2015).

With insects as the primary target pharmacological effects of drugs and their behavioral consequences depict a much more basic biological phenomenon than generally thought. In an evolutionary arms race, basic properties of a bitter taste and severe toxicity, are countered by specialized adaptations for decreased sensitivity (Mello & Silva-Filho, 2002). The drug’s ability to interfere with basic processes of motivation and learning represents an additional layer of defense. Psychostimulant effects, which break the target’s crypsis and generally interfering with fundamental drivers of behavior, appear to offer a formidable impediment. Moreover, forcing the insect into compulsive consumption of the toxins potently interferes with the ability to cope with life’s challenges and shortens life spans (Alcaro & Panksepp, 2011). The fact that many of these compounds produce rewarding effects in invertebrates confirms that the common homocentric diagnostic criteria of addiction may benefit from adoption of a broader perspective (Søvik &

Barron, 2013).

Stimulant drugs such as nicotine, cocaine, and amphetamine increase exploratory behavior in C. elegans (Engleman et al., 2016), which, as in mammals, is mediated via dopaminergic circuits (Carvelli et al., 2010). Drosophila exhibits psycho-stimulation when exposed to lower doses of alcohol, cocaine and nicotine upon repeated use (Wolf and Heberlein

2002). Enhanced exploration in crayfish, expressed in increased locomotion and antennal 29 movements, sensitizes with repeated drug exposure (Nathaniel et al., 2010; Nathaniel et al.,

2012). Termination of amphetamines, cocaine, nicotine and cannabinoids are known to produce withdrawal-like behaviors in planaria (Søvik and Barron 2013). Reinforcing effects of drugs have been demonstrated using conditioned place preference paradigms in planarians (Hutchinson et al., 2015), crayfish (Huber et al., 2011), and fruit flies (Kaun et al., 2011). In planarians, acute mephedrone administration induces cue preferences when subjected to CPP (Ramoz et al., 2012;

Hutchinson et al., 2015; Vouga et al., 2015). Drosophila (Devineni and Heberlein 2009), bees

(Abramson et al., 2005), and crayfish (Datta and Huber, unpublished) self administer drugs.

Once conditioned, flies seek out drug paired stimuli even if these are paired with negative consequences, such as the need to walk over an electrified grid (Kaun et al., 2011). Studies in invertebrates have also started to provide valuable insights into the molecular targets of addictive drugs, including its effects on dopaminergic circuitry in Drosophila and C. elegans (Kaun et al.,

2012; Engleman et al., 2016), and the activation of immediate early gene expression in crayfish

(Nathaniel et al., 2012). The pursuit of drug effects on neural mechanisms in invertebrates promises new insights into the basic functional properties of reward circuits and how drugs of abuse alter their function.

Amenable to a search for the underlying neural mechanisms of behavior, invertebrate preparations have helped advance important discoveries such as neuronal conduction in giant squid (Hodgkin & Huxley, 1945), the function of gap junctions in crayfish (Furshpan and Potter

1959), molecular pathways of learning in Aplysia (Kandel, 2007), and mechanisms underlying circadian rhythms in Drosophila (Konopka, 1987). Due to the ease of experimental manipulations, modularly organized nervous system, absence of blood-brain barrier and well- characterized behavioral paradigms for drug addiction-like behaviors, crayfish continues to be an 30 ideal model to study the addictive potential of any drug. Crayfish share with mammals neurochemical axes for serotonin, catecholamine, and tyramine (Tierney et al., 2003) and enkephalin (Mancillas et al., 1981). Large, individually identifiable neurons, promise a substantial reduction of experimental complexity associated with studying the neuroanatomical targets of drug actions.

Conditioned place preference paradigm (CPP) is used to check the existence and strength of drug-induced reward. If an individual seeks out the environment paired with the drug, it is because of rewarding properties of that drug. Earlier studies in crayfish have used CPP to study seeking, reward, and extinction of drug. Repeated infusions of drug serve as a reward when paired with distinct visual or tactile cues in crayfish. The present work harnesses the experimental advantages of crayfish to examine behavioral effects of synthetic cathinones for learned cure preference. We assess the reward potential of mephedrone and methylone in comparison with their amphetamine analogues 4-MMA and MDMA, respectively. A conditioned place preference paradigm estimates the rewarding potential of each drug. Moreover, extinction trials were designed to then break the pairing between drug and the drug paired environment through extinction trials. A characterization of the persistence of memories for drug-associated cues may offer insights into the mechanisms underlying drug-associated memories, and may advance potential targets for treating addiction-related disorders (Torregrossa & Taylor, 2013).

Materials and Methods

Animal Maintenance

Crayfish were wild caught in the Portage River near Bowling Green, Ohio, and maintained under controlled environmental conditions in an aerated community tank (water temperature 20°C, pH7, 12:12h light: dark cycle), and fed once a week with rabbit chow. A week prior to the 31 experiment, intermolt males (approximately 5-15 grams of body weight) with complete, intact appendages were chosen and isolated in perforated plastic containers. Maintained in large holding trays they received a constant flow of filtered and aerated water.

Surgical procedure for implanting cannula in the pericardial space

Animal were anesthetized in crushed ice for 20 minutes. The cannula was implanted into the anterior end of the pericardial cavity through an incision created using 26.5 gauge needle, slightly lateral of the midline to avoid damaging the heart. A 10 cm section of deactivated, fine- bore, fused silica (Agilent 160-2655, i.d. = 50 µm, o.d. = 350 µm) was implanted into the pericardial sinus (allowing 2 mm to enter the sinus) and affixed to the carapace using superglue and bonding material. Following the surgery, animals were allowed to recover overnight in their holding containers.

Drugs, doses and injection protocols

Mephedrone, methylone, MDMA and 4-MMA were obtained from Cayman Chemical, Ann

Arbor, Michigan. All the chemicals were dissolved in 125mM NaCl and administered systemically at doses 1, 3 and 10 µg/g body weight of crayfish (injection volume 50µl). Control groups received isotonic NaCl solution only. Tygon microbore tubing (Fisher Scientific ND 100-

80, i.d. = 250 um) was used to connect a 0.5m section of deactivated, fine-bore, fused silica needle material (Agilent 160-1010, i.d. = 100 um, o.d. = 190 µm, 0.5m long) to the implanted animal stub on one end and the blunt-tipped needle of a 1ml glass syringe (SGE Analytical

Sciences, Model# 008100) on the other. Mounted above the experimental arena, the syringe pump housed up to eight syringes side by side (Razel R-99E attached with R-ACC-6 Multi

Micro Syringe Adapter) to allow for concurrent drug application to multiple animals (Figure 2b).

Experimental Arena and Video Tracking 32

Training and testing was performed in a circular arena (Diameter = 0.6m) with two soft-textured and two hard substrate quadrants arranged diagonally. When needed, removable plexi-glass barrier was inserted into the arena to restrict the movements of crayfish to a particular quadrant. A digital camera (Microsoft LifeCam Studio, Microsoft, Model #: Q2F-00013, 1080p

HD Sensor), mounted above the experimental arena, provided a live video streams for automated, real time video tracking run on a Apple Macintosh (OS X El Captain, Version

10.11.4, Processor: 3.2 GHz Quad-Core Intel Xeon, Memory: 6GB 1066 MHz DDR3

ECC) Spatial coordinates, orientations and object outlines of the test crayfish were extracted and logged to disk using the open-source, public domain JavaGrinders framework (available for free download at iEthology.com). In a subsequent analysis, the saved coordinates were analyzed to extract spatial descriptives like distance travelled, mean speed, time stationary, etc. (Figure 2d).

For this study, the amount of time spent by crayfish on hard and soft substrate was measured.

Experimental Design - Conditioned Place Preference

Crayfish (n = 104) were randomly assigned to 12 treatment groups (n = 8 per group) - mephedrone, methylone, MDMA and 4-MMA at 1, 3, and 10 µg/g dose each while the thirteenth group (saline-injected) served as control. All possible pairwise combinations of substrate and drug were tested when each treatment group was stratified with half of the individuals experiencing drug paired with soft or hard substrate. The details of stratification and descriptives of each experimental group are included (Table 4). Each individual was subjected to included phases for pre-conditioning (Pre-C), conditioning, the CPP test (Post-C), the extinction pairing, and the extinction test (Post-E). The timeline of the typical CPP experiment is included (Figure

9) We used an unbiased CPP approach by combining equal numbers of individuals with each 33 substrate (i.e., irrespective of initial preference for a substrate by an individual) to examine all pairwise counter-balanced combinations of substrate and drug during conditioning.

Statistical analysis

Data, expressed as means ± standard errors for the amount of time spent in each compartment was compared between pre-C (Day1), post-C (Day6) and post-E (Day 10) outcomes. The initial, pre-conditioning preference for a particular tactile substrate was examined with a one sample t- test (µ = 50%). Repeated measures ANOVA was used to detect significant drug effects on the amount of time spent on the drug-paired substrate with doses as a between-subject variable and day as within-subject (repeated measures) variables. For each drug, the repeated measures

ANOVA detected significance of each of the independent variable (dose and day) along with their interaction (dose:day) on the dependent variable (time spent on drug-paired substrate).

Statistical significance was followed by Dunn Sidak post hoc comparisons. Differences were considered statistically significant at p<0.05.

All statistical analyses were conducted in R (R version 3.3.3 , ) with supplementary installed packages 'ez', 'Deducer',’lsr’, 'ggplot2', 'nlme', 'stats', and 'lme4.'

Repeated measures ANOVA was conducted on time sequence data for the dependent variable

(time spent in drug-paired compartment) using package nlme (Function: lme).

Results

Crayfish show a slight, initial preference for soft substrates

In contrast to our expectation that crayfish will spent equal amount of time on both the substrates, it was found out that crayfish preferred the soft tactile environment (Figure 10) over the hard substrate. During the first day, crayfish spent 54.8 % ± 0.81 (S.E.M.) percent of their 34 time on the soft substrate and 45.2 % ± 0.81 (S.E.M.) on the hard substrate. The preference for soft substrate was statistically significant (one sample t-test (µ = 50%); t-value = 5.98;

P-value = 4.2 X 10-8).

Three days of drug conditioning demonstrate rewarding effects

Figure 11 shows CPP findings for 4-MMA, mephedrone, MDMA and methylone at 1, 3, and 10

µg/g (Table 5). Higher doses (10 µg/g) of drugs induced preference for drug-paired substrate.

Repeated measures ANOVA (Table 6) revealed significant environmental preference for substrate paired with 10 µg/g mephedrone (p < 0.05). Tukey post hoc test showed significant increase in preference for drug-paired compartment during post-C in the crayfish treated with 10

µg/g mephedrone. Crayfish treated with 10 µg/g of mephedrone increased its preference for drug-paired substrate from 47.3 % (pre-C) to 58.1 % (post-C). 10 µg/g of MMA, MDMA, and methylone showed increased preference for drug-paired substrate but the effect did not attain statistical significance. 3 µg/g dose of MMA and MDMA seemed to increase preference for drug-paired substrate, however it did not achieve statistical significance.

The established rewarding effects are relatively unaffected by the extinction pairing sessions

For each of the 10 µg/g drug group, the amount of time spent on the drug-paired substrate during the extinction test (Post-E) on day 10, continued to be elevated when compared to the pre- exposure (Pre-C) on day 1 (Figure 11). For 3µg/g and 1µg/g doses, the sustained preference for drug-paired substrate during the Post-E phase was not apparent. However, none the groups attained statistical significance for the observed trend.

Discussion

The present study was constructed to assess the abuse potential of two synthetic cathinones (mephedrone and methylone) in crayfish and compare their effects with the 35 prototypical drugs (MDMA and 4-MMA respectively) using a conditioned place preference paradigm. The increase in preference for drug-paired substrate after conditioning with mephedrone, methylone, MDMA and 4-MMA strongly indicates that these drugs display rewarding effects and that they may carry significant risks of abuse in all organisms. The rewarding effects of the synthetic cathinones described in the present study are consistent with studies in rodents who readily self-administer synthetic cathinones. Studies involving intracranial self stimulation paradigm have shown that reward circuitry to exert the euphoric and reinforcing effects of mephedrone (Robinson et al.,, 2012), MDPV, and methylone (Watterson and Olive

2014; Lisek et al.,, 2012).

