(Mdma) on Mnemonic and Executive Measures and Serotonergic Neurotoxicity Using Interspecies Effects Scaling

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THE EFFECTS OF 3,4-METHYLENEDIOXYMETHAMPHETAMINE (MDMA) ON MNEMONIC AND EXECUTIVE MEASURES AND SEROTONERGIC NEUROTOXICITY USING INTERSPECIES EFFECTS SCALING

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

THE EFFECTS OF 3,4-METHYLENEDIOXYMETHAMPHETAMIN (MDMA) ON MNEMONIC AND EXECUTIVE MEASURES AND SEROTONERGIC NEUROTOXICITY USING INTERSPECIES EFFECTS SCALING

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

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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.

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.

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

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GENERAL DISCUSSION...... 252

REFERENCES...... 266

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) protein in the hippocampus and amygdala.……...…………………….....91

Table 4. Density of fibers expressing the serotonin transporter (SERT) protein in the diencephalon...... ….101

Table 5. Daily Averaged Mean and (Standard Deviations in paratheses) values for latency to reach platform (s), cumulative distance traveled to platform (cm), and velocity/swim speed of each trial for each drug treatment during the daily sessions for the acquisition phase.…………………………………………………..…...148

Table 6. Mean (Standard Deviations in parentheses) values for search duration (seconds, sec) during each of four 15 second intervals (0-15, 15-30, 30-45, and 45-60 sec) in the 60 second retention probe trial listed for each treatment condition…..……………………...152

Table 7. Daily Averaged Mean (Standard Deviations in parentheses) values for latency to reach platform (s), cumulative distance traveled to platform (cm), and velocity/swim speed of each trial for every drug treatment condition during the daily reversal training session……158

Table 8. Mean (Standard Deviations in parentheses) values for search duration (seconds, sec) across each of four 15 second intervals (0-15, 15-30, 30-45, and 45-60 sec) in the 60 second reversal probe trial listed for each treatment condition ……………………...….161

Table 9. Daily averaged Mean and (Standard Deviation in parentheses) values for latency to reach platform (s), cumulative distance traveled to platform (cm), and velocity/swim speed of each trial for each drug treatment during the three daily sessions in the serial reversal phase………………………………..…………..………165

Table 10. Mean and (Standard Deviations in parentheses) values for total search duration (seconds, sec) in each of four 15 second intervals (0-15, 15-30, 30-45, and 45-60 sec) across the 60 second serial reversal drug challenge probe trial listed for each treatment condition…………………………………...……………...…173

Table 11. Odor and Digging Medium exemplar pairs presented in each intradimensional extradimensional (IED) stage are listed in the table adapted from Birrell and Brown (2000)…………………………..209

Table 12. Mean (Standard Deviations in parentheses) values for total number of trials completed to complete digging establishment and total number of trials to completion, errors, and aborted trials in the first habituation odor simple discrimination (SD1) and the second habituation digging medium simple discrimination (SD2) for each of the treatment conditions…………..…218

Table 13. Mean (Standard Deviations in parentheses) values trials to reach stage completion, errors, and aborted trials for the seven stages of the Intrdimensional Extradimensional (IED) test: Simple Discrimination (SD), Compound Discrimination (CD), Compound Reversal (CD2), Intradimensional shift (ID), Intradimensional Reversal (ID2), Extradimensional shift (ED), Extradimensional reversal (ED2)………………………………………………………….220

Table 14. Mean rank values for median number of trials to complete stage, median errors, and median aborted trials in stages in the intradimensional extradimensional test for which homogeneity of variance could not be assumed.……………………………………. 222

Table 15. Mean (Standard Deviations in parentheses) latencies to complete each trial across treatment conditions across for each habituation and test stage of the intradimensional extradimensional (IED) test: Habituation simple discrimination 1 (HABSD1); Habituation simple discrimination 2 (HABSD2); simple discrimination (SD); compound discrimination (CD); compound discrimination reversal (CD2); intradimensional shift (ID); reversal of intradimensional shift (ID2); extradimensional shift (ED); and reversal of extradimensional shift (ED2)..…………...…………………235 xi

Table 16. Average (Standard Deviations in parentheses) median latencies (seconds) of trials for across each habituation and test stages of the intradimensional extradimensional (IED) test for all treatment conditions: Habituation simple discrimination 1 (HABSD1); Habituation simple discrimination 2 (HABSD2); simple discrimination (SD); compound discrimination (CD); compound discrimination reversal (CD2); intradimensional shift (ID); reversal of intradimensional shift (ID2); extradimensional shift (ED); and reversal of extradimensional shift (ED2).……….……..236

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LIST OF FIGURES

Figure 1. Lightfield photomontage showing the distribution of SERT immunopositive fibers through the rostral frontal cortex for a representative control (A) and MDMA binge high dose (B) subject……………………………………………………………...…….63

Figure 2. High magnification lightfield photomontage through the frontal cortex displaying SERT fiber expression through the midlevel of the ventral orbital (VO) cortex for a representative subject in the control (A), MDMA chronic low dose (B), MDMA binge low dose (C) and MDMA binge high dose rat (D).……………………………………………..…………………..…64

Figure 3. Lightfield photomontage through the lateral frontal cortex displaying SERT fiber expression through the granular (GI) and dorsal agranular (AId) insular cortex for a representative control (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA binge high dose (D) subject.……………………..65

Figure 4. Lightfield photomontage of SERT fiber expression in the caudal ventral medial prefrontal cortex in a representative control (A) and MDMA binge high dose (B) subject………………...... 68

Figure 5. Lightfield photomontage of SERT fiber expression in the anterior cingulate (AC) cortex in a control (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA binge high dose (D) subject…………………………………….………………..…..69

Figure 6. Lightfield photomontage of the pattern of SERT fiber expression in the piriform cortex (PIR), endopiriform nucleus (EPd), and olfactory tubercule (OT) in a representative control (A) and MDMA binge high dose (B) subject………………………...………………...….70

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Figure 7. High magnification lightfield photomontage for a representative case in the control (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA binge high dose (D) illustrating the pattern of SERT fiber expression in the outer layers of the primary somatosensory cortex (SSp)…………………………...………..77

Figure 8. Lightfield photomontage of the pattern of SERT fiber expression in the retrosplenial (RSP) and posterior parietal (PTLp) cortex in a representative control (A) and MDMA binge high dose (B) rat……………………………………………………………..…...…78

Figure 9. Lightfield photomontage of the pattern of SERT fiber expression through the rhinal cortices in a representative control (A) and MDMA binge high dose (B) subject. PERI, ECT, and the lateral entorhinal (ENTl) cortex...... 79

Figure 10. High magnification lightfield photomontage of the pattern of SERT fiber innervation across a midrostral level of the dorsolateral caudate putamen (CP) in a control (A), MDMA chronic low dose (B), MDMA binge low dose (C) and MDMA binge high dose (D) rat……………………………………...…………...81

Figure 11. Lightfield photomontage of the pattern of SERT fiber expression through the a midlevel section of the nucleus accumbens (ACC) in a representative control (A) and MDMA binge high dose (B) rat...... …...82

Figure 12. Lightfield photomontage lightfield of the pattern of SERT fiber expression through coronal sections of the globus pallidus (GP) at the level of the rostral thalamus, in a representative control (A) and MDMA binge high dose (B) rat………………………………………….84

Figure 13. Lightfield photomontage of SERT fiber expression through the septum in a representative control (A) and MDMA binge high dose subject (B)…………………………………………………………..90

Figure 14. Lightfield photomontage of the pattern of SERT fiber expression through the amygdala (AMY) in a representative control (A) and MDMA binge high dose (B) rat………………………………………….92

Figure 15. Lightfield photomontage of the pattern of SERT fiber expression across the dorsal hippocampus in a representative control (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA binge high dose (D) subject………………….…………...... 93

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Figure 16. Lightfield photomontage of the pattern of SERT fiber expression through the subiculum (SUB) in a representative control (A) and MDMA binge high dose (B) rat………….………………………….94

Figure 17. Lightfield photomontage of the pattern of SERT fiber expression through the anterior thalamus in a representative case of control (A) and MDMA binge high dose (B) subject……………...………………..102 .. Figure 18. Lightfield photomontage of SERT fiber expression across the rostral midline thalamus in a representative control (A) and MDMA binge high dose (B) rat………………………………………...103

Figure 19. Lightfield photomontage of the pattern of SERT fiber expression through the lateral dorsal (LD) nucleus of the thalamus in a representative control (A), MDMA chronic low dose (B), MDMA binge low dose (C) and MDMA binge high dose (D) rat…………....…104

Figure 20. Lightfield photomontage of SERT fiber expression through the lateral geniculate nucleus (LGN) complex in a representative control (A) and MDMA binge high dose (B) case………………….…105

Figure 21. Lightfield photomontage through the suprachiasmatic nucleus (SCN) in the hypothalamus for a representative control and MDMAbinge high dose treated subject……………...…………………110

Figure 22. Lightfield photomontage of the pattern of TH fiber expression through the across two levels of the frontal cortex, displaying TH labeled fibers at two levels of the mPFC in a representative control (A; C) and MDMA binge high dose (B; D) rat….……………..111

Figure 23. Lightfield photomontage of TH immunoreactivity across the accumbens (ventral, striatum, ACC) nucleus and olfactory tubercule (OT) in a representative control (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA binge high dose (D) rat…………………………………………….………….112

Figure 24. Lightfield photomontage of TH fiber expression through the dorsal striatum (caudate putamen, CP) in a representative control (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA binge high dose (D) rat………..…………..……113

Figure 25. Scaled illustrative drawing of the water maze apparatus used in experiment 2…………………………..……………………..…136

Figure 26. Outline and timeline of the behavioral paradigms used in the experiment 2...... 137

Figure 27. A: Daily mean values for escape latency to platform (seconds=s; y axis) for each of the drug treatment groups for each of the acquisition phase training sessions (x axis). B: Mean daily values for cumulative distance to platform.……..…………………………..…149

Figure 28. Percent of search time (y axis) spent in each of the four quadrants (x axis) during the retention probe trial following 5 days of training acquisition………………………………………...…150

Figure 29. Schematic illustration of individual probe tracks, displaying the search strategies during the entire 60 second retention probe trial for a representative case for each of the drug treatment conditions: control binge (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA high binge dose (D)……………………..…………………………………………..153

Figure 30. Mean velocity (cm/seconds; y axis.) across each of the three probe trials (retention, reversal, and serial reversal; x axis) for each of the treatment conditions……...……………………………...…157

Figure 31. A: Daily mean values for escape latency to platform in seconds for each of the drug treatment groups for each of the reversal phase training sessions. B: Mean cumulative distant to the platform traveled across daily sessions.…………………………...……159

