THE MODULATORY ROLE OF TAAR1 IN NEUROTOXICITY OF SUBSTITUTED AMPHETAMINES By Nicholas B. Miner A DISSERTATION Presented to the Department of Behavioral Neuroscience and the Oregon Health & Science University School of Medicine In partial fulfillment of the requirements for the degree of Doctor of Philosophy February 2019 School of Medicine Oregon Health & Science University ____________________________________________________________________________ CERTIFICATE OF APPROVAL ___________________________________________ This is to certify that the Ph.D. dissertation of Nicholas B. Miner has been approved ___________________________________________ Mentor – Aaron Janowsky, Ph.D. ___________________________________________ Committee Chair – Tamara Richards, Ph.D. ___________________________________________ Committee Member – Charles Meshul, Ph.D. ___________________________________________ Committee Member – Jennifer Loftis, Ph.D. ___________________________________________ Committee Member – Matthew Lattal, Ph.D. i TABLE OF CONTENTS List of figures and tables ............................................................................................................ iii List of abbreviations .................................................................................................................... v Acknowledgements .................................................................................................................. viii Abstract....................................................................................................................................... x Chapter 1: Introduction ............................................................................................................... 1 1. Methamphetamine and MDMA ........................................................................................ 1 Monoamines .................................................................................................................... 4 2. Neurotoxicity ................................................................................................................... 7 Neurotoxicity models and markers ................................................................................... 9 Neuroinflammation ..........................................................................................................12 Dopamine transporter .....................................................................................................15 Vesicular monoamine transporter 2 ................................................................................16 Oxidative stress ..............................................................................................................18 Thermal response ...........................................................................................................20 3. Substituted Methcathinones ...........................................................................................21 4. TAAR1............................................................................................................................27 Mechanisms of action .....................................................................................................28 Animal models ................................................................................................................34 Interaction with amphetamines .......................................................................................41 Biochemical ..........................................................................................................41 Thermal response .................................................................................................46 Behavior ...............................................................................................................47 5. Dissertation studies ........................................................................................................54 Chapter 2: The combined effects of MDMA and selected substituted methcathinones on measures of neurotoxicity .........................................................................................................60 1. Introduction ....................................................................................................................60 2. Materials and Methods ...................................................................................................61 3. Results ...........................................................................................................................68 4. Discussion ......................................................................................................................75 Chapter 3: TAAR1 regulation of MDMA-induced neurotoxicity ..................................................83 1. Introduction ....................................................................................................................83 2. Methods and Materials ...................................................................................................85 ii 3. Results ...........................................................................................................................89 4. Discussion ......................................................................................................................95 Chapter 4: TAAR1 regulation of methamphetamine-induced neurotoxicity ................................99 1. Introduction ....................................................................................................................99 2. Material and Methods ................................................................................................... 102 3. Results ......................................................................................................................... 106 4. Discussion .................................................................................................................... 117 Chapter 5: The role of biogenic amine transporters on TAAR1 regulation of methamphetamine- induced neurotoxicity .............................................................................................................. 124 1. Introduction .................................................................................................................. 124 2. Material and Methods ................................................................................................... 128 3. Results ......................................................................................................................... 136 4. Discussion .................................................................................................................... 149 Chapter 6: General discussion ................................................................................................ 154 1. Overview ...................................................................................................................... 154 2. Summary of major findings ........................................................................................... 158 3. Clinical implications ...................................................................................................... 174 4. Limitations .................................................................................................................... 176 5. Future directions ........................................................................................................... 177 6. Conclusions .................................................................................................................. 179 Appendices ............................................................................................................................. 181 Appendix 1: additional experiments associated with Chapter 2 ........................................... 181 A1.1. DA ELISA and drug dose ..................................................................................... 181 A1.2. Temperature recordings ...................................................................................... 182 A1.3. [3H]PK11195 binding to TSPO ............................................................................. 183 Appendix 2: additional experiments associated with Chapter 3 ........................................... 185 A2.1. MDMA-induced [3H]DA release optimization experiments .................................... 185 Appendix 3: additional experiments associated with Chapter 4 ........................................... 187 A3.1. TAAR1 modulation of DA and dose response curve pilot ..................................... 187 Appendix 4: Additional experiments associated with Chapter 5 ........................................... 189 A4.1. MA-induced [3H]DA release (superfusion) optimization experiments .................... 189 A4.2. MA-induced [3H]DA uptake optimization experiments .......................................... 193 A4.3. In vivo treated [3H]DA uptake pilot ....................................................................... 196 iii A4.4. In vivo treated [3H]DHTB saturation binding optimization ..................................... 197 References ............................................................................................................................. 204 List of figures and tables Figure 1. Molecular structures for amphetamine related compounds ......................................... 3 Figure 2. Schematic of the actions of