In the current study, comparing the amount of time spent by crayfish treated with substituted amphetamine analogues to the ones treated with synthetic cathinones, lower doses (3

µg/g) of substituted amphetamine analogues are enough to produce increased preference for drug-paired substrates (Figure 12). However, synthetic cathinones produced strong preference for drug-paired substrates only at higher doses (10µg/g). Crayfish treated with 3 µg/g 4-MMA and MDMA increased preference for drug-paired substrate from 48.1% and 50% on Day 1 (Pre-

C) to 59.5% and 63% on Day 6 (Post-C), respectively. However, this increased preference for drug paired substrate was extinguished on Day 10 (Post-E) after the extinction pairing (Figure 6).

Crayfish treated with 10 µg/g mephedrone and methylone spent 59% and 65% on drug paired substrate on Day 6 (Post-C) as against 47% and 51% on Day 1(Pre-C). For 10 µg/g methylone and mephedrone, crayfish continued to prefer the drug paired compartment on Day 10 (Post-E) compared to the Day 1 (Pre-C). Synthetic cathinones seemed to produce rewarding effects at higher doses than those of their substituted amphetamine analogues suggesting that synthetic cathinones have less abuse potential than the amphetamines. The presence of a ketone in the side 36 chain of cathinones make them more hydrophilic than their amphetamine counterparts which may account for lower potency than the amphetamines (Gibbons and Zloh, 2010), a results consistent with self administration studies in mice (Watterson & Olive, 2014).

The current study appears to be the first of its kind to look at persistent reward strength of cathinones and amphetamines following extinction pairings in crayfish. Preferences for drug paired substrates remained elevated for higher doses of drugs suggesting that these drugs can have long term effects even after extinction trials. Studies have shown that environmental cues that are repeatedly associated with a drug are known to promote compulsive drug taking and craving and are a primary trigger of relapse. Our study shows that a relatively simple organism like crayfish can learn to associate environmental cues with anticipation of drug-induced reward.

Despite the advantages that invertebrate models of addiction offer, less studies have evaluated the behavioral effects of synthetic cathinones in invertebrate models. Our results of the

CPP experiment are consistent with the rewarding effects of some known stimulants in crayfish.

Previous studies in crayfish have demonstrated that repeated systemic infusions of various doses of psychostimulants like D-amphetamine, Methamphetamine, morphine, cocaine, and heroine served as a reward when paired with a distinct visual or tactile environment. Crayfish can learn to self-administer D-amphetamine in an operant conditioning paradigm (Datta and Huber, unpublished). Our CPP data strongly suggests that synthetic cathinones and their substituted amphetamine analogues target the neural pathways in crayfish that serve as powerful rewards.

This finding indicates that crayfish represents a competent model for studying the primary sites of psychostimulants to explore the proximate mechanisms and fundamental neurobiological processes that underlie drug addiction in an invertebrate model. 37

At higher doses, mephedrone and 4-MMA were able to produce statistically significant preference for drug paired substrate. Addiction-like behavior in crayfish appears to interfere with monoaminergic circuitry. Crayfish are known to have serotoninergic, dopaminergic, octopaminergic , tyraminergic (Tierney et al., 2003) and enkephalinergic (Mancillas et al.,,

1981) neurons. Recent findings have revealed monoaminergic systems as the cellular and molecular targets of the synthetic cathinones. Synthetic cathinones interact with plasma membrane transporters for dopamine (i.e., DAT), norepinephrine (i.e., NET) and serotonin (i.e.,

SERT) (Baumann et al., 2011; Baumann et al., 2011; Simmler et al., 2012). Mephedrone, methylone, and MDMA are non-selective transporter substrates (i.e., non-selective releasers), while amphetamine is a selective substrate at DAT and NET as shown by release assays. Studies using transporter-transfected cell lines have revealed that mephedrone, methylone induces release of 5-HT similar to MDMA. The potency of drugs of abuse to inhibit the NET and DAT or activate NA and DA systems is associated with the psychostimulant effects and enhanced abuse liability (Rothman et al., 2001). In contrast, relatively increased activation of 5-HT system is linked to reduction in abuse potential and more MDMA-like subjective drug effects. Relative in vitro effects of DAT versus SERT can be used as a predictor of drug characteristics in vivo and vice versa (Simmler et al., 2012). The findings of our study suggest involvement of dopaminergic circuits in producing rewarding effects of these drugs. The knowledge about precise neuroanatomical location of monoaminergic neurons affirms crayfish as an ideal candidate for underpinning the neuropharmacology of drug-induced reward. In mammals, psychostimulants are generally known to interfere with the monoamine chemistry to induce reward when exposed to a distinct visual environment. Our finding that synthetic cathinones and substituted amphetamine analogues are rewarding to an invertebrate system with simple neuronal 38 organization indicates that mammalian drugs of addiction are likely to initiate reward beyond those peculiar to humans.

Future studies are necessary to identify specific brain targets and circuits of synthetic cathinones-associated reward in the brain of crayfish Identification of the precise neural circuits underlying the effects described here, and the exploration of the overlap between synthetic cathinone and substituted amphetamine analogues will be interesting to study in order to characterize similarity and differences between their mode of action. A direct extension of current study involves introduction of a drug priming injection following extinction phase to look at the potential of synthetic cathinones for relapse. Even though crayfish nervous system serves several advantages for easier experimental manipulations, the pharmacokinetics of drugs in crayfish is less known. Knowledge about the metabolites formed following the introduction of these synthetic cathinones along with their half life in crayfish hemolymph will be allow us to understand the temporal component addiction-related behavioral processes in an efficient manner. Recent studies have revealed that crayfish can learn to self-administer drugs (Udita and

Huber, unpublished) and that they can learn to avoid aversive stimulus like shocks (Bhimani &

Huber, 2016). Drugs are consumed by addicts despite its known negative consequences. An extension of current study in combination with the new paradigms that are established is crayfish will allow us to look at the reinforcing effects of drug despite the negative consequences (like shocks).

References

Abramson, C. I., Sanderson, C., Painter, J., Barnett, S., & Wells, H. (2005). Development of an ethanol model using social insects: V. Honeybee foraging decisions under the influence of alcohol. Alcohol , 36(3), 187–193. 39

Alcaro, A., Huber, R., & Panksepp, J. (2007). Behavioral functions of the mesolimbic dopaminergic system: an affective neuroethological perspective. Brain Research Reviews, 56(2),

283–321.

Alcaro, A., & Panksepp, J. (2011). The SEEKING mind: primal neuro-affective substrates for appetitive incentive states and their pathological dynamics in addictions and depression.

Neuroscience and Biobehavioral Reviews, 35(9), 1805–1820.

Alcaro, A., Panksepp, J., & Huber, R. (2011). d-amphetamine stimulates unconditioned exploration/approach behaviors in crayfish: Towards a conserved evolutionary function of ancestral drug reward. Pharmacology, Biochemistry, and Behavior, 99(1), 75–80.

Angoa-Pérez, M., Kane, M. J., Francescutti, D. M., Sykes, K. E., Shah, M. M., Mohammed, A.

M., … Kuhn, D. M. (2012). 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, no–no.

Baumann, M. H., Ayestas, M. A., Partilla, J. S., Sink, J. R., Shulgin, A. T., Daley, P. F., …

Cozzi, N. V. (2011). The Designer Methcathinone Analogs, Mephedrone and Methylone, are

Substrates for Monoamine Transporters in Brain Tissue. Neuropsychopharmacology: Official

Publication of the American College of Neuropsychopharmacology, 37(5), 1192–1203.

Baumann, M. H., Clark, R. D., Woolverton, W. L., Wee, S., Blough, B. E., & Rothman, R. B.

(2011). In Vivo Effects of Amphetamine Analogs Reveal Evidence for Serotonergic Inhibition of

Mesolimbic Dopamine Transmission in the Rat. The Journal of Pharmacology and Experimental

Therapeutics, 337(1), 218–225. 40

Berman, S. M., Kuczenski, R., McCracken, J. T., & London, E. D. (2009). Potential adverse effects of amphetamine treatment on brain and behavior: a review. Molecular Psychiatry, 14(2),

123–142.

Bhimani, R., & Huber, R. (2016). Operant avoidance learning in crayfish, Orconectes rusticus:

Computational ethology and the development of an automated learning paradigm. Learning &

Behavior, 44(3), 239–249.

Budzynska, B., Michalak, A., Frankowska, M., Kaszubska, K., & Biała, G. (2017). Acute behavioral effects of co-administration of mephedrone and MDMA in mice. Pharmacological

Reports: PR, 69(2), 199–205.

Carvelli, L., Matthies, D. S., & Galli, A. (2010). Molecular mechanisms of amphetamine actions in Caenorhabditis elegans. Molecular Pharmacology, 78(1), 151–156.

Cruickshank, C. C., & Dyer, K. R. (2009). A review of the clinical pharmacology of methamphetamine. Addiction , 104(7), 1085–1099.

Devineni, A. V., & Heberlein, U. (2009). Preferential ethanol consumption in Drosophila models features of addiction. Current Biology: CB, 19(24), 2126–2132.

Dichiara, G., & Bassareo, V. (2007). Reward system and addiction: what dopamine does and doesn’t do. Current Opinion in Pharmacology, 7(2), 233–233.

Engleman, E. A., Katner, S. N., & Neal-Beliveau, B. S. (2016a). Caenorhabditis elegans as a

Model to Study the Molecular and Genetic Mechanisms of Drug Addiction. Progress in

Molecular Biology and Translational Science, 137, 229–252.

Engleman, E. A., Katner, S. N., & Neal-Beliveau, B. S. (2016b). Caenorhabditis elegans as a

Model to Study the Molecular and Genetic Mechanisms of Drug Addiction. Progress in

Molecular Biology and Translational Science, 137, 229–252. 41

Fraioli, S., Crombag, H. S., Badiani, A., & Robinson, T. E. (1999). Susceptibility to amphetamine-induced locomotor sensitization is modulated by environmental stimuli.

Neuropsychopharmacology: Official Publication of the American College of

Neuropsychopharmacology, 20(6), 533–541.

Fuentealba, J. A., Gysling, K., & Andrés, M. E. (2007). Increased locomotor response to amphetamine induced by the repeated administration of the selective kappa-opioid receptor agonist U-69593. Synapse , 61(9), 771–777.

Furshpan, E. J., & Potter, D. D. (1959). Transmission at the giant motor synapses of the crayfish.

The Journal of Physiology, 145(2), 289–325.

Gibbons, S., & Zloh, M. (2010). An analysis of the “legal high” mephedrone. Bioorganic &

Medicinal Chemistry Letters, 20(14), 4135–4139.

Hall, D. A., Stanis, J. J., Marquez Avila, H., & Gulley, J. M. (2008). A comparison of amphetamine- and methamphetamine-induced locomotor activity in rats: evidence for qualitative differences in behavior. Psychopharmacology, 195(4), 469–478.

Hodgkin, A. L., & Huxley, A. F. (1945). Resting and action potentials in single nerve fibers. The

Journal of Physiology, 104(2), 176–195.

Huber, R., Panksepp, J. B., Nathaniel, T., Alcaro, A., & Panksepp, J. (2011). Drug-sensitive reward in crayfish: an invertebrate model system for the study of SEEKING, reward, addiction, and withdrawal. Neuroscience and Biobehavioral Reviews, 35(9), 1847–1853.

Hutchinson, C. V., Prados, J., & Davidson, C. (2015). Persistent conditioned place preference to cocaine and withdrawal hypo-locomotion to mephedrone in the flatworm planaria. Neuroscience

Letters, 593, 19–23. 42

Hyman, S. E., Malenka, R. C., & Nestler, E. J. (2006). Neural mechanisms of addiction: the role of reward-related learning and memory. Annual Review of Neuroscience, 29, 565–598.

Jentsch, J. D., & Taylor, J. R. (1999). Impulsivity resulting from frontostriatal dysfunction in drug abuse: implications for the control of behavior by reward-related stimuli.

Psychopharmacology, 146(4), 373–390.