Figure 32. Percent of total search time spent (y axis) in each of the four quadrants (x axis) during the reversal probe trial, where rats were trained to find a hidden submerged platform in the reverse (opposite) SE quadrant as the retention phase ………………………....160

Figure 33. Schematic illustration of individual probe tracks demonstrating search strategies for a representative case in each of the treatment groups: control chronic (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA high binge dose (D) during the 60 second reversal probe trial.… …………………………...164

Figure 34. A: Daily mean values for escape latency to platform for each of the drug treatment groups for the serial reversal phase. B: Daily averaged cumulative distance to the platform for each daily session during the serial reversal phase.…….………………………….168

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Figure 35. Percent of total search time (y axis) spent in each of the four quadrants (x axis) during the serial reversal drug challenge probe trial..……………………………………………………………...169

Figure 36. Schematic illustration of individual probe tracks illustrating search strategies during the serial reversal drug challenge probe test for a representative case of each of the treatment groups: control binge (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA high binge dose (D)..………………………174

Figure 37. Schematic diagram of the behavioral apparatus used in experiment 3…………………………………………….…………...….203

Figure 38. Outline and time of the intradimensional extradimensional (IED) odor texture discrimination paradigm.……………………….. ………..208

Figure 39. Mean number of total trials (y axis) to complete digging establishment for each of the treatment conditions (x axis) during the habituation phase……………………………………………….…...219

Figure 40. Mean total number of trials to reach stage completion (A), errors (B) and aborts (C) for each treatment conditions for each of the two habituation simple discrimination stages: an odor simple discrimination (SD1) and digging medium simple discrimination (SD2). …………………………………………………………….…….221

Figure 41. Mean number of total trials to reach criterion for each of the stages of the intradimensional extradimensional (IED) test……………225

Figure 42. Mean errors committed during each of the seven stages in the intradimensional extradimensional (IED) test……………………….…226

Figure 43. Mean aborted trials during each of the seven stages in the intradimensional extradimensional (IED) test……………………….....227

Figure 44. Performance of rats in the MDMA high binge experimental group across the three reversal stages in the intradimensional extradimensional (IED) test…………………………………………….234

Figure 45. High magnification lightfield photomontage showing the distribution of SERT immunopositive fibers through the prelimbic cortex for a representative control (A) and MDMA binge high dose (B) subject from experiment 3.……………………….237

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Figure 46. Lightfield photomontage showing the distribution of SERT fiber innervation in the dorsal and ventral striatum for a representative subject from the control and MDMA binge high dose condition……………………………………………..……....238

Figure 47. Lightfield photomontage showing the distribution of SERT fiber innervation through the caudal ventral orbital (VLO) cortex for a representative MDMA binge high dose rat from experiment 2 (A) and experiment 3 (B) ……………………………..…246

Figure 48. Lightfield photomontage showing the distribution of SERT fiber innervation in the ventral hippocampus for a representative subject from the MDMA binge high dose group in experiment 2 (A) and experiment 3 (B)…………………………………. …………………....247

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LIST OF ABBREVIATIONS

5,7-DHT…….…………………………..………………………..5,7-dihydroxytryptamine 5-CSRTT…………………………………………………5 choice serial reaction time test 5-HT...... 5-hydroxytryptophan, serotonin ACC...... anterior amygdaloid area ac...... anterior commissure AC, d,v……..………………………anterior cingulate cortex, dorsal and ventral divisions ACC,c,s...... nucleus accumbens, core and shell divisions Ach……………………………………………………………………………acetlycholine AD...... anterodorsal nucleus of the thalamus ADP...... anterodorsal preoptic nucleus AGl….………………………………………………..…..lateral agranular (frontal) cortex AGm………………………………………………….…medial agranular (frontal) cortex AHN...... anterior nucleus of hypothalamus AI,d,p,v………………………agranular insular cortex, dorsal, posterior, ventral divisions AM……………………………………………………...anteromedial nucleus of thalamus ATD…………………………………………………………….acute tryptophan depletion ARH………………………………………………… arcuate nucleus of the hypothalamus AUD…………………………………………………………………….…..auditory cortex AV………………………………………………………anteroventral nucleus of thalamus AVP………………………………………………………...anteroventral preoptic nucleus BDNF…………………………………………...……….brain derived neurotrophic factor BLA………………………………………………………basolateral nucleus of amygdala BMA………………………………….………………….basomedial nucleus of amygdala BST, ad………………………...……bed nucleus of stria terminalis, anterodorsal division CA1,3………………………………………………field CA1 and CA3 of Ammon’s horn CANTAB……………………..Cambridge’s Neuropsychological Test Automated Battery CB…………………………………………………………………………cinguum bundle cc..…………………………………………………………………………corpus callosum ccg…………………………………………………………...genue of the corpus callosum CEA………………………………………………...... central nucleus of amygdala CL……………………………………………...…..central medial nucleus of the thalamus CLA………………………………………………………………………………claustrum CM……………………………………………………..central medial nucleus of thalamus CNS...... central nervous system COA…………………………………...... cortical nucleus of amygdala xix

DA……………………………………………………………………………..…dopamine CP………………………………………………………………………...caudate-putamen DG………………………………………………………….dentate gyrus of hippocampus DI……………………………………………………………….dysgranular insular cortex DLO……………………………………………….…………….dorsolateral orbital cortex DMH...... dorsomedial nucleus of hypothalamus DR…………………………………………………………………….dorsal raphe nucleus ECT……………………………………………………………………….ectorhinal cortex ENT,l,m...... entorhinal cortex, lateral and medial division EPd…………………..……………………………………………….endopiriform nucleus fa…………………………………………..………anterior forceps of the corpus callosum fr…………...………………………………………………………...fasciculus retroflexus FS...... fundus of the striatum GABA………………………………………………………………. γ-Aminobutyric acid GI...... granular insular cortex GLU………………………………………………………………………………glutamate GP...... globus pallidus HF...... hippocampal formation HPC…………………………………………………………………..………hippocampus IA………………………………………………...……intercalated nuclei of the amygdala IAD……………………………………………interoanterodorsal nucleus of the thalamus IED……………………………………………….. intradimensional extradimensional test IAM...... interanteromedial nucleus of thalamus IGL……………………………………………………………….….intergeniculate leaflet IL...... infralimbic cortex isl…………………………………………………………….………….. islands of Cajella IMD...... intermediodorsal necleus of thalamus LA...... lateral nucleus of amygdala LD...... lateral dorsal nucleus of thalamus LGN, d,v...... lateral geniculate nucleus, dorsal, ventral division LH...... lateral habenula LHy...... lateral hypothalamus LM…………………………………………………………...…lateral mammillay nucleus LO...... lateral orbital cortex LP...... lateral posterior nucleus of thalamus LPO...... lateral preoptic area LS, i, l, r...... lateral septum, intermediate, lateral, rostral division MA...... magnocellular preoptic nucleus MB...... mammillary bodies MD, c, l, m...... mediodorsal nucleus of thalamus, central, lateral, medial division MDA………………………………………….…………3,4-methylenedioxyamphetamine MDMA…………………………………………….3,4-methylenedioxymethamphetamine MEA...... medial nucleus of the amygdala MePO………………………………………………………..……median preoptic nucleus METH…………………………………………………………………...methamphetamine xx

MGN...... medial geniculate nucleus of thalamus MH……………………………………………………………………...…medial habenula MM………………………………………………………..…..medial mammillary nucleus MO...... medial orbital cortex MOp, MOs………………………………...……motor cortex primary, secondary division mPFC...... medial prefrontal cortex MPN...... medial preoptic nucleus MPO...... medial preoptic area MR...... median raphe nucleus MS...... medial septum mt...... mammillothalamic tract NDB...... nucleus of diagonal band OFC...... orbital frontal cortex OT...... olfactory tubercle PCA………………………………………………………...……para-chloroamphetamine PAA...... piriform amygdalar area PCN...... paracentral nucleus of thalamus PERI...... perirhinal cortex PF...... parafascicular nucleus of thalamus PFC...... prefrontal cortex PH...... posterior nucleus of hypothalamus PIR...... piriform cortex PL...... prelimbic cortex PM, d, v……………….…………………premammillary nucleus, dorsal, ventral division PO...... posterior nucleus of thalamus POST...... postsubiculum of HF PRE...... presubiculum of HF PTLp...... posterior parietal cortex PT...... parataenial, or paratenial nucleus of thalamus PV,a,p ...... paraventricular nucleus of thalamus, anterior and posterior divisions PVH…………………………………………paraventricular nucleus of the hypothalamus RE...... nucleus reuniens of thalamus RH...... rhomboid nucleus of thalamus RSP, agl, d, v...... retrosplenial cortex, agranular, dorsal, ventral division S-100β………………...…………………………………. S100 calcium binding protein β RT...... reticular nucleus of thalamus SCN...... suprachiasmatic nucleus SERT…………………………………………………..……..serotonin transporter protein SF...... septofimbrial nucleus SI...... substantia innominata slm………………………………………………..………..stratum lacunosum-moleculare slu……………………………………………………………………….... stratum lucidum sm...... stria medullaris SM...... submedial nucleus of thalamus so………………………………………………………………………...…..stratum oriens xxi sp……………………………………………………………………..…….pyramidal layer sr……………………………………………………...…………………..stratum radiatum SSRI…………………………………………………selective serotonin reuptake inhibitor SS, p,s...... somatosensory cortex, primary, secondary division st...... stria terminalis SUB,d,v...... subiculum, dorsal,ventral division SUM...... supramammillary nucleus TEv …………………………………………………….……...temporal association cortex TH…………………………………………………….…………….… tyrosine hyroxylase TMN…………………………………………….…….……….tuberomammillary nucleus TT,d,v ...... taenia tecta, dorsal, ventral divisions TUB……………………………………………………………………..….tuberal nucleus V3...... third ventricle VAL...... ventral anterior nucleus of thalamus VB...... ventral basal nucleus of thalamus VIS...... visual or occipital cortex VISC...... visceral cortex VL...... lateral ventricle VLO...... ventrolateral orbital cortex VM...... ventral medial nucleus of thalamus VMAT…………………………………………...………vesicular monoamine transporter VMH……………………………………...….ventral medial nucleus of the hypothalamus VO...... ventral orbital cortex ZI...... zona incerta

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GENERAL INTRODUCTION

3,4-methlenedioxymethamphetamine (MDMA) is the main constituent of the recreational drug Ecstasy, which is commonly abused in humans (Morgan, 2000; Parrott,

2001). MDMA is classified as a ring substituted amphetamine derivative, whose organic structure is similar to both methamphetamine and mescaline (Green et al, 2003; Lyles and

Cadet, 2003). Similarly, MDMA has a unique psychoactive profile, having both psychostimulant and hallucinogenic properties (Parrott, 2001; Green et al., 2003). In humans, Ecstasy is primarily consumed orally and the effects of the drug begin 20-30 minutes following ingestion. Effects can last up to five hours, with peak activity occurring around 90 minutes after ingestion (Green et al., 2003). The subjective effects of Ecstasy reported by human users include relaxation, euphoria, and a sense of emotional awareness and openness. In the 1980’s, MDMA arose as a street drug, and after a short time, the drug was explored as a therapeutic tool, used in conjunction with psychotherapy to enhance and assist individuals during a therapeutic session. However, due to its escalating abuse and growing evidence for its potential harmful effects, the

Drug Enforcement Agency (DEA) classified MDMA as a Schedule 1 drug in 1985

(Green et al., 2003; Morgan, 2000; Parrott, 2007; Pedersen and Skrondal, 1999).

