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 .................................................................................................