Jiao, D., Liu, Y., Li, X., Liu, J., & Zhao, M. (2015). The role of the GABA system in amphetamine-type stimulant use disorders. Frontiers in Cellular Neuroscience, 9, 162.

Kandel, E. R. (2007). In Search of Memory: The Emergence of a New Science of Mind. W. W.

Norton & Company.

Kaun, K. R., Azanchi, R., Maung, Z., Hirsh, J., & Heberlein, U. (2011). A Drosophila model for alcohol reward. Nature Neuroscience, 14(5), 612–619.

Kaun, K. R., Devineni, A. V., & Heberlein, U. (2012). Drosophila melanogaster as a model to study drug addiction. Human Genetics, 131(6), 959–975.

Kelley, A. E., & Berridge, K. C. (2002). The neuroscience of natural rewards: relevance to addictive drugs. The Journal of Neuroscience: The Official Journal of the Society for

Neuroscience, 22(9), 3306–3311.

Kelly, J. P. (2011). Cathinone derivatives: a review of their chemistry, pharmacology and toxicology. Drug Testing and Analysis, 3(7-8), 439–453.

Konopka, R. J. (1987). Genetics of Biological Rhythms in Drosophila. Annual Review of

Genetics, 21(1), 227–236.

Koob, G. F., & Le Moal, M. (2005). Plasticity of reward neurocircuitry and the “dark side” of drug addiction. Nature Neuroscience, 8(11), 1442–1444. 43

Lisek, R., Xu, W., Yuvasheva, E., Chiu, Y.-T., Reitz, A. B., Liu-Chen, L.-Y., & Rawls, S. M.

(2012). Mephedrone (’bath salt') elicits conditioned place preference and dopamine-sensitive motor activation. Drug and Alcohol Dependence, 126(1-2), 257–262.

Mancillas, J. R., McGinty, J. F., Selverston, A. I., Karten, H., & Bloom, F. E. (1981).

Immunocytochemical localization of enkephalin and substance P in retina and eyestalk neurons of lobster. Nature, 293(5833), 576–578.

Marusich, J. A., Grant, K. R., Blough, B. E., & Wiley, J. L. (2012). Effects of synthetic cathinones contained in “bath salts” on motor behavior and a functional observational battery in mice. Neurotoxicology, 33(5), 1305–1313.

McNamara, R. K., Logue, A., Stanford, K., Xu, M., Zhang, J., & Richtand, N. M. (2006). Dose– response analysis of locomotor activity and stereotypy in dopamine D3 receptor mutant mice following acute amphetamine. Synapse , 60(5), 399–405.

Mello, M. O., & Silva-Filho, M. C. (2002). Plant-insect interactions: an evolutionary arms race between two distinct defense mechanisms. Brazilian Journal of Plant Physiology, 14(2), 71–81.

Motbey, C. P., Hunt, G. E., Bowen, M. T., Artiss, S., & McGregor, I. S. (2011). Mephedrone (4- methylmethcathinone, “meow”): acute behavioral effects and distribution of Fos expression in adolescent rats. Addiction Biology, 17(2), 409–422.

Nathaniel, T. I., Panksepp, J., & Huber, R. (2010). Effects of a single and repeated morphine treatment on conditioned and unconditioned behavioral sensitization in Crayfish. Behavioral

Brain Research, 207(2), 310–320.

Nathaniel, T. I., Panksepp, J., & Huber, R. (2012). Alteration of c-Fos mRNA in the accessory lobe of crayfish is associated with a conditioned-cocaine induced reward. Neuroscience

Research, 72(3), 243–256. 44

Orsini, C., Buchini, F., Piazza, P. V., Puglisi-Allegra, S., & Cabib, S. (2004). Susceptibility to amphetamine-induced place preference is predicted by locomotor response to novelty and amphetamine in the mouse. Psychopharmacology, 172(3), 264–270.

Ramoz, L., Lodi, S., Bhatt, P., Reitz, A. B., Tallarida, C., Tallarida, R. J., … Rawls, S. M.

(2012). Mephedrone (“bath salt”) pharmacology: insights from invertebrates. Neuroscience, 208,

79–84.

Robinson, J. E., Agoglia, A. E., Fish, E. W., Krouse, M. C., & Malanga, C. J. (2012).

Mephedrone (4-methylmethcathinone) and intracranial self-stimulation in C57BL/6J mice: comparison to cocaine. Behavioral Brain Research, 234(1), 76–81.

Robinson, T. E., & Berridge, K. C. (1993). The neural basis of drug craving: an incentive- sensitization theory of addiction. Brain Research. Brain Research Reviews, 18(3), 247–291.

Rothman, R. B., Baumann, M. H., Dersch, C. M., Romero, D. V., Rice, K. C., Carroll, F. I., &

Partilla, J. S. (2001). Amphetamine-type central nervous system stimulants release norepinephrine more potently than they release dopamine and serotonin. Synapse , 39(1), 32–41.

Salahpour, A., Ramsey, A. J., Medvedev, I. O., Kile, B., Sotnikova, T. D., Holmstrand, E., …

Caron, M. G. (2008). Increased amphetamine-induced hyperactivity and reward in mice overexpressing the dopamine transporter. Proceedings of the National Academy of Sciences of the United States of America, 105(11), 4405–4410.

Schultz, W. (2002). Getting formal with dopamine and reward. Neuron, 36(2), 241–263.

Shortall, S. E., Macerola, A. E., Swaby, R. T. R., Jayson, R., Korsah, C., Pillidge, K. E., … King,

M. V. (2013). Behavioral and neurochemical comparison of chronic intermittent cathinone, mephedrone and MDMA administration to the rat. European Neuropsychopharmacology: The

Journal of the European College of Neuropsychopharmacology, 23(9), 1085–1095. 45

Simmler, L. D., Buser, T. A., Donzelli, M., Schramm, Y., Dieu, L.-H., Huwyler, J., … Liechti,

M. E. (2012). Pharmacological characterization of designer cathinonesin vitro. British Journal of

Pharmacology, 168(2), 458–470.

Sorg, B. A. (2012). Reconsolidation of drug memories. Neuroscience and Biobehavioral

Reviews, 36(5), 1400–1417.

Søvik, E., & Barron, A. B. (2013). Invertebrate models in addiction research. Brain, Behavior and Evolution, 82(3), 153–165.

Taylor, J. R., Olausson, P., Quinn, J. J., & Torregrossa, M. M. (2009). Targeting extinction and reconsolidation mechanisms to combat the impact of drug cues on addiction.

Neuropharmacology, 56 Suppl 1, 186–195.

Tierney, A. J., Kim, T., & Abrams, R. (2003). Dopamine in crayfish and other crustaceans: distribution in the central nervous system and physiological functions. Microscopy Research and

Technique, 60(3), 325–335.

Torregrossa, M. M., Corlett, P. R., & Taylor, J. R. (2011). Aberrant learning and memory in addiction. Neurobiology of Learning and Memory, 96(4), 609–623.

Torregrossa, M. M., & Taylor, J. R. (2013). Learning to forget: manipulating extinction and reconsolidation processes to treat addiction. Psychopharmacology, 226(4), 659–672.

Varela, M. J., Brea, J., Loza, M. I., Maldonado, R., & Robledo, P. (2011). Sensitization to

MDMA locomotor effects and changes in the functionality of 5-HT2A and D2 receptors in mice.

Behavioral Pharmacology, 22(4), 362–369.

Vouga, A., Gregg, R. A., Haidery, M., Ramnath, A., Al-Hassani, H. K., Tallarida, C. S., …

Rawls, S. M. (2015). Stereochemistry and neuropharmacology of a “bath salt” cathinone: S- 46 enantiomer of mephedrone reduces cocaine-induced reward and withdrawal in invertebrates.

Neuropharmacology, 91, 109–116.

Watterson, L. R., & Olive, M. F. (2014). Synthetic cathinones and their rewarding and reinforcing effects in rodents. Advances in Neuroscience (Hindawi), 2014, 209875.

Weinstein, A. M., Rosca, P., Fattore, L., & London, E. D. (2017). Synthetic Cathinone and

Cannabinoid Designer Drugs Pose a Major Risk for Public Health. Frontiers in Psychiatry /

Frontiers Research Foundation, 8, 156.

Wink, M. (2015). Modes of Action of Herbal Medicines and Plant Secondary Metabolites.

Medicines (Basel, Switzerland), 2(3), 251–286.

Wolf, F. W., & Heberlein, U. (2002). Invertebrate models of drug abuse. Journal of

Neurobiology, 54(1), 161–178. 47

CHAPTER 3: UNCONDITIONED AND TERMINATION EFFECTS OF SYNTHETIC

CATHINONES IN CRAYFISH: COMPARISON TO SUBSTITUTED AMPHETAMINE

ANALOGS

Introduction

Synthetic cathinones have gained popularity among drug users due to low cost, potency and widespread availability resulting from an unclear legal regulatory status. Cathinones are structurally similar to amphetamines and appear to share some of the behavioral consequences.

In humans, cathinone increases alertness, euphoria, sensory stimulation, hyperthermia, heart rate, respiration, and blood pressure. Apart from the classic stimulatory effects, cathinones are also known to produce MDMA-like hallucinogenic effects. Strong craving following termination of synthetic cathinones is reported by high proportion of synthetic cathinone users (Kelly, 2011). At present little is known about the pharmacology, physiological effects, behavioral consequences, and abuse potential of this rapidly evolving class of substances.

Stimulant drugs are known to elicit unconditioned behavioral responses and increased locomotion to enhance exploration and approach behaviors. These drugs are able to gain control and enhance exploration and approach behaviors when the brain fails to differentiate if the reward circuitry is activated by natural rewards or falsely triggered by the drugs of abuse.

Several lines of evidence suggest that the rewarding properties of drugs of abuse in mammals originate from stimulation of neural processes involved in activation of the appetitive states.

Generally, drugs of abuse promote unconditioned behavioral responses similar to those in mammals, including distinct stereotyped behaviors and increased locomotor activity. In mammals, distinct withdrawal symptoms are observed following termination of drug. Acting primarily as psychostimulants, unconditioned responses to cocaine, amphetamine, and 48 cathinones elicit increased locomotor responses during the first exposure (Dougherty and

Ellinwood 1981), an effect which generally enhances with repeated use. Termination of drug is thought to produce different withdrawal behaviors that follow a distinctive temporal or long term course. Studies suggest that withdrawal can modulate reward seeking of drug reinforcers in mammalian models of drug addiction.

Mammals exposed to psychostimulant drugs display comparable changes in behavior to those observed in human drug users, offering a set validated model systems for the study of addiction. Studies have shown that various doses (3,10 and 30 mg/kg) of methylone and mephedrone were able to produce increase in exploration, circling, hyperactivity, salivation, head weaving, head circling and stereotyped compulsive movements in mice (Kelly 2011;

Marusich et al. 2012). Furthermore, sensitization of stereotyped movements was seen on repeated mephedrone injections in rats (Shortall et al. 2013; Lisek et al. 2012). There is no focused research on the withdrawal syndromes and dependence produced due to termination of synthetic cathinones (Prosser and Nelson 2011).

Recently, the neural consequences of drugs of addiction have received increasing attention in invertebrate models. Various stimulant drugs including amphetamines, nicotine, cocaine produce increased exploration in C. elegans (Engleman, Katner, & Neal-Beliveau,

2016), which is mediated via dopaminergic circuits (Carvelli, Matthies, & Galli, 2010). D- amphetamine, methamphetamine, morphine, and cocaine enhances exploration in crayfish, with increased locomotion and antennal movements. Termination of amphetamines, cocaine, nicotine and cannabinoids are known to produce withdrawal-like behaviors in planaria (Søvik & Barron,

2013). In crayfish, withdrawal-like behaviors such as squirming, twisting posture, antennae clinging are seen after termination of morphine (Imeh-Nathaniel et al. 2014). Termination of 49 morphine injections also resulted in decrease of locomotion, rearing and antennae movements

(Imeh-Nathaniel et al. 2014). Acute mephedrone administration produced stereotyped C-shaped movements and displayed shift in preference when subjected to CPP in Planaria (Ramoz et al.