1 Studies conducted in the 1980’s and 1990’s found that peripheral injections of

MDMA and other related amphetamine analogues, such as p-chloroamphetamine (PCA) produced long term depletion of serotonin stores and subsequently, serotonergic axonal degeneration in rats and primates. While acute effects were observed across all monoaminergic systems, only the serotonin system exhibited long-term deleterious effects (Mamounas and Molliver, 1988; Mamounas et al., 1991; O’Hearn et al., 1988;

Ricaurte et al., 1988a). Doses given to animals in these studies, however, were extremely high and do not necessarily reflect doses taken by recreational drug users (for review, see

Baumann et al, 2007). Ecstasy users often consume MDMA by ‘stacking’ pills.

Stacking refers to ingesting several pills every few hours across an occasion of use.

Thus, stacking or binge consumption may affect the serotonergic system in a manner similar to that observed in animals given high doses.

Studies on the possible neurotoxicity of Ecstasy in humans have produced conflicting results. Both neuropsychological and neuroimaging studies have found deficits in cognition, in the binding of the serotonin transporter protein (SERT), and changes in the expression of serotonin receptors (Buchert et al., 2007; Fox et al., 2001,

2002; Quednow et al., 2006, 2007; Reneman, et al., 2001, 2002; Thomasius et al., 2003,

2006). Ecstasy users across these studies varied extensively on polydrug use, drug histories, and abstinence periods. Additionally, Ecstasy pills are known to contain a number of additional active pharmacological agents including cocaine, methamphetamine, caffeine, ephedrine, and ketamine (Sang Leung and Cottler, 2008;

Vogels et al., 2009). Therefore, determining causation of any measurable deficits in human Ecstasy users remains inconclusive. Several studies, however, have positively

2 correlated both the dose and cumulative lifetime use of Ecstasy with the extent of cognitive deficits in recreational users (de Sola Llopis, 2008; Fox et al., 2001; Golding et al., 2007; Quednow et al., 2005, 2007).

Presently, a number of approved clinical studies conducted by the United States

Food and Drug Administration (FDA) are examining the therapeutic avenues for MDMA that may assist in the treatment of psychiatric and physiological disease. Investigations include the role of MDMA in the treatment of posttraumatic stress disorder (PTSD) and terminal cancer (Parrott, 2007). However, it is still unclear how neurotoxic MDMA and its active metabolites may be to the nervous system, and more over, how the drug may affect a premorbidly compromised central nervous systems (CNS). Concomitant polydrug use, psychiatric disorders, and the morphological and molecular effects associating with the disease of addiction may augment MDMA’s impact on the CNS. It is necessary to continue preclinical research on the neurotoxicity of the drug to determine long-term effects on cognitive and emotional processes.

In order to evaluate the effects of MDMA on the mammalian nervous system, it is first imperative to discuss the serotonergic system. The primary site of action of all amphetamines is the presynaptic monoaminergic transporter protein, however MDMA is selectively neurotoxic to serotonergic nerve terminals whereby amphetamine and methamphetamine are neurotoxic to dopamine, norepinephrine, and serotonin (Brown and Molliver, 2000; Cadet et al., 2007). In addition, the pharmacokinetic, physiological, behavioral, and neurotoxic properties of MDMA and related compounds will be outlined.

3 Anatomy of the serotonergic innervation of the forebrain

Serotonin (5-hydroxytryptamine; 5-HT) is a monoamine classified as an indoleamine transmitter substance found ubiquitously throughout the central nervous system, peripheral nervous system, and the enteric gastrointestinal system. Nine raphe nuclei, located within the pontine and medullary brainstem formation, are the source of the serotonergic innervation of the CNS. 5-HT neurons located in these nuclei, send ascending and descending axonal projections that outline specific 5-HT innervation patterns throughout the forebrain, brainstem, and periphery (Jacobs and Azmitia, 1992;

Lowry et al., 2008a; Vertes and Crane, 1999; Vertes and Linley, 2007, 2008). The dorsal raphe (DR) and median raphe (MR) are two midline pontine brainstem nuclei, which contain a heterogeneous cell population. DR and MR contain a large number of

5-HT neurons, whose fibers are responsible for the majority of 5-HT innervation of the forebrain. Each of these nuclei however differ in both their morphology and in the forebrain structures that they innervate, with minimal overlap between the two. Rather than exerting a defined role in one or few adaptive behaviors, serotonin is known to modulate a number of behaviors and vital functions including sleep, feeding, nociception, thermoregulation, locomotor activity, respiration, reward, mood, and cognition (Fields et al., 1991; Hensler, 2006; Jacobs and Azmitia, 1992; Jacobs and Fornal, 1999; Meneses,

1999; Robbins, 2005; Vertes and Linley, 2007, 2008; Cools et al., 2008; Monti and

Jantos, 2008; Sanford et al., 2008; Dayan and Huys, 2009).

The DR is a large nucleus located from the caudal mesencephalon extending through the pontine tegmentum. DR contains the greatest number of serotonergic cells in comparison to any other raphe nuclei. Several subdivisions within the DR nucleus

4 produce a topographical innervation of the forebrain, whereby certain subdivisions project to specific regions of the brain that are functionally homogeneous (Lowry et al.,

2008a). For example, the interfascicular DR has anatomical connections involved with structures involved in thermoregulation whilst the dorsomedial DR has interconnections involved in the modulation of mood and stress (Lowry et al., 2008b; Lowry et al., 2009).

The DR innervates several subcortical targets including: the dopaminergic substantia nigra pars compacta and the ventral tegmental area, the lateral and supramammillary nuclei of the hypothalamus, dorsal striatum (caudate putamen), ventral striatum (the nucleus accumbens), lateral septum, thalamic nuclei including the anteroventral and anteromedial nuclei, lateral thalamus, all midline thalamic nuclei, mediodorsal nucleus, and intralaminar nuclei of the thalamus, the bed nucleus of the stria terminalis, the basal and lateral nuclei of the amygdala, ventral pallidum, claustrum, and cholinergic basal forebrain nucleus the substantia innominata, the magnocellular and lateral preoptic area, and the lateral geniculate nucleus and intergeniculate leaflet. In addition, the DR provides the entire neocortex with serotonergic terminal fibers, including all sensory and sensory association cortices, as well as significant innervation of limbic cortices including the medial prefrontal cortex (mPFC), orbital frontal cortex

(OFC), the insular cortical divisions, and the entorhinal cortex. The DR nucleus also projects significantly to the hippocampus, though tracing studies have determined DR fibers are directed primarily at the dorsal hippocampus (Molliver, 1987; Morin and

Meyer-Bernstein, 1999; Peyron, et al., 1998; Vertes, 1991; Vertes and Linley, 2007,

2008).

5 The MR is located ventral to the DR, and contains a heterogenous cell population similar to DR, which includes a dense number of 5-HT containing cells, though smaller in number in comparison to DR. MR targets primarily subcortical forebrain structures that do not receive strong innervation from the DR. These include the medial zona incerta, medial mammillary bodies, supramammillary nucleus, posterior nucleus of the hypothalamus, ventral tegmental nucleus, midline and intralaminar thalamic nuclei, lateral habenula, suprachiasmatic nucleus, medial septum and nucleus of the diagonal band (horizontal and vertical divisions), and nucleus accumbens. While the MR projections to sensory cortical areas are sparse, MR does send dense projections to the entorhinal and perirhinal cortices, as well as lightly innervates the prefrontal cortex. In addition, the MR is the major source of serotonergic influence on the hippocampal formation, sending widespread dense projections to both dorsal and ventral hippocampal formation proper, including the CA3 and CA1 divisions of Ammon’s horn, the dentate gyrus, and subiculum (Molliver, 1987; Leranth and Vertes, 1999; Morin and Meyer-

Bernstein, 1999; Vertes et al., 1999; Vertes and Linley, 2007, 2008; Vertes and Martin,

1988;).

In cases, where DR and MR do innervate the same brain structures, they do so in a manner that is topographically complementary. For example, both the DR and MR provide serotonergic information to the septum. However their input remains segregated as DR projects solely to the intermediate lateral septum, where as MR projects only to the medial septum and lateral septum (Vertes and Linley, 2007, 2008). Additionally, both the DR and MR project to the nucleus accumbens however recent anatomical and morphological investigations have illustrated that while DR mainly innervates the core of

6 accumbens, MR directs its terminal fibers to the shell (Brown and Molliver, 2000; Vertes and Linley, 2008).

In addition to their relatively distinct projections to forebrain structures, DR and

MR also differ with respect to the morphology of their axon terminal endings. Axons originating from DR consist of thin, fine fibers with small varicosities that are heteromorphic in shape. MR fibers are thick with terminal buttons with a spherical beaded shape with large varicosities (Molliver, 1987; Molliver et al., 1990; Halladay et al., 2004). As discussed later, among the reported differences in the morphology of these two axons, DR and MR fibers are classically known to be differentially vulnerable to the neurotoxic effects of amphetamine analogues (Kosofsky and Molliver, 1987; Mamounas and Molliver, 1988; Molliver et al., 1990). This may be indicative of 5-HT neuronal differences in molecular or cellular constituents and processes in DR and MR as well.

These stark molecular, morphological, and anatomical differences between DR and MR suggest likely distinct functional contributions of each nucleus to behavior.

Serotonergic neurons in the ascending raphe nuclei are surrounded by a heterogenous cell population. The DR and MR also contain a large number of dopaminergic, glutamatergic, and GABAergic neurons and 5-HT neurons within these nuclei colocalize a number of neuropeptides and neurosteroids (Trulson et al., 1985;

Melander et al., 1986; Charara and Parent, 1998; Kozicz et al., 1998; Day et al., 2004;

Otake, 2005; Valentino and Commons, 2005; Waselus and Van Bockstaele, 2007; Lowry et al., 2008a). This diverse array of neural signaling associated with serotonergic activation may be responsible for its complex and indirect behavioral contributions.

