THE EFFECTS OF 3,4-METHYLENEDIOXYMETHAMPHETAMINE (MDMA) ON MNEMONIC AND EXECUTIVE MEASURES AND SEROTONERGIC NEUROTOXICITY USING INTERSPECIES EFFECTS SCALING by Stephanie Brooke Linley A Dissertation Submitted to the Faculty of The Charles E. Schmidt College of Science In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Florida Atlantic University Boca Raton, FL May 2011 Copyright by Stephanie Brooke Linley 2011 ii THE EFFECTS OF 3,4-METHYLENEDIOXYMETHAMPHETAMIN (MDMA) ON MNEMONIC AND EXECUTIVE MEASURES AND SEROTONERGIC NEUROTOXICITY USING INTERSPECIES EFFECTS SCALING by Stephanie Brooke Linley This dissertation was prepared under the directions of the candidate's dissertation advisor, Dr. Katherine M. Hughes, Department ofPsychology, and has been approved by the members of her supervisory committee. It was submitted to the faculty of the Charles E. Schmidt College of Science and was accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy. SUPERVISORY COMMITTEE: ~~ Katherine M. HUghe~~Ji. Dissertation Advisor ~~~. Robert W. Stacldiian Jr., Ph.D. ~/~~tA..~L~LJl.!:7---L~ i David L. Wolgin, Ph.D. tft-'--- l e-o Rui Tao, Ph.D. Barry T. Ross n? Ph.D. Date Dean, Graduate Studies iii ACKNOWLEDGEMENTS I would like to thank my dissertation advisor, Dr. Katherine M. Hughes, for her wisdom, support, and guidance. I would like to thank Dr. Robert W. Stackman Jr for serving as a core committee member, and for his support in the set up, development, and execution of Experiment 2. I would like to thank Dr. David L. Wolgin, for serving as my third core committee member, and for generously allowing me the use of his laboratory space for behavioral testing. I would like to thank my two additional committee members, Dr. Michael A. King, Associate Scientist at the University of Florida Department of Pharmacology, and Dr. Rui Tao for their time and insightful comments. I am also grateful to Dr. Robert P. Vertes, for sharing his laboratory space and equipment during the completion of the experiments. I would like to share my genuine appreciation to the graduate students, postdoctoral fellows, and undergraduate DIS students who played a critical role in assisting me with several aspects of graduate student life and research: Dr. Walter B. Hoover, III, Eric D. Buerger, Michael Guidi, Amy B. Peebles, Michelle L. Steigerwald, and Courtney Graham. This dissertation would not be possible without the fiscal and emotional support of my parents, George and Anna Linley. I am grateful to my family and friends who stood by me through this process, especially my grandparents Dee and Rita Goodman, my brother George W. Linley III, and Jeffrey Douglass Pereboom. iv ABSTRACT Author: Stephanie Brooke Linley Title: The effects of 3,4-methylenedioxymethamphetamine (MDMA) on mnemonic and executive measures and serotonergic neurotoxicity using interspecies effects scaling. Institution: Florida Atlantic University Dissertation Advisor: Dr. Katherine M. Hughes Degree: Doctor of Philosophy Year: 2011 3,4-methlenedioxymethamphetamine (MDMA), the main constituent of Ecstasy, is a ring-substituted amphetamine commonly abused in recreational users. High doses of MDMA determined by allometric scaling produce serotonin (5-HT) axon deneveration. Studies suggest that this interspecies scaling does not reflect human use. An ‘effects’ scale comparing similar behavioral and physiological effects between species has been postulated as more accurate for translational studies. Experiment 1 examined the effects of MDMA on serotonergic forebrain innervation using immunohistochemical labeling targeting the serotonin transporter protein (SERT). Experiments 2 and 3 examined low and high doses of MDMA on spatial memory, prefrontal functioning, and serotonergic v neurotoxicity using ‘effects’ scaling. Long Evans rats were given MDMA regimens of: chronic low dose (daily injections of 1.5 mg/kg for 10 days); binge low dose (2 days of 4 x 1.5 mg/kg spaced 2 hours apart), binge high dose (2 x 7.5 mg/kg spaced 2 hours apart). Acquisition, retention, and spatial reversal (SR) were measured in a water maze task. A 2.0 mg/kg MDMA drug challenge was then given prior to a serial spatial reversal (SSR) task to assess performance while under the effect of the drug. Attentional set shifting and behavioral flexibility were assessed in an intradimensional extradimensionl (IED) task using odor/texture discriminations. MDMA chronic and binge low doses did not impair water maze or IED performance and produced no reductions in SERT expression. MDMA binge high dose resulted in significant reductions of SERT density in the prefrontal cortex, striatum, cortical mantle, hippocampus, amygdala, and many thalamic nuclei. Despite prominent 5-HT denervation, water maze