2012; Hutchinson et al., 2015; Vouga et al. 2015). Above mentioned studies suggest that diversity of invertebrate nervous system can be utilized to explore the basic functional properties of reward circuits and how drugs of abuse alter their function. Featuring highly modular nervous systems with relatively few, large neurons, invertebrate models are amenable to a search for the underlying neural mechanisms of behavior. Despite the distinct advantages invertebrate models may offer for the study of addiction, evaluations of the behavioral effects of synthetic cathinones have remained rare.

The primary goal of this study was to explore the proximate effects of synthetic cathinones (methylone and mephedrone) in comparison to known stimulant drugs (4-MMA and

MDMA). We explored the unconditioned effects of the drugs at various doses. We also investigated the withdrawal-like behaviors and distance travelled by crayfish upon termination of the drug. Thus, current study explores the unconditioned and termination effects of mephedrone,

4-MMA, MDMA and methylone.

Materials and Methods

Animal Maintenance

Crayfish were wild caught in the Portage River near Bowling Green, Ohio, and maintained under controlled environmental conditions in an aerated community tank (water temperature 20°C, pH7, 12:12h light: dark cycle), and fed once a week with rabbit chow. A week prior to the experiment, intermolt males (approximately 5-15 grams of body weight) with complete, intact 50 appendages were chosen and isolated in perforated plastic containers. Maintained in large holding trays they received a constant flow of filtered and aerated water.

Surgical procedure for implanting cannula in the pericardial space

Animal were anesthetized in crushed ice for 20 minutes. The cannula was implanted into the anterior end of the pericardial cavity through an incision created using 26.5 gauge needle, slightly lateral of the midline to avoid damaging the heart. A 10 cm section of deactivated, fine- bore, fused silica (Agilent 160-2655, i.d. = 50 µm, o.d.=350 um) was implanted into the pericardial sinus (allowing 2 mm to enter the sinus) and affixed to the carapace using superglue and bonding material. Following the surgery, animals were allowed to recover overnight in their holding containers. Table 7 shows the specific details about the descriptives of crayfish for each experimental group.

Drugs, doses and injection protocols

Mephedrone, methylone, MDMA and 4-MMA were obtained from Cayman Chemical, Ann

Arbor, Michigan. Mephedrone, methylone, MDMA and 4-MMA were dissolved in 125mM

NaCl and administered systemically at doses 1, 3 and 10 µg/g body weight of crayfish (injection volume 50µl). Control groups received isotonic NaCl solution only. Tygon microbore tubing

(Fisher Scientific ND 100-80, i.d. = 250 µm) was used to connect a 0.5m section of deactivated, fine-bore, fused silica needle material (Agilent 160-1010, i.d. = 100 µm, o.d. = 190 µm, 0.5 m long) to the implanted animal stub on one end and the blunt-tipped needle of a 1ml glass syringe

(SGE Analytical Sciences, Model# 008100) on the other. Mounted above the experimental arena, the syringe pump housed up to eight syringes side by side (Razel R-99E attached with R-ACC-6

Multi Micro Syringe Adapter) to allow for concurrent drug application to multiple animals

(Figure 2b). 51

Experimental Arena and Video Tracking

Training and testing was performed in a circular arena (Diameter = 0.6m) with two soft-textured and two hard substrate quadrants arranged diagonally. When needed, removable plexi-glass barrier was inserted into the arena to restrict the movements of crayfish to a particular quadrant.

A digital camera (Microsoft LifeCam Studio, Microsoft, Model #: Q2F-00013, 1080p HD

Sensor), mounted above the experimental arena, provided a live video streams for automated, real time video tracking run on a Apple Macintosh (OS X El Captain, Version 10.11.4,

Processor: 3.2 GHz Quad-Core Intel Xeon, Memory: 6GB 1066 MHz DDR3 ECC) Spatial coordinates, orientations and object outlines of the test crayfish were extracted and logged to disk using the open-source, public domain JavaGrinders framework (available for free download at iEthology.com). In a subsequent analysis, the saved coordinates were analyzed to extract spatial descriptives like distance travelled, mean speed, time stationary, etc.

Unconditioned and termination effects - Experimental Design

Crayfish (N=104) were randomly distributed in 12 treatment groups - mephedrone, methylone,

MDMA and 4-MMA at 1,3, and 10 µg/g dose each while the thirteenth group (vehicle-injected) served as control. In order to study the unconditioned effects, behaviors elicited during the conditioning trials (day 3,4, and 5) were observed and to investigate the drug termination effects, locomotion and specific withdrawal-like behaviors during the extinction pairing (day 7,8, and 9) were considered. The timeline of the typical experiment for studying unconditioned and termination effects is included (Figure 9)

Statistical analysis

Data were expressed as means ± standard errors. Repeated measures ANOVA was used to detect significant effects of each drug at various doses on the distance travelled on different days (day 52

7, 8, and 9). For each drug, the repeated measures ANOVA detected significance of each of the independent variable (dose, day) along with their interaction (dose:day) on the dependent variable (total distance travelled). Statistical significance was followed by Tukey post hoc comparisons. Differences were considered statistically significant at p<0.05.

All statistical analyses were conducted in R (R version 3.3.3 , ) with supplementary installed packages such as: 'ez', 'Deducer',’lsr’, 'ggplot2', 'nlme', 'stats', and 'lme4.'

Repeated measures ANOVA was conducted on time sequence data for the dependent variable

(distance travelled) using packages nlme (Function: lme) and stats (Function: t.test).

Results

Unconditioned effects of the drugs

Table 8 provides information about the description of each of the unconditioned behavior in detail. Apart from significant locomotor effects (Chapter 1), Crayfish consistently displayed different behavioral patterns throughout the trial when administered with mephedrone, methylone, 4-MMA and MDMA. When placed in the arena, treatment as well as saline groups quickly approached the closest arena wall and followed its curve. They explored the edges of arena with antennae and with occasional rearing which decreased towards the end of the trial.

Individuals injected with MDMA typically showed grooming, increased movement of antennae and antennules, claw closure, circling, bouts of locomotion following prolonged immobility, claw waving, rearing, extended posture, stereotyped movements. Generally, crayfish follow the edge of the arena and rarely turns in exactly opposite direction before encountering the corner of the arena. Individuals injected with MDMA showed circling behavior which was the tendency to turn suddenly in the opposite direction by rotating around their own axis. These individuals showed increased activity for short amount of time between prolonged inactivity. During the 53 prolonged inactivity, these individuals showed stereotyped movements of legs without any walking. Crayfish treated with MDMA produced claw closure where it would pinch with the help of the claws. Another interesting behavior that was seen after injecting MDMA was claw waving where, crayfish would move one or both its claws in and out. Unlike higher doses of

MDMA, 1µg/g showed depressant-like effect where, individuals would stay immobile in corners of the arena for a long period of time. Individuals injected with methylone typically showed bouts of locomotion following prolonged immobility, rearing, grooming, extended posture, stereotyped movements and antennae movements. The stereotyped movements increased in frequency over the course of the trial. Methylone at 10 µg/g also produced aversive behaviors like backward walking, tail flips, tremors, and uncoordinated leg movements. 4-MMA elicited behaviors like forward walking, claw waving, rearing, grooming, extended posture, stereotyped movements and antennae movements. Individuals treated with 4-MMA also showed rapid upward movement of whole body when 4-MMA was being administered. Mephedrone also promoted behaviors like claw waving, forward walking, rearing, grooming, extended posture, stereotyped movements and antennae movements. Frequency of claw waving increased during the course of trial for mephedrone. Unconditioned behaviors such as circling, claw waving, stereotyped movements, extended postures, tail flips, backward walking were unique to the treatment groups and were not observed in saline individuals.

Termination effects of the drugs

Some of the behaviors that were observed after termination of MDMA, 4-MMA, mephedrone and methylone included twisted posture, static posture, squirming, uncoordinated leg movements similar to tremors, and immobility for a long period of time. In addition to these behaviors, individuals treated with MDMA, 4-MMA and mephedrone when exposure to the arena also 54 showed claw waving. Claw waving was one of the unique unconditioned behavior that was observed following drug administration and it was unexpected to observe this behavior after termination of the drug. However, claw waving after termination of drug suggests that these drugs might have long term effects. Another possible explanation is that exposure to the arena that was earlier paired with the drug resulted in retrieving the memories that produced subjective effects of these drugs. Figure 13 shows the total distance travelled on Day 7, 8, and 9 for 4-

MMA, mephedrone, MDMA and methylone at 1,3, and 10 µg/g (Table 9). Repeated measures

ANOVA (Table 10) revealed significant differences in total distance travelled across days 7, 8, and 9 for MDMA and 4-MMA and a significant Dose*Day interaction effect for methylone. Post hoc Tukey test did not reveal significant difference in locomotion on different days. Figure 14 shows the total distance travelled following drug administration (i.e. average of total distance travelled on day 3, 4, and 5) and after drug termination (i.e. average of total distance travelled on day 7, 8, and 9) by each experimental group. No distinct effect on locomotion was seen during drug termination when compared to locomotion following drug administration.

Discussion

In this study, we demonstrate that crayfish can provide critical insights into the complex interactions of behaviors and neurochemical changes during drug addiction. It is intriguing that a relatively less complex organism like crayfish can elicit a range of behaviors in response to mammalian drugs of abuse.

In our study, all the chemicals produced distinct unconditioned behaviors when drugs were administered for the first time. Earlier studies have shown that crayfish explores novel arena as evident by increase in distance travelled and exploratory behaviors (Nathaniel,

Panksepp, and Huber 2010b; Imeh-Nathaniel et al. 2016). All the chemicals produced 55 exploratory behaviors similar to the behaviors that are seen when crayfish is placed in a novel arena. However, MDMA was able to produce circling behavior similar to that seen in rodents

(Kelly 2011; Marusich et al. 2012). One of the unique unconditioned behavior seen after MDMA infusion was claw closure. Generally, crayfish uses its claws to capture, manipulate, and process prey, as well as for defense and for aggressive intraspecific interactions and displays (Claussen et al. 2007). Serotonin is the primary monoamine known to modulate agonistic behaviors in crayfish (Panksepp and Huber 2002; Huber et al. 1997; Panksepp et al. 2003). The fact that crayfish showed claw closing suggests that MDMA may activate serotonergic circuitry in crayfish which is consistent with the known pharmacology of MDMA. Another noteworthy behavior typically seen in response to mephedrone, 4-MMA and MDMA was claw waving.

Individuals moved either one or both of their claws in and out when injected with 4-MMA, mephedrone and occasionally for MDMA. This behavior is similar to the head weaving or head circling behavior seen in rodents (Kelly 2011; Marusich et al. 2012). Synthetic cathinones typically produced bouts of locomotion following long period of inactivity which might be because of MDMA-like hallucinogenic properties of these drugs (Kelly 2011; Baylen and

Rosenberg 2006). This remarkable similarity in the unconditioned behaviors seen for crayfish and rodent drug addiction models suggests these drugs act on same neural targets.

In mammals, dependence of drug is evident by distinct withdrawal symptoms that are observed following termination of drug. Termination of drug is thought to produce different behaviors that follow a distinctive temporal or long term course. Studies suggest that withdrawal can modulate reward seeking of drug reinforcers in mammalian models of drug addiction.

Termination of MDMA, 4-MMA, mephedrone and methylone produced twisted posture, static posture, squirming, uncoordinated leg movements similar to tremors, and immobility for a long 56 period of time. In addition to these behaviors, individuals treated with MDMA, 4-MMA and mephedrone when exposure to the arena also showed claw waving. It was unexpected to observe claw waving behavior after termination of the drug. However, claw waving seen during termination of drug might suggest that these drugs produced long lasting motor effects. Another possible explanation can be that exposure to the arena that was earlier paired with the drug resulted in retrieving the memories associated with drugs and these drug-associated memories in turn produced subjective effects of these drugs. Significant differences in locomotion were observed following termination of the drug across days 7-9 suggesting the presence of long lasting effects. Studies have shown that crayfish decreases locomotion and exploratory behaviors upon termination of morphine. Our results did not show decrease in locomotion during termination of drugs.