7 Even more intricate is the nature of receptor proteins which serotonin acts upon.

The biochemical and electrophysiological properties of serotonergic activity and the affinity of 5-HT drug targets are dependent upon the localization and profiles of 5-HT receptors in the central nervous system (Mengod et al., 2006; Fink and Gothert, 2007).

There have been over 14 receptors identified within the central nervous system, each belonging to one of 6 families. Additionally some of these receptors have multiple biologically active isoforms which vary within the mammalian population (Mengod et al., 2006; Hannon and Hoyer, 2008).

Serotonergic modulation of emotion

Since the introduction of reserpine and monoamine oxidase inhibitors in the treatment of psychiatric conditions, serotonin has been known to play a well-versed role in mood and affect (Cools et al., 2008; Dayan and Huys, 2009; Lopez-Munoz and Alamo,

2009). Today, 5-HT has been implicated in a number neurological disorders, temperament, and emotional states including depression, bipolar disorder, schizophrenia, obsessive compulsive disorder, stress, anxiety, aggression, and suicide (Lesch and

Merschdorf, 2000; Bondy et al., 2006; Miklowitz and Johnson, 2006; Geyer and

Vollenweider, 2008; Leonard, 2006; Goddard et al., 2008; Popova, 2008; Remington,

2008; Carrillo et al., 2009). Both the serotonin transporter and several 5-HT receptor proteins are current drug targets to treat many of these psychiatric diseases (Schmidt et al., 1995; Thase, 1999; Cipriani et al., 2005; Reynolds et al., 2005). However, the molecular mechanism behind the efficacy of 5-HT drugs has not been thoroughly elucidated as it is likely that serotonin works in combination with a broad range of molecular, genetic, and other chemical factors to modulate emotionality and improve

8 neurological conditions (Hariri and Holmes, 2006; Schosser and Kasper, 2009). Carver et al. (2008) stated “there is an interaction among causal influences, leading some but not others to experience depression in the context of serotonergic deficits.” That is, the etiology of depression and other mood disorders is not solely serotonergic, but a constellation of genetic and neurochemical factors, in which serotonergic dysfunction can often correspond to affect changes.

Serotonin plays a role in depression. The most widespread and effective treatment for individuals suffering from monopolar depressive episodes is selective serotonin reuptake inhibitors (SSRIs), drugs that specifically bind to and block the presynaptic serotonin transporter, thus increasing serotonergic functioning. Hypoactivity of serotonin and other monoamines, and the dysreglation of their receptor proteins are central to the serotonin hypothesis of depression (Nemeroff and Owens, 2009). To understand the acute effects of serotonin on mood and affect, researchers use acute tryptophan depletion (ATD) to study behavior. Studies in humans show that tryptophan depletion has a negative impact on affect, however, increases in depressed mood following ATD are usually most apparent in patients with histories of antidepressant therapy and depressive episodes, but not healthy volunteers (Booji et al., 2002; Ruhe et al., 2007). In rodents, the home cage emergence test measures anxiogenesis and the forced swimming test is modeled to demonstrate “behavioral despair.” ATD rats did not show increases in these measures, thus acute 5-HT blunting does not impact overall affective behaviors in rodents (Lieben et al., 2004).

While the efficacy of antidepressants has been proven, their therapeutic actions are not felt by patients until several weeks into treatment. The etiology of depression is

9 still relatively unknown. It is likely that serotonin works within its receptor profiles, many of which are expressed polymorphically, to modulate several systems. Serotonin is known to act as a neurotrophic factor. It promotes brain derived neurotrophic factor

(BDNF) and s100 calcium binding protein β (s100β), both of which are known to facilitate neuronal growth. (Eriksen et al., 2002; Cowen, 2007; Martinowich and Lu,

2008). Enhanced serotonergic function increases hippocampal neurogenesis while lesions to the serotonin system produce decreases in cell proliferation in the hippocampus

(Brezun and Daszuta, 1999, 2000a, 2000b; Banasr et al., 2004; Benninhoff et al., 2010).

Studies have shown chronic antidepressant treatment promotes neurotrophic substances, increases axonal and dendritic sprouting, and increases hippocampal neurogenesis

(Russo-Neustadt et al., 2004; Russo-Neustadt and Chen, 2005; Bachis et al., 2008; Wang et al., 2008). It has been hypothesized that the efficacious actions of antidepressant therapy may be the result of increases in neuronal growth and dendritic sprouting in key limbic areas, rather than a direct increase in serotonin levels in the CNS (Castren, 2004;

Homberg et al., 2010; Tanti and Belzung, 2010).

There is an inverse relationship between 5-HT’s role in depression and in stress and anxiety. 5-HT is released during stressful situations and anxiety, in return may be related to an excess release of serotonin. Serotonin is involved in the release of the stress hormone corticotropin releasing factor, the modulation of adrenocorticotropic hormone

(ACTH), and other neurohormones of the HPA axis (Leonard, 2005; Jorgensen, 2007).

In addition to SSRIs and benzodiazepines, patients with anxiety related disorders may be

prescribed 5-HT1A (autoreceptor) agonists to blunt serotonergic activity at the raphe nuclei. Chronic stress reduces hippocampal neurogenesis and impairs both long-term 10 potentiation and long term depression, thus serotoninergic treatments may combat these impairments (Joels et al., 2004; Buwalda et al., 2005).

Serotonin’s role in executive and mnemonic processes

Research conducted over the last decade has implicated serotonin in the modulation of attentional and cognitive processes known to be reliant on the prefrontal cortex and associated structures. Executive functioning is a term used for numerous higher order behaviors including attention, decision-making and goal selection, behavioral flexibility, behavioral inhibition, impulsivity, and temporal organization. All of these assist in the ability to select and respond to appropriate stimuli that guide behavior (Dalley et al., 2004). The prefrontal cortex (PFC), including the ventromedial, orbital, and insular divisions, is part of a neuroanatomical loop including the dorsal and ventral striatum, ventral pallidum, and thalamus, which is involved in the processing of attentional, motivational, and cognitive representations within the CNS (Chudasama and

Robbins, 2006; Boulougouris and Tsaltas, 2008). Additionally, this prefrontal cognition is organized in both a neurochemically and regionally specific manner in which discrete behavioral functions are regulated via specific neurotransmitter acting on local subregions within the PFC (Robbins, 2005; Chudasama and Robbins, 2006; Chamberlain et al., 2006; Robbins and Roberts, 2007; Robbins and Arnsten, 2009).

Serotonergic innervation of the prefrontal cortex originates primarily from the 5-

HT neurons in the DR, and as such, executive functioning is dependent upon the DR’s inputs to PFC and reciprocal top down control of DR from PFC (Peyron et al., 1998;

Vertes, 1991; Hoover and Vertes, 2007; Vertes and Linley, 2007, 2008; Goncalves et al.,

2009). Proper homeostatic levels of 5-HT are crucial in cognitive processes such as

11 decision-making, behavioral flexibility, behavioral inhibition and impulsivity (Clark et al., 2004; Chudasama and Robbins, 2006; Robbins and Roberts, 2007). Each of these behaviors is dependent upon 5-HT input to specific cortical subregions of the prefrontal cortex. For example, 5-HT input to the medial prefrontal cortex (mPFC) modulates decision making and impulsivity, while the orbitofrontal cortex is involved in behavioral flexibility (Chudasama and Robbins, 2006; Clark et al., 2004; Robbins and Roberts,

2007).

Attention is the ability to attend to pertinent information and disregard irrelevant internal and external stimuli in order to make an appropriate selection and guide behavior

(Boulougouris and Tsaltas, 2008). Impulsivity is defined as an inability to inhibit one’s behavior in order to select an appropriate choice. It can be measured by premature motor responses, or selection of a stimulus without properly assessing all possible outcomes

(Dalley et al., 2008). Decision-making is associated with the latter definition of impulsivity, and is associated with the ability to guide one’s behavior to select an appropriate choice out of several outcomes to provide the best response. Morgan et al.

(2005) remarked “Effective decisions involving uncertain benefits and penalties require decision-makers to integrate different reinforcemental cues including the size of possible gains (rewards), the size of possible losses (punishments), and the probability of these outcomes.” Deficits in decision-making can be revealed by an animal’s selection of an operant choice with disregard to possible outcomes, including punishment associated with that choice. This is seen through impairments in behavioral inhibition in paradigms that require an animal to wait during a delay for a more profitable reward. Rather, impaired animals select the immediate (and less salient) reward (Clark et al., 2004).

12 Decision-making and impulsivity in the rat can be measured by using behavioral paradigms such as the 5 choice serial reaction time test (5-CSRTT) and delayed discounting tasks. In the 5-CSRTT, rats are placed in an arena which has five ports.

Through training, rats learn to correspond a reward with a location in space indexed by the presentation of a light stimulus. The presentation of a light above one port is followed by a delay, after which a rat can select the appropriate port using a nose poke to obtain a reward. This paradigm is utilized to assess visuospatial attention (accurate responses), impaired vigilance and motivation (omitted responses), and impulsivity

(premature responses) following neuropharmacological manipulations (Robbins, 2002;

Bari et al, 2008). In delay discounting tasks, rats are exposed to paired reward delay contingencies, which vary by size of reward and length of delay. Delay discounting refers to a decrease in the salience of a reward in comparison to its time of delivery. Rats that choose smaller more immediate rewards, in lieu of a larger reward associated with a longer delay, are thought to have impairments in decision-making and thus make impulsive choices (Winstanley et al., 2004; Mar and Robbins, 2007)

Intracerebroventricular or local injections of 5,7-dihydroxytryptamine (5,7-DHT), a serotonergic neurotoxin, infused directly into mPFC increases impulsive motor choices in the 5-CSRTT as well as impulsive selections in delay discounting tasks. Additionally,

5-HT is increased in mPFC during performance of these tasks (Clark et al., 2004; Dalley et al., 2004, 2008; Winstanley et al., 2006; Chudasama and Robbins, 2006; Robbins and

Roberts, 2007; Robbins, 2005; Cools et al., 2008). Van der Plasse et al. (2007) found that serotonergic denervation of the medial PFC impaired decision-making in relationship to the salience of an appetitive reward.

13 As mentioned above, serotonergic input into the orbital frontal cortex (OFC) is crucial to behavioral flexibility and reversal learning. 5-HT lesions of the OFC in primates result in perseveration in choice response (Clarke et al., 2004, 2005, 2007).