performance was unaffected. Selective impairment in behavioral flexibility on the IED test was found. This suggests that low doses of MDMA do not produce long-term deleterious effects. But, high doses of MDMA taken in ‘binges’ produces widespread loss of forebrain SERT fiber innervation and significant impairments in reversal learning, while leaving attentional set shifting and spatial navigation unscathed. vi TABLE OF CONTENTS LIST OF TABLES………………………………………………………………………...x LIST OF FIGURES..........................................................................................................xiii LIST OF ABBREVIATIONS ..........................................................................................xix GENERAL INTRODUCTION……….……………………………………………….......1 Anatomy of the serotonergic innervation of the forebrain.....................................4 Serotonergic modulation of emotion .....................................................................8 Serotonin’s role in executive and mnemonic processes ....……..........................11 Pharmacokinetics and acute effects of MDMA and its metabolites in rats….....16 Behavioral effects of MDMA and its metabolites in rats………………………19 Effects of MDMA on cognition and emotion…………………………………..20 MDMA neurotoxicity……………………………………………………..……31 Rationale………………………………………………………………………..37 EXPERIMENT 1………………………………………………………………………...40 Introduction……………………………………………………………................40 Materials and Methods……………………………………………...…………...51 Subjects……………………………………………………………..…....51 Drugs……………………………………………………………………..52 Histology ……………………………………………………………..….52 SERT immunohistochemistry………………………..……..……52 TH immunohistochemistry ………………….….…...……..……54 Photomicroscopy and data analysis ….……………………………..…...55 Results………………………………….……………………………………….56 Serotonergic innervation of the forebrain……….…………………..... ...57 Prefrontal cortex…………………………………………..…... ...57 Piriform cortex, endopiriform nucleus, and olfactory tubercule…66 Remaining cortical mantle………………….………..……..…....71 Dorsal and ventral striatum and globus pallidus……………...….74 Basal forebrain and claustrum……………...……...………..…...83 Hippocampal formation and amygdala ……………………….....87 Thalamus……..………………………………………….…….…89 Hypothalamus…………………………………………..………106 Cathecolaminergic innervation…………………………………………107 vii Discussion..……………………………………………………………………..114 EXPERIMENT 2 ……………………............................................................................124 Introduction…..…………………………………………………......................124 Materials and Methods………………………..……………...………………..133 Subjects…………………………………………………………..…......133 Dose regimen ………………………………………………………..…133 Drugs………………………………………………………………..…..134 Apparatus ………………………………………………………………134 Procedure ………………………………………………………………135 Spatial memory ………………………………………………...135 Spatial reversal task ……………………………………………139 Serial Reversal and drug challenge ……………………………139 Histological analysis …………………………………………………...140 Data analysis …………………………………………………………...147 Results…………………………………………………………………………..142 Physiological effect of MDMA ……………………………………..…142 Behavioral Testing …………………………………………………..…143 Acquisition and retention ………………………………………143 Reversal task ………………………………………………...…154 Serial reversal drug challenge ………………………….………162 Immunohistochemistry ………………………………………………...171 Discussion ……………………………………………………………………...171 EXPERIMENT 3 ………………………………………………………………………188 Introduction …………………………………………………………………….188 Materials and Method …………………………………………………….……200 Subjects ……………………………………………………………...…200 Drugs ……………………………………………………...……………201 Apparatus ………………………………………………………………202 Procedure ………………………………………………………………204 Habituation ……………………………………………………..204 IED testing ……………………………………………………..206 Immunohistochemical analysis ………………………………………...211 Data analysis …………………………………………………………...211 Results ………………………………………………………………………….214 Physiological effects of MDMA …………………………………….…214 Behavioral Testing ……………………………………………..………215 Digging establishment/habituation ………………………….…215 Odor texture IED test ………………………………………..…216 Latencies ……………………………………………………….230 Immunohistochemistry ……………………………………………...…232 Discussion…...…………………………………………………………………..239 viii GENERAL DISCUSSION..............................................................................................252 REFERENCES.................................................................................................................266 ix LIST OF TABLES Table 1. Density of fibers expressing the serotonin transporter (SERT) protein in the cortex……………………………………….......................62 Table 2. Density of fibers expressing the serotonin transporter (SERT) protein in the basal forebrain………….………………………………....80 Table 3. Density of fibers expressing the serotonin transporter (SERT)