Results from our studies are consistent with the unconditioned and withdrawal-like effects seen for other stimulants in crayfish. Various drugs of abuse like morphine, cocaine, D- amphetamine produce distinct stereotyped behaviors in crayfish (Alcaro, Panksepp, and Huber

2011a; Nathaniel, Huber, and Panksepp 2012); (Alcaro, Panksepp, and Huber 2011a)(Alcaro,

Panksepp, and Huber 2011a; Nathaniel, Huber, and Panksepp 2012). D-amphetamine enhances exploration in crayfish, with increased locomotion, rearing and antennal movements ((Alcaro,

Panksepp, and Huber 2011a)). In crayfish, withdrawal-like behaviors such as squirming, twisting posture, antennae clinging are seen after termination of morphine (Imeh-Nathaniel et al. 2014).

Termination of morphine injections also resulted in decrease of locomotion, rearing and antennae movements (Imeh-Nathaniel et al. 2014). Very few studies have looked at the effect of termination and ability to cause dependence by synthetic cathinones. Our data strongly suggests that synthetic cathinones and their substituted amphetamine analogues target the neural pathways 57 in crayfish that serve as to control locomotor response. This finding indicates that crayfish represents a competent model for studying the primary sites of psychostimulants to explore the proximate mechanisms and fundamental neurobiological processes that underlie drug addiction in an invertebrate model.

The potency of drugs of abuse to modulate dopaminergic systems is associated with the psychostimulant effects and enhanced abuse liability (Rothman et al. 2001). In contrast, relatively increased activation of 5-HT system is linked to reduction in abuse potential and more

MDMA-like subjective drug effects. Our results show distinct behaviors like circling, claw waving that were elicited by injections of MDMA and methylone which is consistent with the effects these chemicals are known to produce as shown in other studies as well. The fact that crayfish showed claw closing suggests that MDMA may activate serotonergic circuitry in crayfish which is consistent with the known pharmacology of MDMA. Relative in vitro effects of DAT versus SERT can be used as a predictor of drug characteristics in vivo and vice versa.

The findings of our study speculates involvement of serotonergic circuits in producing distinct subjective effects as seen for MDMA and methylone. The knowledge about precise neuroanatomical location of monoaminergic neurons affirms crayfish as an ideal candidate for underpinning the neuropharmacology of drug-induced reward.

Future studies are necessary to identify specific brain targets and circuits of synthetic cathinones-associated reward in the brain of crayfish. Because doses as low as 1µg/g could produce distinct effects on locomotion, it would be necessary to look at the effect on locomotion for doses lower than 1 µg/g. Identification of the precise neural circuits underlying the effects described here, and the exploration of the overlap between synthetic cathinone and substituted amphetamine analogues will be interesting to study in order to characterize similarity and 58 differences between their mode of action. Even though crayfish nervous system serves several advantages for easier experimental manipulations, the pharmacokinetics of drugs in crayfish is less known. Knowledge about the metabolites formed following the introduction of these synthetic cathinones along with their half life in crayfish hemolymph will be allow us to understand the temporal component addiction-related behavioral processes in an efficient manner. Use of specific monoamine antagonists would be helpful to pinpoint the role of each monoamine in psychostimulation, sensitization and other components of drug-induced reward.

References

Abramson, C. I., Sanderson, C., Painter, J., Barnett, S., & Wells, H. (2005). Development of an ethanol model using social insects: V. Honeybee foraging decisions under the influence of alcohol. Alcohol , 36(3), 187–193.

Alcaro, A., & Panksepp, J. (2011). The SEEKING mind: primal neuro-affective substrates for appetitive incentive states and their pathological dynamics in addictions and depression.

Neuroscience and Biobehavioral Reviews, 35(9), 1805–1820.

Alcaro, A., Panksepp, J., & Huber, R. (2011). d-amphetamine stimulates unconditioned exploration/approach behaviors in crayfish: Towards a conserved evolutionary function of ancestral drug reward. Pharmacology, Biochemistry, and Behavior, 99(1), 75–80.

Alvarez Alvarado, R., Porras Villalobos, M. G., Calderón Rosete, G., Rodríguez Sosa, L., &

Aréchiga, H. (2005). Dopaminergic modulation of neurosecretory cells in the crayfish. Cellular and Molecular Neurobiology, 25(2), 345–370.

Angoa-Pérez, M., Kane, M. J., Francescutti, D. M., Sykes, K. E., Shah, M. M., Mohammed, A.

M.,Kuhn, D. M. (2012). Mephedrone, an abused psychoactive component of “bath salts” and 59 methamphetamine congener, does not cause neurotoxicity to dopamine nerve endings of the striatum. Journal of Neurochemistry, no–no.

Baumann, M. H., Ayestas, M. A., Partilla, J. S., Sink, J. R., Shulgin, A. T., Daley, P. F., …

Cozzi, N. V. (2011). The Designer Methcathinone Analogs, Mephedrone and Methylone, are

Substrates for Monoamine Transporters in Brain Tissue. Neuropsychopharmacology: Official

Publication of the American College of Neuropsychopharmacology, 37(5), 1192–1203.

Baumann, M. H., Clark, R. D., Woolverton, W. L., Wee, S., Blough, B. E., & Rothman, R. B.

(2011). In Vivo Effects of Amphetamine Analogs Reveal Evidence for Serotonergic Inhibition of

Mesolimbic Dopamine Transmission in the Rat. The Journal of Pharmacology and Experimental

Therapeutics, 337(1), 218–225.

Berman, S. M., Kuczenski, R., McCracken, J. T., & London, E. D. (2009). Potential adverse effects of amphetamine treatment on brain and behavior: a review. Molecular Psychiatry, 14(2),

123–142.

Boehm, S. L., Goldfarb, K. J., Serio, K. M., Moore, E. M., & Linsenbardt, D. N. (2007). Does context influence the duration of locomotor sensitization to ethanol in female DBA/2J mice?

Psychopharmacology, 197(2), 191–201.

Budzynska, B., Michalak, A., Frankowska, M., Kaszubska, K., & Biała, G. (2017). Acute behavioral effects of co-administration of mephedrone and MDMA in mice. Pharmacological

Reports: PR, 69(2), 199–205.

Carvelli, L., Matthies, D. S., & Galli, A. (2010). Molecular mechanisms of amphetamine actions in Caenorhabditis elegans. Molecular Pharmacology, 78(1), 151–156.

Cruickshank, C. C., & Dyer, K. R. (2009). A review of the clinical pharmacology of methamphetamine. Addiction , 104(7), 1085–1099. 60

Devineni, A. V., & Heberlein, U. (2009). Preferential ethanol consumption in Drosophila models features of addiction. Current Biology: CB, 19(24), 2126–2132.

DiChiara, G., & Bassareo, V. (2007). Reward system and addiction: what dopamine does and doesn’t do. Current Opinion in Pharmacology, 7(2), 233–233.

Dougherty, G. G., Jr, & Ellinwood, E. H., Jr. (1981). Chronic D-amphetamine in nucleus accumbens: lack of tolerance or reverse tolerance of locomotor activity. Life Sciences, 28(20),

2295–2298.

Engleman, E. A., Katner, S. N., & Neal-Beliveau, B. S. (2016). Caenorhabditis elegans as a

Model to Study the Molecular and Genetic Mechanisms of Drug Addiction. Progress in

Molecular Biology and Translational Science, 137, 229–252.

Fass, J. A., Fass, A. D., & Garcia, A. S. (2012). Synthetic Cathinones (Bath Salts): Legal Status and Patterns of Abuse. The Annals of Pharmacotherapy, 46(3), 436–441.

Fraioli, S., Crombag, H. S., Badiani, A., & Robinson, T. E. (1999). Susceptibility to amphetamine-induced locomotor sensitization is modulated by environmental stimuli.

Neuropsychopharmacology: Official Publication of the American College of

Neuropsychopharmacology, 20(6), 533–541.

Fuentealba, J. A., Gysling, K., & Andrés, M. E. (2007). Increased locomotor response to amphetamine induced by the repeated administration of the selective kappa-opioid receptor agonist U-69593. Synapse , 61(9), 771–777.

Hall, D. A., Stanis, J. J., Marquez Avila, H., & Gulley, J. M. (2008). A comparison of amphetamine- and methamphetamine-induced locomotor activity in rats: evidence for qualitative differences in behavior. Psychopharmacology, 195(4), 469–478. 61

Hodgkin, A. L., & Huxley, A. F. (1945). Resting and action potentials in single nerve fibers. The

Journal of Physiology, 104(2), 176–195.

Huber, R., Panksepp, J. B., Nathaniel, T., Alcaro, A., & Panksepp, J. (2011). Drug-sensitive reward in crayfish: an invertebrate model system for the study of SEEKING, reward, addiction, and withdrawal. Neuroscience and Biobehavioral Reviews, 35(9), 1847–1853.

Hutchinson, C. V., Prados, J., & Davidson, C. (2015). Persistent conditioned place preference to cocaine and withdrawal hypo-locomotion to mephedrone in the flatworm planaria. Neuroscience

Letters, 593, 19–23.

Imeh-Nathaniel, A., Adedeji, A., Huber, R., & Nathaniel, T. I. (2016). The rewarding properties of methamphetamine in an invertebrate model of drug addiction. Physiology & Behavior, 153,

40–46.

Jiao, D., Liu, Y., Li, X., Liu, J., & Zhao, M. (2015). The role of the GABA system in amphetamine-type stimulant use disorders. Frontiers in Cellular Neuroscience, 9, 162.

Kalivas, P. (2004). Glutamate systems in cocaine addiction. Current Opinion in Pharmacology,

4(1), 23–29.

Kalivas, P. W., & Stewart, J. (1991). Dopamine transmission in the initiation and expression of drug- and stress-induced sensitization of motor activity. Brain Research. Brain Research

Reviews, 16(3), 223–244.

Kandel, E. R. (2007). In Search of Memory: The Emergence of a New Science of Mind. W. W.

Norton & Company.

Karila, L., Billieux, J., Benyamina, A., Lançon, C., & Cottencin, O. (2016). The effects and risks associated to mephedrone and methylone in humans: A review of the preliminary evidences.

Brain Research Bulletin, 126(Pt 1), 61–67. 62

Kaun, K. R., Azanchi, R., Maung, Z., Hirsh, J., & Heberlein, U. (2011). A Drosophila model for alcohol reward. Nature Neuroscience, 14(5), 612–619.

Kelley, A. E., & Berridge, K. C. (2002). The neuroscience of natural rewards: relevance to addictive drugs. The Journal of Neuroscience: The Official Journal of the Society for

Neuroscience, 22(9), 3306–3311.

Kelly, J. P. (2011). Cathinone derivatives: a review of their chemistry, pharmacology and toxicology. Drug Testing and Analysis, 3(7-8), 439–453.

Konopka, R. J. (1987). Genetics of Biological Rhythms in Drosophila. Annual Review of

Genetics, 21(1), 227–236.

Kuczenski, R., & Segal, D. S. (2001). Locomotor effects of acute and repeated threshold doses of amphetamine and methylphenidate: relative roles of dopamine and norepinephrine. The Journal of Pharmacology and Experimental Therapeutics, 296(3), 876–883.

Lessov, C. N., & Phillips, T. J. (1998). Duration of sensitization to the locomotor stimulant effects of ethanol in mice. Psychopharmacology, 135(4), 374–382.

Lisek, R., Xu, W., Yuvasheva, E., Chiu, Y.-T., Reitz, A. B., Liu-Chen, L.-Y., & Rawls, S. M.

(2012). Mephedrone (’bath salt') elicits conditioned place preference and dopamine-sensitive motor activation. Drug and Alcohol Dependence, 126(1-2), 257–262.

Mancillas, J. R., McGinty, J. F., Selverston, A. I., Karten, H., & Bloom, F. E. (1981).

Immunocytochemical localization of enkephalin and substance P in retina and eyestalk neurons of lobster. Nature, 293(5833), 576–578.

Marusich, J. A., Grant, K. R., Blough, B. E., & Wiley, J. L. (2012). Effects of synthetic cathinones contained in “bath salts” on motor behavior and a functional observational battery in mice. Neurotoxicology, 33(5), 1305–1313. 63

May, J., Andrade, J., Panabokke, N., & Kavanagh, D. (2004). Images of desire: Cognitive models of craving. Memory , 12(4), 447–461.