Perseveration is the inability to ignore previously rewarded stimuli to stop responding to an incorrect choice and failure to learn and select a new rewarded choice. Reversal learning can be tested in a number of behavioral paradigms, most notably the Wisconsin

Card Sorting Task and its automated version, Cambridge’s Neuropsychological

Automated Test Battery (CANTAB) language independent Intradimensional

Extradimensional (IED) test. In these primate versions of the task, visual percepts are used as discriminative stimuli. In the rodent analogue, odors and digging media are used.

Subjects must learn first to create an attentional set, selecting the correct perceptual domain (an odor or tactile exemplar), which are presented together. Correct choice is food-rewarded, based on a criteria of perceptual rules, and rats who form an attentional set learn to ignore the irrelevant percept. Behavioral flexibility is assessed when subjects are required to respond to the previously incorrect choice and to ignore the last rewarded choice. Lastly, attentional set shifting is assessed when subjects must shift their attention to new rules regarding the percepts and their exemplars, and disregard old rules established to guide choice selection (Fray and Robbins, 1996; Birrell and Brown, 2000;

Brown and Bowman, 2002; McAlonan and Brown, 2003).

Serotonergic innervation of the orbital frontal cortex (OFC) in primates is crucial to cognitive flexibility. Neurotoxic depletions of 5-HT in the OFC impaired reversal learning in the IED task, but left other attentional measures unimpaired. These lesioned primates perseverated in responding to the choices that were originally correct; but were

14 no longer rewarded. However, decreases in dopamine and norepinephrine in the OFC leave reversal learning intact. Catecholaminergic innervation of other subregions of the prefrontal cortex play a role in the attentional tasks in the IED. Lastly, acute tryptophan depletion in healthy volunteers produces impairments in reversal learning in the IED task

(as reviewed in Clark et al., 2004). In rodents, excitotoxic lesions of the OFC produce impairments in reversal learning (McAlonan and Brown, 2003). Antagonism of the 5-

HT2A, 5-HT2C, 5-HT6, 5-HT7 and agonism of 5-HT1A receptors are all involved in reversal learning in rodent paradigms (Hatcher et al., 2005; Boulougouris et al., 2008;

Boulougouris and Robbins, 2010; McLean et al., 2009). These studies draw clear distinction of the functional activity of 5-HT across subdivisions of the PFC.

Homeostatic levels of serotonin contribute to working memory and long-term memory formation (Cassaday et al., 2003; Fernandez-Perez et al., 2005; Hritcu et al.,

2007). However, 5-HT’s role in these memory processes is complex, and involves both postsynaptic receptor effects and interactions with other neurotransmitter systems, including a modulatory interplay between serotonin and acetylcholine (Cassel and

Jeltsch, 1995; Steckler and Sahgal, 1995). The activation of 5-HT1A receptors stimulates the release of acetlycholine, and may share an inverse relationship to mnemonic performance (Lehmann et al., 2002a, 2002b; Filip and Bader, 2009).

In humans, the direct involvement of 5-HT in memory is primarily assessed through tryptophan depletion and enhancement. These studies report no differences in verbal or spatial working memory following a tryptophan depletion challenge in healthy volunteers (Riedel et al., 1999; Luciana et al., 2001; Harrison et al., 2004). Most acute tryptophan depletion (ATD) studies in humans fail to find a strong relationship between

15 depletion of 5-HT as measured by tryptophan plasma levels and impairments in learning and memory. While two studies showed impairments in learning using a visual verbal learning task in healthy ATD controls (Schmitt et al., 2000; Scholtissen et al., 2006); most studies using healthy volunteers have yielded insignificant results using paired associates tasks, pattern recognition tests, and spatial memory tasks (Park et al., 1994;

Luciana et al., 2001; Rubinsztein et al., 2001; Hughes et al., 2003; Roiser et al., 2007).

Therefore, serotonergic challenge to already hindered transmitter systems may augment deficits associated with reduced 5-HT function.

In rodents, long-term hippocampal dependent memory has been assessed using a number of spatial and nonspatial paradigms. The ability to orient and navigate oneself through an environment is reliant upon spatial memory. It can be assessed using appetitive or aversive maze tasks whereby animals are trained to navigate and find a target location based on internal vestibular and external allothetic environmental cues. In rats, serotonergic lesions alone do not produce large impairments in spatial acquisition or memory, however when serotonergic transmission is disrupted concomitantly with other transmitter systems, or concurrent stress is applied, spatial learning can be significantly affected (Normile et al., 1990; Lehmann et al., 2002a; Dyer and Cain, 2007; Kenton et al., 2008; Zhou et al., 2008).

Pharmacokinetics and acute effects of MDMA and its metabolites in rats

MDMA, its active metabolite, 3,4-methylenedioxyamphetamine (MDA), and other amphetamine analogues including p-chloroamphetamine (PCA) and fenfluramine exert their primary action by facilitating monoaminergic release through interactions with presynaptic transporter proteins (Berger et al., 1992; Lyles and Cadet, 1993; Green et al.,

16 2003). The methylendioxy ring in MDMA is responsible for the drug’s greater affinity for, and potency of, serotonin (5-HT) release as opposed to other monoamines, such as dopamine and norepinephrine (Bauman et al., 2007). As measured by microdialysis, acute MDMA administration produces a strong rapid increase in extracellular serotonin in forebrain regions including the medial prefrontal cortex, hippocampus, and dorsal and ventral striatum (Green et al., 2003; Lyles and Cadet, 2003; Simantov 2004).

The mechanism of action for 5-HT release is through non-exocytotic mechanisms at the presynaptic transporter protein. MDMA binds to and is taken up internally into the terminal, where it produces conformational changes of the serotonin presynaptic receptor

(SERT) protein and causes calcium independent 5-HT release via reversal transport of the vesicular monoamine transporter 2 (VMAT) and SERT (Lyles and Cadet, 2003).

MDMA, MDA, and PCA induced 5-HT release can be attenuated with pretreatment of fluoxetine or other selective serotonin reuptake inhibitors (SSRIs) (Berger et al., 1992;

Green et al., 2003; Lyles and Cadet, 2003; Sanchez et al., 2001).

MDMA also inhibits the activity of tryptophan hydroxylase (TPH), the rate- limiting enzyme in the synthesis of 5-HT. Thus, MDMA administration inhibits tryptophan hydroxylase, depletes central 5-HT stores, and reduces the concentration of 5-

HT for days to several weeks (Green et al., 2003). High doses of MDMA produce a reduction of TPH2 immunoreactivity, which correlated to a loss of SERT expressing fibers (Bonkale and Austin, 2008). MDMA also acutely inhibits the synaptosomal and vesicular uptake of 5-HT (Bogen et al., 2003). Enhancement of serotonergic activity following PCA and MDMA administration also occurs through the binding of MDMA to

17 several serotonergic receptors including the 5-HT1 and 5-HT2 receptor families (Battaglia and De Souza, 1989, Green et al., 2003; Sarkar and Schmued, 2010).

MDMA produces a significant increase in extracellular dopamine (DA) concentration in forebrain regions including the striatum and nucleus accumbens (Green et al., 2003; Lyles and Cadet, 2003; Gudelsky and Yamamoto, 2008). DA release, however, may not be directly mediated through the presynaptic transporter protein.

MDMA most likely enters the presynaptic terminal via diffusion rather than reversal transport. Increases in the extracellular [DA] following MDMA administration are

associated with both 5-HT release and 5-HT2 receptor activation. This has been demonstrated by a reduction in extracellular [DA] following pretreatment with fluoxetine

or ritanserin, a 5-HT2A/2C antagonist, prior to MDMA administration. MDMA impairs the vesicular uptake of DA (Bogen et al., 2003). There is an acute reduction in DA availability hours following administration of MDMA, but this fully recovers in rats and primates, illustrating that MDMA does not produce long-term depletion of DA stores.

Additionally, there is no long-term damage to DA axon fibers, as witnessed by in vivo, western blot, and immunohistochemical studies. For dopamine and the dopamine transporter. Mice are unique among mammalian species in that they display a selective dopaminergic neurotoxicity in response to MDMA (Armstrong and Noguchi, 2004;

Colado et al., 2004; Green et al., 2003; Gudelsky and Yamamoto, 2008). In summary,

MDMA does not exert long-term effects on DA system.

MDMA also exerts acute actions on other transmitter systems as well. This includes a facilitatory effect on norepinephrine and histamine. However, similar to DA, there is no long term depletion or other detrimental actions on these ligand sites (Lyles

18 and Cadet, 2003; Green et al., 2003). Studies have also noted that MDMA produces an increase in acetylcholine in the prefrontal cortex, striatum, and hippocampus through interactions with muscarinic 1 and 2 receptors as well as indirect mechanisms through the

activation of H1, D1, and 5-HT2 receptors (Nair and Gudelsky, 2006a, 2006b; Fischer et al. 2000; Gudelsky and Yamamoto, 2008).

Behavioral effects of MDMA and its metabolites in rats

Administration of MDMA produces massive 5-HT release within the CNS, which results in hyperactivity and hyperlocomotion. In addition, there is a dose-dependent relationship between MDMA and the severity of symptoms associated with serotonin toxicity or the ‘serotonin syndrome.’ High doses of MDMA result in behavioral symptoms characteristic of serotonin toxicity. Serotonin syndrome occurs from a large toxic increase in extracellular [5-HT], first identified as a result of interactions of 5-HT drugs including monoamine oxidase inhibitors (MAOI). In rats, behavioral symptoms of serotonin syndrome include: hyperthermia, piloerection, increased salivation, pupil dilation, head weaving, forepaw treading, hind limb splaying, ejaculation, increased defecation, and straub tail (Gillman, 2005; Green et al., 2003; Isbister et al., 2007;

Kalueff et al., 2008). 5-HT selective amphetamine derivatives also produce tachycardia,

which is thought to arise from interactions with both peripheral 5-HT2 receptors as well as sympathetic noradrenergic stimulation (Baumann et al., 2007). MDMA induced 5-HT toxicity may generate potentially lethal hyperthermia. If extreme hyperthermia is experienced, rats can become ataxic, catalytic, experience convulsions and may even die

(Green et al, 2003; Lyles and Cadet, 2003; Bauman et al., 2007).

19 The subjective effects of the Ecstasy include overall enhanced mood, euphoria, a higher sensitivity or intimacy with others, and an enhanced awareness of one’s owns emotions. This, however, is coupled by a number of negative physiological symptoms such as tachycardia, bruxism, muscle ataxia, and impaired cognition. Only in rare cases, is serotonin toxicity witnessed in human abusers, and manifests in hyperthermia, which may cause organ failure, cardiac arthymias, and death (Lyles and Cadet, 2003).