McNamara, R. K., Logue, A., Stanford, K., Xu, M., Zhang, J., & Richtand, N. M. (2006). Dose– response analysis of locomotor activity and stereotypy in dopamine D3 receptor mutant mice following acute amphetamine. Synapse , 60(5), 399–405.

Mello, M. O., & Silva-Filho, M. C. (2002). Plant-insect interactions: an evolutionary arms race between two distinct defense mechanisms. Brazilian Journal of Plant Physiology, 14(2), 71–81.

Motbey, C. P., Hunt, G. E., Bowen, M. T., Artiss, S., & McGregor, I. S. (2011). Mephedrone (4- methylmethcathinone, “meow”): acute behavioral effects and distribution of Fos expression in adolescent rats. Addiction Biology, 17(2), 409–422.

Nathaniel, T. I., Panksepp, J., & Huber, R. (2010). Effects of a single and repeated morphine treatment on conditioned and unconditioned behavioral sensitization in Crayfish. Behavioral

Brain Research, 207(2), 310–320.

Nathaniel, T. I., Panksepp, J., & Huber, R. (2012). Alteration of c-Fos mRNA in the accessory lobe of crayfish is associated with a conditioned-cocaine induced reward. Neuroscience

Research, 72(3), 243–256.

Orsini, C., Buchini, F., Piazza, P. V., Puglisi-Allegra, S., & Cabib, S. (2004). Susceptibility to amphetamine-induced place preference is predicted by locomotor response to novelty and amphetamine in the mouse. Psychopharmacology, 172(3), 264–270.

Paulson, P. E., Camp, D. M., & Robinson, T. E. (1991). Time course of transient behavioral depression and persistent behavioral sensitization in relation to regional brain monoamine concentrations during amphetamine withdrawal in rats. Psychopharmacology, 103(4), 480–492. 64

Prosser, J. M., & Nelson, L. S. (2011). The Toxicology of Bath Salts: A Review of Synthetic

Cathinones. Journal of Medical Toxicology: Official Journal of the American College of Medical

Toxicology, 8(1), 33–42.

Ramoz, L., Lodi, S., Bhatt, P., Reitz, A. B., Tallarida, C., Tallarida, R. J., … Rawls, S. M.

(2012). Mephedrone (“bath salt”) pharmacology: insights from invertebrates. Neuroscience, 208,

79–84.

Robinson, T. E., & Berridge, K. C. (1993). The neural basis of drug craving: an incentive- sensitization theory of addiction. Brain Research. Brain Research Reviews, 18(3), 247–291.

Robinson, T. E., & Berridge, K. C. (2003). Addiction. Annual Review of Psychology, 54, 25–53.

Robinson, T. E., Yager, L. M., Cogan, E. S., & Saunders, B. T. (2014). On the motivational properties of reward cues: Individual differences. Neuropharmacology, 76, 450–459.

Rosenbaum, C. D., Carreiro, S. P., & Babu, K. M. (2012). Here today, gone tomorrow…and back again? A review of herbal marijuana alternatives (K2, Spice), synthetic cathinones (bath salts), kratom, Salvia divinorum, methoxetamine, and piperazines. Journal of Medical

Toxicology: Official Journal of the American College of Medical Toxicology, 8(1), 15–32.

Rothman, R. B., Baumann, M. H., Dersch, C. M., Romero, D. V., Rice, K. C., Carroll, F. I., &

Partilla, J. S. (2001). Amphetamine-type central nervous system stimulants release norepinephrine more potently than they release dopamine and serotonin. Synapse , 39(1), 32–41.

Salahpour, A., Ramsey, A. J., Medvedev, I. O., Kile, B., Sotnikova, T. D., Holmstrand, E., …

Caron, M. G. (2008). Increased amphetamine-induced hyperactivity and reward in mice overexpressing the dopamine transporter. Proceedings of the National Academy of Sciences of the United States of America, 105(11), 4405–4410. 65

Shortall, S. E., Macerola, A. E., Swaby, R. T. R., Jayson, R., Korsah, C., Pillidge, K. E., … King,

M. V. (2013). Behavioral and neurochemical comparison of chronic intermittent cathinone, mephedrone and MDMA administration to the rat. European Neuropsychopharmacology: The

Journal of the European College of Neuropsychopharmacology, 23(9), 1085–1095.

Simmler, L. D., T. A. Buser, M. Donzelli, Y. Schramm, L-H Dieu, J. Huwyler, S. Chaboz, M. C.

Hoener, and M. E. Liechti. (2012) Pharmacological Characterization of Designer Cathinonesin

Vitro. British Journal of Pharmacology 168 (2): 458–70.

Søvik, E., & Barron, A. B. (2013). Invertebrate models in addiction research. Brain, Behavior and Evolution, 82(3), 153–165.

Steketee, J. (2003). Neurotransmitter systems of the medial prefrontal cortex: potential role in sensitization to psychostimulants. Brain Research Reviews, 41(2-3), 203–228.

Tabor, J. N., & Cooper, R. L. (2002). Physiologically identified 5-HT2-like receptors at the crayfish neuromuscular junction. Brain Research, 932(1-2), 91–98.

Tierney, A. J., Kim, T., & Abrams, R. (2003). Dopamine in crayfish and other crustaceans: distribution in the central nervous system and physiological functions. Microscopy Research and

Technique, 60(3), 325–335.

Vanderschuren, L. J. M. J., Louk J M, & Christopher Pierce, R. (2009). Sensitization Processes in Drug Addiction. In Current Topics in Behavioral Neurosciences (pp. 179–195).

Varela, M. J., Brea, J., Loza, M. I., Maldonado, R., & Robledo, P. (2011). Sensitization to

MDMA locomotor effects and changes in the functionality of 5-HT2A and D2 receptors in mice.

Behavioral Pharmacology, 22(4), 362–369. 66

Vezina, P. (2004). Sensitization of midbrain dopamine neuron reactivity and the self- administration of psychomotor stimulant drugs. Neuroscience and Biobehavioral Reviews, 27(8),

827–839.

Vouga, A., Gregg, R. A., Haidery, M., Ramnath, A., Al-Hassani, H. K., Tallarida, C. S., …

Rawls, S. M. (2015). Stereochemistry and neuropharmacology of a “bath salt” cathinone: S- enantiomer of mephedrone reduces cocaine-induced reward and withdrawal in invertebrates.

Neuropharmacology, 91, 109–116.

Watterson, L. R., & Olive, M. F. (2014). Synthetic cathinones and their rewarding and reinforcing effects in rodents. Advances in Neuroscience (Hindawi), 2014, 209875.

Weinstein, A. M., Rosca, P., Fattore, L., & London, E. D. (2017). Synthetic Cathinone and

Cannabinoid Designer Drugs Pose a Major Risk for Public Health. Frontiers in Psychiatry /

Frontiers Research Foundation, 8, 156.

Wink, M. (2015). Modes of Action of Herbal Medicines and Plant Secondary Metabolites.

Medicines (Basel, Switzerland), 2(3), 251–286.

Wolf, F. W., & Heberlein, U. (2002). Invertebrate models of drug abuse. Journal of

Neurobiology, 54(1), 161–178. 67

APPENDIX A: FIGURES AND TABLES

Figure 1: Structural similarity of synthetic cathinones and substituted amphetamines: Aside from the Beta-keto moiety of cathinones, methylone and mephedrone are analogous to MDMA and 4-

MMA respectively. Red colored line represents the phenylethylamine backbone present in synthetic cathinones and their amphetamine equivalents. The only point of difference between synthetic cathinones (methylone and mephedrone) and their amphetamine (MDMA and MMA) equivalents is the beta-keto-oxy group (blue) present on cathinones. 68

Figure 2: The experimental setup for tracking activities of crayfish. (A) Surgical implantation of cannula: A 10 cm section of deactivated, fine-bore, fused silica (Agilent 160-2655, i.d. = 50 µm, o.d. = 350 µm) was implanted into the pericardial sinus (allowing 2 mm to enter the sinus) and reinforced with superglue and bonding material. The position of implanted cannula (red dot) was consistent across all the crayfish to avoid biases because of change in location of implantation.

(B) Injecting drugs and recording behavior of crayfish using a video camera: An overhead syringe pump allowed continuous infusion (50 µl for 5 minutes) of drug into the pericardial space of crayfish via implanted cannula. A digital camera (Microsoft LifeCam, Mfg. Part: Q2F-

00013, CDW Part: 2835954, UNSPSC: 45121520) mounted above the experimental arena was used to record behavioral activities of crayfish. (C) Digitization of recording signal: The signal from camera was then streamed to a video digitizer on a powered Macintosh computer. (D)

Video tracking of crayfish: Video tracking was accomplished using freeware JAVA program

(available at http://caspar.bgsu.edu/~software/java/JavaGrinders.html). Various spatial characteristics like distance travelled, mean speed, time stationary, and time spent on hard/soft substrate were extracted for analyzing effects of drugs. 69

Day 1 à Pre-Conditioning: Crayfish was allowed to move freely

throughout the arena without any drug administered

Day 2 à Locomotion in quadrant (drug free state)

Day 3 à First drug injection

Day 4 à Second drug injection

Day 5 à Third drug injection

Figure 3: Experimental design for psychomotor stimulation and sensitization: On Day 1, the test crayfish was allowed to move freely throughout the experimental arena (without the partition).

Locomotion of crayfish in a quadrant was recorded on day 2 by placing the removable partition.

Crayfish received its first drug injection on day 3 either during morning or evening on soft or hard substrate in random order. Increased locomotion during the drug treatment on Day 3 was considered as psychomotor stimulation. On day 4 and day 5, crayfish received its second and third drug injection respectively. Increased locomotion during repeated drug treatment (day 4 and day 5) served as a measure of sensitization of psychomotor response. On day 3, 4, and 5, the injection cannula was attached to the tubing and directly connected to crayfish. Crayfish was then gently placed into the desired quadrant, followed by a continuous drug/saline injection for the first 5 minutes of the 75 minute conditioning session. 70

Figure 4: Locomotion (total distance travelled) during repeated drug injections: Lower doses (1 and 3 µg/g) of 4-MMA, MDMA, mephedrone and methylone showed increase in total distance travelled on days 3,4 and 5 in comparison to the saline group (grey bars) while higher doses generally had depressant-like effect. When total distance travelled was considered, the sensitization of locomotor response was not very apparent upon repeated drug administrations on day 4 and day 5.

71

Figure 5: Effect of repeated 4-MMA drug injections at doses 1, 3 and 10 µg/g on locomotion:

Crayfish were injected with 1, 3 and 10 µg/g of 4-MMA (red) on day 3 (first drug injection), day

4 (second injection) and day 5 (third injection). Saline group (grey) received saline on day 3,4 and 5. Drug/Saline was administered during the initial five minutes (highlighted pink area) of the trial. Each vertical panel represents the distance travelled by each dose of 4-MMA upon repeated drug injections (across different days). Horizontal panel represents the distance travelled by every dose of 4-MMA for each drug injection. 1 µg/g 4-MMA produces distinct increase in locomotion when injected for the first time in crayfish. Upon repeated 1µg/g injections, the duration of psychostimulation sensitizes. First 3µg/g injection produces increase in locomotion but repeated injections cause depression of locomotion. 10µg/g injections produce depression of locomotion. 72

Figure 6: Effect of repeated mephedrone drug injections at doses 1,3 and 10 µg/g on locomotion:

Crayfish were injected with 1, 3 and 10 µg/g of mephedrone on day 3 (first drug injection), day 4

(second injection) and day 5 (third injection). Saline group received saline on day 3, 4 and 5.

Each vertical panel represents the distance travelled by each dose of mephedrone upon repeated drug injections (across different days). Horizontal panel represents the distance travelled by every dose of mephedrone for each drug injection. 1 µg/g mephedrone produces increase in locomotion for a brief time when injected for the first time in crayfish. Upon repeated 1 µg/g injections, the duration of psychostimulation sensitizes. Similar pattern is observed for 3 µg/g and 10 µg/g.