Effects of MDMA on cognition and emotion

The primary aim of studying the emotional, attentional and mnemonic processes after MDMA administration is to assess the detrimental effects of the drug. Ecstasy users are not always aware of the potential dangers, as the drug is abused in club settings, and not taken habitually, thus drug tolerance and physical dependence is not normally noted as in other drug addictions. Early studies conducted in the 80’s and 90’s, which emphasized the dangerous and persistent neurotoxic effects of the drugs were not circumspect of the interspecies dose scaling of the drug. More recent studies reflecting on the behavioral and physiological effects, and pharmacokinetics of the MDMA indicate that earlier reports did not use comparable doses that reflected human use (Baumann et al., 2007).

The effects of Ecstasy on emotional states, cognition, and memory vary dramatically from study to study, often resulting in conflicting findings. These include: deficits, no deficits in Ecstasy users; no changes in neuronal markers in imaging studies; or abnormalities in imaging studies using 5-HT and 5-HT receptors as markers. Changes in brain activity noted by functional imaging studies have not always been positively correlated with changes in behavior (Gouzoulis-Mayfrank et al., 2000; Reneman et al.,

20 2001, 2002; Fox et al., 2002; Cowan et al., 2003; Thomasius et al., 2003, 2006; Daumann et al., 2005; Quednow et al., 2006, 2007; Buchert et al., 2007; Jager et al., 2008).

It is important to consider the following confounds whenever reviewing the literature on MDMA in human use. First, Ecstasy use is typically associated with previous exposure to and concomitant use of other drugs including, nicotine, caffeine, alcohol, delta-9-tetrahydrocannabinol (THC), cocaine, amphetamine, methamphetamine, lysergic acid (LSD), psilocybin mushrooms, and other drugs. It is well known that some of these substances produce distinct changes in mood and memory (Marshall et al., 2007;

Scott et al., 2007; Solouij and Battisti, 2008; Pattij et al., 2008; Heifets and Castillo,

2009). In polydrug users, cognitive deficits may be attributed to other substances or be a result of the synergistic effects of MDMA and other drugs. Second, exposure to both

Ecstasy and other drugs is varied across each participant in any given experiment. Not only does lifetime use of the drug (cumulative dose) fluctuate among each subject, but the length of abstinence or last use of both Ecstasy and other drugs differs across individuals. One individual may be tested two months from their last use, another two weeks prior to their last use, and another two days. The frequency of use and pattern of consumption is also a critical factor and is widely uncontrolled. For example, lifetime use of one individual may be 100 pills over four years, while another may be 100 Ecstasy tablets over 4 months, showing an increase in the frequency and number of pills consumed over time. One individual may consume only 2 pills on one occasion, while another may participate in binge consumption or ‘stacking’ pills, taking several every few hours in an occasion. Users are usually lumped into groups based on loose similarities of their drug histories, which are not always equivalent and add to variance.

21 Third, there is a strong association between drug abuse and the comorbidity of other psychiatric conditions including depression, bipolar disorder, schizophrenia, and other psychoses, and the presence of such illnesses may not be overtly apparent, even with the effort to prescreen and exclude those with DSM criteria. A link between Ecstasy polydrug use and psychopathology has also been identified in users (Morgan et al., 2002).

In this instance of whether the chicken or the egg came first, not only are subjects likely to be exposed to pharmacological agents used to treat these conditions, but the diseases themselves are likely to impact both behavior and cognition. Lastly, several genetic polymorphisms exist for serotonin transporter, the primary target of MDMA. Recent studies have concluded that different alleles of SERT may increase vulnerability to psychiatric illnesses. Furthermore, variable neurotoxic effects of Ecstasy in sample populations may be associated by different SERT allelic variants (Roiser et al., 2005,

2006a, 2006b, 2007; Uher and McGuttin, 2009). Additionally, there are genetic polymorphisms of liver enzymes which may be associated with differences in toxicity of

Ecstasy (Schiffano, 2004). This suggests that across any given sample population in a study, different individuals may tolerate Ecstasy differently. It is likely that each of these factors contributes to the plethora of discrepancies within the literature on Ecstasy’s neuropsychological effects.

MDMA’s effects on mood and affect also fluctuate across studies. Some reports have not found a direct correlation between Ecstasy use and changes in mood or anxiety

(Morgan, 1998; Dafters et al., 1999; Murphy et al., 2006; de Win et al., 2006). Others report long-term changes in depression, anxiety, and aggression, in current and former

Ecstasy users (Verheyden et al., 2003; Thomasius et al., 2003; Lamers et al., 2006).

22 However, none of these studies excluded use of other psychoactive drugs. For instance, the presence of emotional disturbances in Ectsasy users has been controlled when polydrug use or prior lifetime exposure is taken into account (Daumann et al., 2001,

2004; Morgan et al., 2002; Roiser and Sahakian, 2004). Studies have found THC to be a better predictor of anxiety, depression, and psychopathology in polydrug Ecstasy users than Ecstasy itself (Medina and Shear, 2007; Morgan et al., 2002). While this study grouped individuals by lifetime use, no measurements were used to examine the length of abstinence of illicit drugs in all groups.

The persistence of psychological problems in Ecstasy users was addressed in a longitudinal study by Thomasius et al. (2006) who followed both current and abstinent polydrug Ecstasy users over 1 ½ years to determine if problems persisted, worsened, or were alleviated over time. They found no large differences in psychopathology in current or previous Ecstasy polydrug users in comparison to controls over time, however those with severe psychological deficits in initial studies did not return for follow up visits

(Thomasius et al., 2003). Therefore, another caveat may be in keeping track of sample populations most vulnerable to the deleterious effects of the drug. Acute detrimental changes of increased depressed mood were also noted within a few days following

Ecstasy use, perhaps reflecting withdrawal from the drug (Parrott and Lasky, 1998;

Curran and Travill, 1997). Fisk et al. (2009) found Ecstasy users to self-report increased depression, anxiety, increased irritability, sleep deprivation and other sleep impairments.

Interestingly, this study found a positive association between the presence of psychological deficits and impairments in prospective and self reported memory function.

23 In rats, MDMA has an acute and persistent effect on anxiety like behavior and emotional states (Green et al., 2003). Morley and McGregor (2000) found low to moderate doses of MDMA increased anxiety and social interaction. In addition, a follow up study that tested anxiety 3 months post MDMA exposure found persistent increased anxiety, and decreases in social interaction (Morley et al., 2001). The differences between acute and long-term effects of the drug may be associated with hyperlocomotion produced by the physiological effects of MDMA. MDMA (10 mg/kg) produced acute anxiogenic behavior in the elevated plus maze, which dissipated two weeks later, despite significant reductions in [5-HT] in the hippocampus (Sumnall et al., 2004). However, a larger binge dosing of a total of 30 mg/kg MDMA in one day did produce deficits in the elevated plus maze ten days following treatment in periadolescent rats, which coincided with decreased 5-HT amygdalar concentration using HPLC methods. (Faria et al., 2006).

This may indicate a developmental association between anxiety like behaviors and

MDMA.

Green and McGregor (2002) found different strains of rat performed differently on an elevated plus maze following MDMA treatment. Wistar rats displayed long term increased anxiety in response to a high MDMA dose, however Dark Agouti rats showed only an initial anxiogenic response. Scoring performance on the elevated plus maze using a mean rank order was used as a behavioral phenotype to categorize rats into high anxiety and low anxiety groups. Low and high anxiety Wistar rats received 5 consecutive treatments of MDMA 5 mg/kg or saline, followed by a 5 mg/kg MDMA drug challenge. The chronically treated MDMA rats showed increases in time spent in open arms regardless of group assignments, while a treatment group interaction was

24 found in saline rats given a drug challenge (Ludwig et al. 2008). This experiment emphasizes the need for a categorization in both phenotypes and genotypes when assessing the effects of MDMA on behavior.

PCA administration produced anxiogenesis as measured using the open field test and social interaction test (Harro et al., 2001; Kanarik et al., 2008; Tonissaar et al., 2008).

Both MDMA and PCA produced increases in immobility in the forced swimming test, the paradigm sensitive to antidepressant properties of psychotropic drugs in animal models. As such, serotonergic lesioning with these drugs may produce changes in the affective states (Harro et al., 2001; McGregor et al., 2003; Haidkind et al., 2004; Tonisaar et al., 2008). Chronic fluoxetine treatment administered concomitantly with MDMA treatment reduced anxiogenesis and immobility in the forced swimming test (Thompson et al., 2004). In summary, while there are conflicting data regarding the possible long- term effects of MDMA and related compounds, evidence suggests that there is an acute anxiogenic response to the drug.

Similarly, the effects of MDMA and related compounds on measures of cognition and memory have produced a large body of conflicting data, which are largely dependent upon dose, task, and memory system evaluated in rats. Several studies have found very high doses of MDMA are associated with deficits in spatial learning and memory.

Sprague et al. (2003) found a total amount of 40mg/kg MDMA marginally impaired spatial memory retention in the Morris water maze (MWM), yet had no effect on spatial learning. MDMA treated animals exhibited a decreased preference for searching in the target quadrant during a probe test. This was correlated with a significant decrease in [5-

25 HT] in the hippocampus measured by high-pressure liquid chromatography (HPLC) methods.

Similarly, deficits in spatial memory retention and path integration have been noted following high binge doses of MDMA (Able et al., 2005; Skelton et al. 2008).

High doses of MDMA (15 mg/kg x 4 injections every two hours for a single day) in

Sprague Dawley rats did not effect spatial learning acquisition or reversal learning, however deficits in the retention of place learning were found. MDMA treated rats also illustrated deficits in path integration in the Cincinnati water maze (Able et al., 2005). In a follow up study, Skelton et al. (2008) found both multiple (several weeks of 4 x 15 mg/kg) and single binge dose regimens produced similar deficits in path integration however there were no deficits in spatial memory retention. When the platform size was decreased to 75% however, there were increases in swim paths to target location.

Deficits in retention of place learning during a probe trial have been observed following

4 x 7.5 mg/kg MDMA (Cunningham et al., 2009). While the lasting effects of the drug on place learning have been contradictory, MDMA given immediately before MWM training sessions increased latencies and produced recall deficits (Arias-Cavieres et al.,

2010; Camarasa et al., 2008).

Kay et al. (2009) assessed the acute effects of variable low and moderate doses of

MDMA and scopolamine on spatial reference memory and working memory using a delayed match to sample radial 8 arm maze. This study found that MDMA in moderate doses (3-4 mg/kg) impaired accuracy on trials, with a trend to commit more reference memory errors than working memory errors. Latencies to complete each trial were also affected, with lower doses producing a decrease in the amount of time to select an arm

26 and increased latencies following the larger more moderate doses, which indicate locomotor or motivational functioning may be modulated during this appetitive task (Kay et al. 2009).

The effects of MDMA and related compounds on other forms of nonspatial hippocampal dependent learning are even less consistent. Novel object recognition is a task thought to be reliant upon the hippocampus and other temporal structures. Rats show a natural bias to explore novel objects. During a sample session, two identical objects are placed in opposite corners of a square arena. Following a delay, during the test session, a novel object is introduced along with the presentation of the familiar object. Rats who “remember” the familiar object, will show a preference in exploration for the novel object. Those who do not will perform at chance, exploring both objects equally. Impairments in novel object recognition have been noted 3 months following high binge dose regimen of 8 x 5 mg/g MDMA in adult and periadolescent rats

((McGregor et al. 2003; Piper and Meyer 2004).

Most studies, however, have failed to find any observable impairment in object recognition. Methamphetamine which degrades both dopaminergic and serotonergic terminals in the forebrain significantly impairs performance on object recognition (OR) however PCA, selectively neurotoxic to 5-HT, did not (Belcher et al., 2005; Marshall et. al., 2005). Similarly, multiple injections of 15 mg/kg MDMA also had no effect on novel object recognition in rats (Able et al., 2005; Skelton et al., 2008). Differences may be attributed to variations in the protocol and delays used within each experiment, and period of testing. MDMA given prior to the sample session decreased attentional set to the objects during the sample session (Piper and Meyer, 2005).

27 Aversive avoidance learning is also a paradigm used to measure memory. Doses of 20 mg/kg MDMA and high doses of 3,4-methylenedioxyethamphetamine (MDEA) given during a training session acutely impaired avoidance during the test session in a passive avoidance task. No deficits persisted when tested a week later (Barrioneuvo et al., 1999). Galindo et al., (2008) found that 10mg/kg PCA significantly impaired acquisition and recall of an aversive stimulus in a classic one-way active avoidance task.

However, if the strength of the aversive stimuli (millivolts, mV) was increased to a more noxious level, these PCA treated rats learned the association. These results suggest high doses of amphetamine analogues may disrupt a number of distinct hippocampal dependent learning processes, however leave other nonspatial forms of learning unscathed.

As mentioned above, serotonin plays a prominent role in a number of frontal cognitive measures including behavioral inhibition, reversal learning, and decision making, but each of these measures are modulated by different cortical areas as well as influenced by additional transmitter systems (Clark et al., 2004; Robbins, 2005;

Chudasama and Robbins, 2006; Robbins and Roberts, 2007). As such, systemic injections of MDMA and analogues, which produce widespread damage to serotonergic fibers, should negatively impede each of these functions. Interestingly, few studies have examined the effects of amphetamine analogues which target the serotonin transporter on prefrontal processes. Masaki et al. (2006) gave rats neurotoxic doses of PCA and tested them on a visual discrimination go/no go and reversal task. Deficits in both the acquisition and reversal versions of the task were exhibited.

28 Byrne et al. (2000) tested rats on acquisition of delayed reinforcement using a fixed ratio lever press task with rats given variable low and moderate doses of MDMA immediately preceding the test. MDMA increased the number of omitted responses, which reflected impairment in learning, but also may have represented a motivational deficit. In a second experiment using the same task, the long-term effects of high doses

(2 x 20mg/mg) were assessed and ¼ of the MDMA treated rats could not successfully acquire the delayed reinforcement task (Byrne et al. 2000).

Rats that chronically self-administered MDMA showed increased impulsivity on the 5-CSRRT immediately following cessation of the drug. However, during later post withdrawal test sessions, rats showed a decrease in impulsive responding and exhibited impairments in attention and vigilance (Dalley et al., 2007). This is consistent with the studies that assessed impulsivity using a visual discrimination delayed match to sample lever press task; either immediately or months after a total of 15 mg/kg MDMA. While there were initial impairments in selection immediately following MDMA treatment, over time MDMA rats regained performance similar to controls (Saadat et al., 2006).

Thus while local manipulations of [5-HT] in the medial prefrontal cortex may result in discrete manipulations of executive functioning in animal models, chronic administration of MDMA over time may result in more extensive changes in neurochemistry which may affect overall affective or motivational states.

Harper et al. (2005, 2006) found evidence that even low doses of MDMA (2-

3mg/kg) subacutely produced deficits in accuracy of choice using a delayed match to sample paradigm, but these deficits were delay independent (Harper et al., 2005). The overall impairment in accuracy of choice was the result of proactive interference of trials

29 across a testing session, and in fact, introducing an intertrial interval delay in between each trial improved performance. MDMA treated rats displayed more severe impairments in reference memory, than working memory, on a radial arm amze task, suggesting that this system may remain intact, as least while the animal is under the influence of MDMA (Kay et al., 2009).

To model human consumption of MDMA, it is imperative to examine the potential concomitant effects of MDMA with other psychoactive compounds. For example, most human studies examining the effects of Ecstasy on cognition emphasize the caveat that Ecstasy users abuse a number of psychoactive substances, including marijuana, cocaine, amphetamine, methamphetamine, nicotine, alcohol and various hallucinogens (Parrott, 2002). Each of these substances has been reported to exert changes in cognition and memory. Studies have found endocannabinoids and their agonists modulate synaptic plasticity and may impair executive functioning, learning, and memory recall (Ranganathan and D’Souza, 2006; Zhu, 2006; Pattij et al., 2008).

Young et al. (2005) examined the effects of concomitant administration of

MDMA and THC on learning in a double Y maze task, which tests both reference memory via spatial discrimination and working memory using a delayed spatial alternation paradigm. Low and moderate doses of MDMA alone, did not impair performance on either task. Low, moderate, and high doses of THC did produce deficits in spatial working memory. Additive deficits in spatial working memory, more severe than those seen with THC treatment alone, were witnessed in rats who were administered

MDMA and THC concomitantly (Young et al., 2005). This suggests, that much like human consumption, polydrug use may produce additive and deleterious effects on

30 cognition, however MDMA ingestion did not. However, the results of these studies illustrate the acute effects of combined use of THC and MDMA on mnemonic functions, but no conclusions can be drawn from possible long-term effects following chronic simultaneous use of the substances.

McGregor and colleagues (see Clemens et al., 2007a for review) have conducted a series of experiments looking at the independent and concomitant effects of MDMA and methamphetamine (METH). METH is highly neurotoxic, producing destruction of fiber terminals originating from dopamine, norepinephrine and serotonin neurons (Brunswick et al., 1991). Clear additive effects of the drugs on hyperthermia and affective behaviors including social interaction and anxiety were noted (Clemens et al., 2004; 2006, 2007b).

The effects of amphetamine, cocaine, ethanol, and heroin were assessed with an elevated plus maze using Lister hooded rats, pretreated with a high dose of MDMA.

MDMA acutely produced significant anxiogenic behavior, but this behavior was absent following the administration of the other psychoactive drugs. MDMA pretreated rats which received ethanol exhibited anxiolytic behaviors compared to controls, with an increase in open arm entries. Overall, there were no long lasting changes in affect measured between the interaction of MDMA and these other abused drugs (Sumnall et al., 2004).

MDMA Neurotoxicity

Early studies found high doses of PCA and MDMA given to animals produced profound acute and long term detrimental effects on the serotonergic system. Peripheral injections of MDMA at doses of 10mg/kg and greater produce short term depletion of 5-

HT stores followed by long term depletion of 5-HT due to morphological damage to axon

31 terminals (for review, see Green et al., 2003; Baumann et al., 2007). Additionally,

Ecstasy use in humans often results in cognitive deficits and dysregulation of the serotonergic system measured by imaging studies. However, cognitive deficits are not always positively correlated with this deregulation of serotonergic markers recorded in imaging studies (Fox et al., 2002; Morgan, 2000; Reneman et al., 2000, 2002; Parrott,

2001).

However, current research suggests that these high doses of MDMA given in earlier studies may not be comparable to doses of human intake. Allometric dose scaling from animals to humans used in previous studies may not have been accurate model.

Allometry is the study of changes in an organism related to their body size. More moderate doses comparable to human patterns of drug taking may not result in the same amount of damage (Baumann et al., 2007; de la Torre and Farre, 2004; Easton and

Marsden, 2006). Current investigations are seeking to determine the effects of MDMA in therapeutic use in the treatment of posttraumatic stress disorder and in terminal cancer patients. Thus, it is crucial to assess possible neurotoxicity across a wide spectrum of discriminatory doses (Doblin, 2002; Parrott, 2007). Controlled human clinical studies have eliminated the adverse side effects of MDMA, which are persistently found in sample populations of recreational drug users. This suggests MDMA given intermittently to people who do not abuse Ecstasy or other recreational drugs may not produce significant neurotoxicity or behavioural or physiological impairments in the CNS

(Baumann et al., 2007).

As stated, MDMA administration generates a sizeable increase in extracellular 5-

HT concentration by binding to the serotonin transporter protein. Once taken up into the

32 terminal, the drug facilitates release of serotonin via reversal transport of 5-HT through the transporter receptor protein, carrying 5-HT against its concentration gradient.

MDMA also inhibits tryptophan hydroxylase for several days following drug administration and can be measured up to two weeks following drug treatment. These two MDMA facilitated actions produce a substantial depletion of 5-HT widespread through several forebrain structures that persisted for several weeks (Cadet et al., 2007;

Lyles and Cadet, 2003; Baumann et al., 2007).

MDMA, PCA, methamphetamine, and related amphetamine analogues produce degeneration of serotonergic axon terminal fibers in both rats and nonhuman primates.

Specifically, these amphetamine compounds target the fine 5-HT axon fibers which primarily originate from the DR. After one to two weeks following peripheral administration of MDMA and other amphetamine analogues, autoradiography and immunohistochemical studies, using both 5-HT and SERT as markers, found significant loss of DR fine axonal 5-HT fibers throughout the forebrain whilst the large beaded axons stemming from MR appeared unscathed. The remaining labelled fine axon fibers appeared swollen and fragmented. Interestingly, these studies have found no retrograde loss of 5-HT labelled cell bodies in the DR and MR in drug treated animals (O’Hearn et al., 1988; Molliver and Mamounas, 1988; Molliver et al., 1990; Ricaurte et al., 1988a).

In humans, it is unclear how neurotoxic Ecstasy may be to the CNS. Studies conducted by Ricaurte and colleagues have found decreases in levels of 5-HIAA in

MDMA users. Additionally, several fMRI and radiolabeled ligand PET imaging studies found decreases in SERT and changes in other 5-HT postsynaptic receptors (Ricaurte et al., 1988b; Reneman et al. 2002; Cadet et al., 2007). Reductions in 5-HT function did not

33 directly correlate with prominent neuropsychological deficits. Cognitive deficits witnessed in Ecstasy users who remained abstinent for long periods, exhibited recovery of function in 5-HT imaging measures (Thomasius et al., 2003, 2006; Zakzanis et al.,

2007).