73

Figure 7: Effect of repeated MDMA drug injections at doses 1, 3 and 10 µg/g on locomotion:

Crayfish were injected with 1, 3 and 10 µg/g of MDMA on day 3 (first drug injection), day 4

(second injection) and day 5 (third injection). Saline group received saline on day 3,4 and 5.

Each vertical panel represents the distance travelled by each dose of MDMA upon repeated drug injections (across different days). Horizontal panel represents the distance travelled by every dose of MDMA for each drug injection. Upon repeated 1 µg/g injections, increased locomotion is seen for a brief amount of time; however, first drug injection produced depression of locomotion. 3 µg/g produced psychostimulation and the effect was maintained on repeated drug injections. A spike in locomotion was observed during initial ~8 minutes of the trial and then the locomotion faded out. Repeated 10 µg/g injections produced long lasting effects on locomotion and the increase in locomotion was evident approximately 15 minutes after the start of trial. 74

Figure 8: Effect of repeated methylone drug injections at doses 1, 3 and 10 µg/g on locomotion:

Crayfish were injected with 1, 3 and 10 µg/g of methylone on day 3 (first drug injection), day 4

(second injection) and day 5 (third injection). Saline group received saline on day 3, 4 and 5.

Each vertical panel represents the distance travelled by each dose of methylone upon repeated drug injections (across different days). Horizontal panel represents the distance travelled by every dose of methylone for each drug injection. Repeated 1 µg/g methylone produces a slight increase in locomotion for a brief time. First 3 µg/g injection produced increased locomotion and the locomotion remained higher for the repeated drug injections. 10 µg/g did not produce any distinct effect in locomotion. 75

Figure 9: Schematic illustration of the experimental design used for drug-induced reward.

The experimental design comprised of five phases: pre-exposure that explored the spatial activities of crayfish, the conditioning, the CPP test, the extinction pairing, and the extinction test. Day two recorded crayfish locomotion restricted to a single quadrant with removable partitions. During the conditioning trials (day 3, 4, and 5), the infusion cannula was connected to the crayfish and the crayfish was gently placed into the desired quadrant. The test individual experienced a 5 minute drug/saline infusion at the beginning of each 75 minute conditioning session. The conditioning sessions were conducted twice a day (morning and afternoon) with 75 minute treatments per day in random order. Each animal received three consecutive days of substrate consistent drug/saline injections (days 3, 4, & 5) days. We used an unbiased CPP approach by combining equal numbers of individuals with each substrate (i.e., irrespective of initial preference for a substrate by an individual) to examine all pairwise counter-balanced combinations of substrate and drug during conditioning. The control group consisted of crayfish who received saline on both (hard and soft) substrates. The morning and afternoon sessions were 76 separated by 6 hours to permit maximum drug clearance. For the CPP (post-C) test on day six, the partition was removed and crayfish were gently placed at the center of the arena allowing them free access to the whole (soft and hard substrates) arena for 75 minutes. The amount of time spent on each substrate during the CPP test (post-C) was recorded. Increased time spent in the paired environment served as a measure of preference for the specific stimulus, whereas a decrease in time spent indicated an aversion (conditioned place aversion). During the extinction pairing, the injection cannula was again attached to the tubing and directly connected to crayfish.

Crayfish was then gently placed into the desired quadrant, followed by a continuous saline injection for the first 5 minutes of the 75 minute extinction pairing session. During the extinction pairing phase, each animal was confined to one substrate during the morning session and to the other in the afternoon session and individuals received saline injections on both the substrates in random order. Each animal received saline injections for 3 consecutive (day7, 8, and 9) days. For the extinction test (on day10), the partition was removed and crayfish were gently placed at the center of the arena allowing them free access to the whole (soft and hard substrates) arena for 75 minutes. The amount of time spent on each substrate during the extinction (post-E) test was recorded. Increased time spent in the paired environment served as a measure of preference for the specific stimulus, whereas a decrease in time spent indicated an aversion (conditioned place aversion). No injections were given on day of the CPP and extinction test, thus maintaining the same procedure as that used during the preliminary baseline test of exploring the spatial activities of crayfish. Crayfish were allowed free access to the entire aquarium for 75 minutes.

77

Figure 10: Unconditioned preference of crayfish. Crayfish (n=92) seemed to prefer the soft- textured compartment during the pre-exposure phase (before the conditioning trials). Our expectation that crayfish will spent equal amount of time on soft and hard substrate was proved to be false. The preference for soft substrate was statistically significant (one sample t-test (mu =

50%); t-value = 5.98; P-value = 4.2 X 10-8).

78

Figure 11: Synthetic cathinones and their substituted amphetamine analogues show increased preference for drug-paired substrate at 10µg/g. Data are expressed as means percentage of time spent on each substrate S.E.M. Vertical panels depicts the preference for drug paired substrate on

Pre-C (pre-conditioning on Day1), Post-C (post-conditioning on day 6) and post-E (post- extinction on day 10) for each of the drug - 4-MMA, mephedrone, MDMA and methylone.

Horizontal panel depicts the preference for drug paired substrate on Pre-C (pre-conditioning on day1), Post-C (post-conditioning on day 6) and post-E (post-extinction on day 10) for each concentration (1,3 and 10 µg/g) of the drug. Increased preference for drug-paired substrate on day 6 (Post-C) is apparent for 10 µg/g dose of each drug. At 3µg/g dose, there is increased preference for drug paired substrate during post-C for 4-MMA, MDMA and methylone but not mephedrone. At 1 µg/g dose of each drug, increased preference for drug paired substrate during post-C is not seen.

79

Figure 12: Lower doses of substituted amphetamine analogues are able to produce rewarding effects equivalent to higher doses of cathinones. Each vertical panel represents the amount of time spent on drug-paired compartment on Pre-C, Post-C and Post-E for 1,3, and 10 µg/g doses of each drug. 3µg/g dose of 4-MMA and MDMA increased preference for drug-paired substrate by 11.4 % and 13% respectively. 10 µg/g of mephedrone and methylone increased preference for drug-paired substrate by 12 % and 13% respectively.

80

Figure 13: Locomotion (total distance travelled) during termination of drug: Repeated measures

ANOVA revealed significant differences in total distance travelled on days 7, 8, and 9 for

MDMA and 4-MMA and a significant Dose*Day interaction effect for methylone. Post hoc

Tukey test did not reveal significant difference in locomotion on different days.

81

Figure 14: Total distance travelled following drug administration and drug termination:

No changes in locomotor activity were observed during drug termination in comparison to the locomotion when drug was administered.

82

Tables:

Treatment Dose Number of Mean Sex Mean carapace

(µg/g) crayfish weight ± Standard length ± Standard

error (g) error (cm)

Saline N/A 8 8 ± 0.76 Male 6.9 ± 0.4

4-MMA 1 8 7.1 ± 0.85 Male 7.8 ± 0.2

3 6 5.9 ± 0.42 Male 6.7 ± 0.2

10 8 9.6 ± 1.5 Male 7.4 ± 0.3

Mephedrone 1 7 8.4 ± 1.16 Male 7.7 ± 0.3

3 8 6.4 ± 0.39 Male 7.1 ± 0.2

10 8 9 ± 1.46 Male 7.5 ± 0.6

MDMA 1 7 9 ± 1.15 Male 6.9 ± 0.2

3 6 9.8 ± 1.4 Male 7.9 ± 0.4

10 6 11.1 ± 0.24 Male 8.3 ± 0.3

Methylone 1 6 8.2 ± 0.79 Male 6.9 ± 0.1

3 6 5.6 ± 0.57 Male 7 ± 0.1

10 8 9.3 ± 1.51 Male 8.1 ± 0.5 83

Table 1: Specific details of crayfish for psychostimulation and sensitization experiment: Crayfish

(n = 104) were randomly assigned to 13 experimental groups (n = 8 per group) - one control and

12 treatment groups. The control group received saline while each of the treatment group received mephedrone, methylone, MDMA or 4-MMA at 1,3, and 10 µg/g dose. During the course of the experiment, few crayfish died and so were not included in the analysis.

84

Days MMA Mephedrone MDMA Methylone Sal

1 3 10 1 3 10 1 3 10 1 3 10

3 5.4 5.0 2.5 3.7 4.7 5.1 3.2 5.0 4.4 4.2 8.8 4.6 4.3

± 1.3 ± 1.6 ± 0.2 ± 0.4 ± 0.6 ± 0.9 ± 0.6 ± 1.0 ± 0.6 ± 1.2 ± 4.0 ± 0.7 ± 0.6

4 4.4 1.9± 3 4.3 5.6 4 4.5 6.8 5.2 4.1 4.8 3.3 3.7

± 0.6 0.3 ± 0.4 ± 0.4 ± 1.2 ± 0.5 ± 0.9 ± 1.0 ± 1.0 ± 1.3 ± 1.2 ± 0.5 ± 0.5

5 5.3 2.9 4.9 4 3.8 5.6 4.2 4.7 4.4 3.3 4.2 3.2 3.5

± 1.3 ± 1 ± 1.5 ± 0.6 ± 0.7 ± 0.9 ± 0.8 ± 0.5 ± 0.7 ± 1.1 ± 0.6 ± 0.2 ± 0.5

Table 2. Descriptive statistics (mean ± S.E.M) for measures of total distance travelled (in pixels) during day 3, 4 and 5 for control and treatment groups. The actual distance values are 104 times the values given in table. Treatment groups were administered with

MDMA, 4-MMA, methylone and mephedrone at 1, 3, and 10 µg/g and control group with saline. 85

MDMA

Source df SS MS F p

Dose 3 22.1 7.3 2.62056 0.075

Day 2 6.8 3.4 12.02155 <.0001***

Segment 5 14 2.8 9.89101 <.0001***

Day: Segment 10 3 0.3 1.07816 0.3778

Dose: Segment 15 3.5 0.2 0.83261 0.6411

Dose: Day 6 14.8 2.5 8.69981 <.0001***

Dose:Day:Segment 30 5.9 0.2 0.69919 0.8829

Treatments 71 70.1 1 5

Error 391 110.6 0.2

Total 462 180.7

Methylone

Source df SS MS F p

Dose 3 21.5 7.2 1.06609 0.382

Day 2 12.6 6.3 23.29117 <.0001***

Segment 5 7.6 1.5 5.61664 0.0001*** 86

Day: Segment 10 0.3 0 0.10767 0.9997

Dose: Segment 15 2.9 0.2 0.7221 0.7625

Dose: Day 6 11.1 1.8 6.83299 <.0001***

Dose:Day:Segment 30 3.6 0.1 0.44487 0.9956

Treatments 71 59.6 0.8 4

Error 408 110.2 0.2

Total 479 169.8

4-MMA

Source df SS MS F p

Dose 3 75 250 0.756264 0.5296

Day 2 21 105 3.687566 0.0259*

Segment 5 17.1 0.3 1.202394 0.3072

Day: Segment 10 8.9 0.9 0.31241 0.9779

Dose: Segment 15 25.3 1.7 0.591321 0.882

Dose: Day 6 111.3 18.5 6.514988 <.0001***

Dose:Day:Segment 30 25 0.8 0.293727 0.9999 87

Treatments 71 283.6 4 1.4

Error 408 1161 2.8

Total 479 1444.6

Mephedrone

Source df SS MS F p

Dose 3 182.4 60.8 3.012 0.0474*

Day 2 8.1 4 1.07619 0.3418

Segment 5 20.2 4 1.07449 0.3737

Day: Segment 10 14.7 1.5 0.39142 0.9504

Dose: Segment 15 6.8 0.5 0.12121 1

Dose: Day 6 27.6 4.6 1.2219 0.2936

Dose:Day:Segment 30 46.1 1.5 0.40837 0.998

Treatments 71 305.9 4.3 1.2

Error 459 1726 3.7

Total 530 2031.9

88

Table 3: Repeated measures ANOVA on the distance travelled (pixel) during 5 minute time segment for each drug at 1,3 and 10 µg/g doses on day 3, 4 and 5: Significant differences were observed for Day, Segment and Dose: Day for MDMA and methylone. Significant Day and

Dose: Day effect was seen for individuals treated with 4-MMA. Significant effect of dose was observed for mephedrone. Significance. Codes: *** p≤0.001; ** 0.001

‘.’ 0.05

Treatment Dose Drug Number Mean Sex Mean carapace

(µg/g) paired of weight ± Standard length ± Standard

substrate crayfish error (g) error (cm)

Saline N/A N/A Male 6.9 ± 0.4 8 8 ± 0.76

4-MMA Male 7.6 ± 0.2 1 Soft 4 7.35 ± 1.47

Hard Male 7.9 ± 0.4 4 6.83 ± 1.08

Male 6.3 ± 0.2 3 Soft 3 6.13 ± 0.58

Hard Male 7.2 ± 0.3 3 5.57 ± 0.69

Male 6.7 ± 0.2 10 Soft 4 9.45 ± 2.54 89

Hard Male 8.1 ± 0.2 4 9.65 ± 2.02

Mephedrone Male 8.3 ± 0.2 1 Soft 3 7.67 ± 2.13

Hard Male 7.2 ± 0.3 4 8.88 ± 1.5

Male 6.6 ± 0.2 3 Soft 4 6.65 ± 0.66

Hard Male 7.6 ± 0.2 4 6.18 ± 0.5

Male 9 ± 0.2 10 Soft 4 8.88 ± 2.15

Hard Male 5.9 ± 0.2 4 9.05 ± 2.31

MDMA Male 6.9 ± 0.4 1 Soft 3 8.33 ± 1.79

Hard Male 6.9 ± 0.3 4 9.43 ± 1.7

Male 8.1 ± 0.4 3 Soft 3 9.83 ± 2.4

Hard Male 7.7 ± 0.7 3 9.77 ± 2.02

Male 8.7 ± 0.3 10 Soft 2 11.27 ± 0.23 90

Hard Male 7.8 ± 0.2 4 10.97 ± 0.43

Methylone Male 6.9 ± 0.2 1 Soft 3 8.6 ± 1.11

Hard Male 6.8 ± 0.1 3 7.7 ± 1.31

Male 6.9 ± 0.2 3 Soft 3 5.6 ± 0.85

Hard Male 7 ± 0.2 3 5.63 ± 0.94

Male 6.9 ± 0.3 10 Soft 4 9.7 ± 2.32

Hard Male 9.3 ± 0.3 4 8.9 ± 2.26

Table 4: Stratification and descriptives of crayfish for conditioned place preference: All male intermolt crayfish with intact appendages were selected and randomly assigned to 13 experimental groups – Control (saline) and 1,3 and 10 ug/g of 4-MMA, mephedrone, MDMA and methylone.

91

Days MMA Mephedrone MDMA Methylone

1 3 10 1 3 10 1 3 10 1 3 10

52.2 ± 48.1 ± 51.3 ± 48.4 ± 48.9 ± 47.4 ± 51.2 ± 49.6 ± 45 ± 47.5 ± 51.8 ± 51.7 ± Pre-C 3.32 4 1.6 1.8 3.2 3.4 3.8 9.7 3.6 3.5 5.3 5.3

52.2 ± 59.5 ± 56.1 ± 46.9 ± 46.7 ± 59.4 ± 50.4 ± 63 ± 48.7 ± 48 ± 57.7 ± 65.7 ± Post-C 1.5 7.7 4.4 1.2 1.4 4.1 2.5 7.7 1 1.78 6.8 6.8

44 ± 7 46.9 ± 53 ± 47.7 ± 51.9 ± 53.3 ± 45.7 ± 47.3 ± 51.2 ± 54.5 ± 57.3 ± 48.11 ± Post-E 1.9 6.7 1.4 2.8 3.4 2.8 6.1 7.3 5.8 4.9 6.8

Table 5: Descriptive statistics (mean ± S.E.M) for measures of amount of time spent on drug paired substrate (in percentage) during

pre-C, post-C and post-E days for each dose of treatment groups 92

MDMA

Degrees of freedom Sum of squares Mean squares F value P value

Dose 2 263.7 131.8 0.869 0.5285

Day 2 393 196.5 0.885 0.3829

Dose: Day 4 716 179 0.806 0.475

Treatment 8 1372.7 171.5 0.772

Error 32 7109 222.1

Total 40 8481.7

Methylone

Degrees of freedom Sum of squares Mean squares F value P value

Dose 2 369 184.6 0.72 0.501

Day 2 584 292 1.716 0.195 93

Dose: Day 4 580 144.9 0.852 0.503

Treatment 8 1533 191.625 1.125

Error 34 5787 170.2

Total 42 7320

4-MMA

Degrees of freedom Sum of squares Mean squares F value P value

Dose 2 85.3 42.66 0.391 0.682

Day 2 636 318.2 1.979 0.152

Dose: Day 4 509 127.3 0.792 0.538

Treatment 8 1230.3 153.7875 0.956

Error 38 6110 160.8

Total 46 7340.3

Mephedrone 94

Degrees of freedom Sum of squares Mean squares F value P value

Dose 2 301.3 150.65 1.927 0.172

Day 2 77.5 38.77 1.014 0.3718

Dose: Day 4 466.8 116.71 3.053 0.0275 *

Treatment 8 845.6 105.7 2.765

Error 40 1529 38.22

Total 48 2374.6

Table 6: Repeated measures ANOVA for amount of time spent on the drug paired substrate during pre-C, post-C and post-E for 1, 3 and 10 ug/g of MDMA, methylone, 4-MMA and mephedrone. Significant environmental preference for substrate paired with 10 µg/g mephedrone

(p < 0.05) is seen. Tukey post hoc test showed significant increase in preference for drug-paired compartment during post-C in the crayfish treated with 10 µg/g mephedrone.

95

Treatment Dose Number of Mean Sex Mean carapace

(µg/g) crayfish weight ± Standard length ± Standard

error (g) error (cm)

Saline N/A 8 8 ± 0.76 Male 6.9 ± 0.4

4-MMA 1 8 7.1 ± 0.85 Male 7.8 ± 0.2

3 6 5.9 ± 0.42 Male 6.7 ± 0.2

10 8 9.6 ± 1.5 Male 7.4 ± 0.3

Mephedrone 1 7 8.4 ± 1.16 Male 7.7 ± 0.3

3 8 6.4 ± 0.39 Male 7.1 ± 0.2

10 8 9 ± 1.46 Male 7.5 ± 0.6

MDMA 1 7 9 ± 1.15 Male 6.9 ± 0.2

3 6 9.8 ± 1.4 Male 7.9 ± 0.4

10 6 11.1 ± 0.24 Male 8.3 ± 0.3

Methylone 1 6 8.2 ± 0.79 Male 6.9 ± 0.1

3 6 5.6 ± 0.57 Male 7 ± 0.1

10 8 9.3 ± 1.51 Male 8.1 ± 0.5

96

Table 7: Specific details of crayfish for unconditioned and termination effects experiments:

Crayfish (n = 104) were randomly assigned to 13 experimental groups (n = 8 per group) - one control and 12 treatment groups. The control group received saline while each of the treatment group received mephedrone, methylone, MDMA or 4-MMA at 1,3, and 10 µg/g dose. During termination trials, all groups received saline. During the course of the experiment, few crayfish died and so were not included in the analysis.

Unconditioned Behavior Description Produced by behavior

Forward walking Moves forward anywhere in the arena Saline, MDMA, 4-

MMA, methylone,

mephedrone

Backward walking Moves backward anywhere in arena Higher (10µg/g) doses

of methylone

Tail Flips Rapidly contracts abdominal segments to Higher (10µg/g) doses

propel backwards. Usually lasts for a short of methylone

duration of time (few milliseconds)

Rearing Stands on last pair of walking legs with other Saline, MDMA, 4-

pairs of legs trying to climb the wall of the MMA, methylone,

arena mephedrone 97

Exploring Uses antennae to gather tactile information of Saline, MDMA, 4-

the arena walls MMA, methylone,

mephedrone

Grooming Uses walking legs to clean different regions of Saline, MDMA, 4-

the body MMA, methylone,

mephedrone

Bouts of Moves rapidly for a short duration of time MDMA, 4-MMA, locomotion followed by long period of inactivity mephedrone

Claw waving Moves one or both the claws in and out. At MDMA, 4-MMA,

times, both the claws are swayed from one side mephedrone

to another.

Upward Moves its whole body upwards 4-MMA movements

Claw closure Closes claw similar to the pinching MDMA

Circling Rotates around itself using walking pair of legs. MDMA

Turns suddenly in opposite direction.

Extended posture Extends its claws anteriorly producing a MDMA, 4-MMA,

stretched posture methylone, mephedrone 98

Stereotyped Does not move in space but pairs of legs MDMA, 4-MMA, movements produce uncoordinated movements methylone, mephedrone

Immobility Does not move at all and sits at one place Lower doses (1 µg/g) of

usually in corners for long period of time. Does MDMA and methylone

not move any of the appendages

Table 8: Ethogram describing unconditioned behavioral responses to MDMA, 4-MMA, mephedrone and methylone. 99

MMA Mephedrone MDMA Methylone Saline

Day 1 3 10 1 3 10 1 3 10 1 3 10

5 ± 3.6 ± 3.4 ± 4.7 ± 4.7 ± 4.3 ± 4.9 ± 6.2 ± 5 ± 3.8 ± 4.5 ± 4.1 ± 4.3 ± 7 0.6 0.6 0.4 0.4 0.5 0.5 0.6 0.8 0.6 0.6 0.9 0.2 0.8

3.8 ± 3.8 ± 3.7 ± 3.5 ± 4.2 ± 4.8 ± 4.6 ± 6 ± 3.6 ± 4 ± 4.1 ± 4.1 ± 3.2 ± 8 0.5 0.5 0.3 0.3 0.5 0.5 0.8 0.7 0.3 0.6 0.7 0.3 0.5

3.9 ± 3.3 ± 2.9 ± 4.3 ± 3.6 ± 4.8 ± 4.4 ± 5.3 ± 4.3 ± 3.5 ± 5.6 ± 4.6 ± 3.5 ± 9 0.7 0.5 0.3 0.5 0.3 0.4 0.8 0.7 0.6 0.5 1.1 0.2 0.6

Table 9: Descriptive statistics (mean ± S.E.M) for measures of total distance travelled (in pixels) during day 7,8 and 9 for control and treatment groups. The actual distance values are 104 times the values given in table. All the groups received saline during extinction trials i.e. on day 7, 8 and 9. 100

MDMA

Degrees of freedom Sum of squares Mean squares F value P value

Dose 3 505.6 168.5 1.467 0.2497

Day 2 102 50.9 4.25525 0.0202

Dose: Day 6 45.9 7.6 0.63955 0.698

Treatments 11 653.5 59.4 4.95

Error 46 551.2 12

Total 57 1204.7

Methylone

Degrees of freedom Sum of squares Mean squares F value P value

Dose 3 160.9 53.3 0.58177 0.6327

Day 2 35.4 17.7 2.0304 0.1424

Dose: Day 6 123 20.5 2.34965 0.0454

Treatments 11 319.3 29 3.3

Error 48 418.8 8.7 101

Total 59 738.1

4-MMA

Degrees of freedom Sum of squares Mean squares F value P value

Dose 3 81.9 27.3 0.3494 0.7899

Day 2 60 34.5 4.3537 0.0186

Dose: Day 6 61.3 10.2 1.28986 0.2808

Treatments 11 203.2 18.5 2.3

Error 46 364.9 7.9

Total 57 568.1

Mephedrone

Degrees of freedom Sum of squares Mean squares F value P value

Dose 3 124 41.3 0.80519 0.502

Day 2 51.5 25.7 2.65363 0.0796

Dose: Day 6 119.1 19.8 2.0427 0.0756

Treatments 11 294.6 26.8 2.8

Error 54 524.7 9.7 102

Total 65 819.3

Table 10: Repeated measures ANOVA for total distance travelled on Day 7, 8, and 9 for 4-

MMA, mephedrone, MDMA and methylone at 1,3, and 10 µg/g. Repeated measures ANOVA revealed significant differences in total distance travelled across days 7, 8, and 9 for MDMA and

4-MMA and a significant Dose*Day interaction effect for methylone. Post hoc Tukey test did not reveal significant difference in locomotion on different days.