The disruption of the serotonergic system following abnormally high doses of

MDMA and PCA does not necessarily produce definitive evidence that these compounds are, by their very nature, neurotoxic. For example, a study conducted by Kalia et al.

(2000) compared the effects of high doses of MDMA and fenfluramine with intracerebroventricular administration of 5,7-DHT, and several selective serotonin reuptake inhibitors (SSRIs) on the serotonin system via immunohistochemical methods.

SSRIs, given to rats at 10x or greater the effective dose, produced swollen and disoriented axons terminals, similar to that observed with high doses of MDMA and fenfluramine (Kalia et al., 2000; Baumann et al., 2007). Therefore, the negative deformations of 5-HT fibers by MDMA, witnessed in preclinical and human Ecstasy studies could reflect abnormally high doses of the drug which may not occur when the dose is given in a clinical setting at a discriminatory or effective dose.

Other neural markers are used to assess the possible neurotoxicity of amphetamine compounds. Silver positive staining and increases in glial activity measured by increases in the expression of glial acidic fibrillary protein (GFAP) are two markers used to assess neurotoxicity. Both of these markers were correlated with loss of serotonergic cells and subsequent terminal fibers following administration of the 5-HT neurotoxin 5,7-DHT. However, PCA and MDMA induced 5-HT fiber degeneration did not positively correlate with increases in GFAP or silver positive staining. Increases in

34 GFAP following intracerebroventricular (i.c.v.) 5,7-DHT administration in rats was absent following peripheral administration of MDMA, as measured by Western Blot immunoreactivity of GFAP and [SERT] for several dissected brain regions (Wang et al.,

2004; Baumann et al., 2007 for review). Lastly, both audioradiography and immunohistochemical studies have found that 5-HT axonal fiber degradation following peripheral administration of MDMA and PCA does not produce retrograde neuronal death (Baumann et al., 2007).

There have been several studies conducted to determine the exact mechanisms by which MDMA and related compounds promote serotonergic axotomy in rodents and primates. While it is not entirely known, the destruction of 5-HT terminals is thought to be a product of both 5-HT and DA activation, including lethal hyperthermia, the oxidative effects of metabolites which produce free radicals, the production of quinones, and excitotoxicity (Cadet et al., 2007).

In the case of MDMA, the drug is taken up into 5-HT terminals, where its metabolites generate free radicals. This is evident by a number of studies which emphasize a reduction in vitamin E and ascorbic acid in response to MDMA treatment.

Application of free radical scavengers and anti oxidant agents such as ascorbic acid attenuated axonal degeneration following amphetamine treatment. (see Green et al., 2003 for review). These free radicals are responsible for the production of oxidative agents which interrupt a number of cellular processes and produce 5-HT terminal degradation

(Lyles and Cadet, 2003; Green et al., 2003; Cadet et al., 2007; Yamamato and

Raudensky, 2008). Similar increases in oxidative activity have also recently been recorded following administration of PCA in rats (Kanarik et al., 2008). When DA is

35 released via application of MDMA, it can be taken up by serotonergic terminals, where it is metabolized and reduced to quinones, which contribute to the oxidative stress of the terminals. Studies have found that lesioning dopaminergic neurons with 6- hydroxydopamine or pretreating rats to block dopamine release, significantly attenuated the neurotoxic effects of the serotonergic system by MDMA, partially and most likely by blunting the hyperthermic response to the drug (Green et al., 2003; Lyles and Cadet,

2003; Cadet et al., 2007). But Cadet et al. (2007) points out that the DA hypothesis of

MDMA induced neurotoxicity is not wholly supported because it “does not account for the fact that MDMA can damage 5-HT terminals in areas of the brain such as the hippocampus that are almost devoid of DA terminals.”

All amphetamines produce hyperthermia. In MDMA, the hyperthermic response is directly related to the neurotoxicity of the drug. While 5-HT and its receptors play a vital role in thermoregulation, hyperthermia produced by MDMA and other amphetamine

analogues appears to be interrelated with DA release, mediated by 5-HT2 activation. The

SSRI fluoxetine, attenuates long-term neurotoxicity is associated with MDMA, but does not affect the temperature increase caused by treatment of amphetamine analogues (Lyles and Cadet, 2003; Cadet et al., 2007;Yamamoto and Raudensky, 2008; Kiyatkin, 2005).

By contrast, pretreatment using D1 receptor antagonist SCH 23390, diminished increases in [DA] in response to treatment with both PCA and MDMA and successfully inhibited the hyperthermic response (Sugimoto et al., 2001). This suggests that in the case of

MDMA, the hyperthermic response related to the drug is correlated with increases in dopaminergic release. Importantly, if the hyperthermic response to MDMA is diminished, there is significant reduction in the neurotoxic effects induced by the drug.

36 Hyperthermia promotes the production of free radicals and plays a crucial role in the involvement of denaturing of proteins, enzymes, and mitochondrial function, which produce significant 5-HT terminal damage (Green et al., 2003; Cadet et al., 2007).

Rationale

Despite early preclinical and neuropsychological studies, which suggest MDMA and other related compounds are neurotoxic and produce damage to the serotonin system, there is current research being conducted in clinical trials assessing MDMA as a therapeutic tool for the treatment of some psychiatric and physiological disorders and biological disease. It is likely that recreational abuse of MDMA in polydrug users and its use in a clinical setting will affect the nervous system differentially. While Ecstasy users do not often ingest MDMA at doses comparable to those used in earlier animal studies, they do engage in ‘stacking,’ which is consuming a number of pills across an evening during one occasion. This escalation in dose over several hours could be detrimental physiologically and lead to damaging effects comparable to those observed with higher doses. It is imperative, then, to understand the full scope of this compound in preclinical models, and how it may interact with central 5-HT function.

Previous work has found that high doses of MDMA and PCA and other amphetamine analogues produce significant depletion of central 5-HT and axonal degeneration of the fine 5-HT axon fibers that innervate limbic and cortical forebrain structures including the hippocampus, amygdala, prefrontal cortex, nucleus accumbens, and dorsal striatum given to Sprague Dawley (SD) and other albino strains (Battaglia et al., 1987; Brown and Molliver, 2000; Mamounas and Molliver, 1988; Mamounas et al.,

1991; Molliver et al., 1990; O’Hearn et al., 1988; Ricaurte et al., 1988). By comparision,

37 SD rats treated with these large doses of the drug have shown impariments in cognitive and mnemonic paradigms (Able et al., 2006; Cunningham et al., 2009; Morley et al.,

2001; Skleton et al., 2008; Sprague et al., 2003). While several studies have found that

MDMA has a subacute effect on performance of spatial and working memory tasks, it is unknown whether more moderate doses of the drug will produce long-term effects that will have a drastic effect on 5-HT regulation in the CNS. Moreover, whether or not such an effect will manifest itself in a behavioral problem belying a cognitive deficit is unknown.

Baumann and colleagues found that low doses of MDMA (1.5mg/kg) do not produce a significant loss of [5-HT] or significantly decrease the expression of the serotonin transporter or other markers of neurotoxicity such as GFAP. However, doses of 1.25-1.50 mg/kg in both rats and humans produce comparable physiological effects, suggesting that these doses are more relevant to human intake (see Baumann et al., 2007 for review). MDMA may have therapeutic efficacy in the treatment of terminal cancer and post traumatic stress disorder. Little is known about the behavioral deficits that may be found in relationship to rather smaller doses of the drug.

The current experiments are designed to obtain a full spectrum of the extent of 5-

HT fiber damage using immunohistochemical analysis across several dose regimens of

MDMA modeling more accurate representations of interspecies dosing in Long Evans

(LE) rats. LE rats were selected rather than Sprague Dawley (SD) rats for two reasons.

Currently, few laboratories use LE rats to test the effects of MDMA (Camarasa et al.,

2008; Cassel et al., 2004, 2007; Hamida et al., 2006, 2007, 2008, 2009; Riegert et al.,

2008; Jones et al., 2010). LE rats demonstrate superior performance on the behavioral

38 paradigms used in the follow series of experiments. LE rats have increased visual acuity in comparison to albino strains. LE rats demonstrated decreased latencies and better performance on MWM tasks by comparison to SD rats (Prusky, 2002; Tonkiss et al.,

1992). Pigmented rats also exhibited better attentional performance on a three choice serial reaction time test as compared to albino strains (Broersen and Uylings, 1999). LE rats performed better on the intradimensional extradimensional (IED) task used in

Experiment 3. SD rats committed more aborted trials and had a higher task failure rate than the LE strain (Linley and Hughes, unpublished data; S.J. O’Dell, personal communication). Secondly, while effects on the physiological and behavioral effects of the drug exist, to date, there are no studies, which examine the extent of monoaminergic neurotoxocity with immunohistochemistry on LE rats.

This series of experiments examined the long-term effects of multiple low and high dose regimens of MDMA on LE rats in order to 1.) establish possible 5-HT neurotoxic effects of the drugs and 2.) any correlated behavioral deficits. Neurotoxicity was assessed using immunohistochemical methods for monoaminergic innervation of the forebrain. Serotonin toxicity and axonal denervation was analyzed using the antisera for the serotonin transporter. As a comparative measure, the effects of MDMA on catecholamines was assessed using the antisera for tyrosine hyroxylase. The long-term effects of MDMA given in these multiple dose regimens were tested using two separate behavioral tasks that are comparable to human cognitive measures. These tests measure hippocampal dependent spatial memory, and prefrontal measures including attentional set, attentional set shifting, and behavioral flexibility.

EXPERIMENT 1

INTRODUCTION

Over the last thirty years, the central tenet of research reports studying the effects of MDMA and Ecstasy on the mammalian nervous system has sought to determine if the detrimental toxicity of the drug outweigh any possible therapeutic avenues MDMA may possess. Due to its rampant and escalated use by recreational drug users, the Food and

Drug Administration classified the drug as a Schedule 1 drug in 1985. Early studies in rodents and primates utilizing Western blot protein assays, HPLC, and immunohistochemical methods have found MDMA and its metabolites produced a loss of serotonergic fibers throughout the forebrain.

Some of the earliest studies conducted by Molliver and colleagues (O’Hearn et al., 1988) studied the lasting effects of neurotoxicity of MDMA and its metabolite, 3,4- methylenedioxyamphetamine (MDA) using Sprague Dawley (SD). Typical motor and physiological behaviors associated with serotonin toxicity were found following subcutaneous (s.c.) MDMA or MDA injections administered at a dose of 20 mg/kg every twelve hours for 4 days. Two weeks after their last injection, rats’ brain tissue was examined with immunohistochemical methods. In addition, several animals were sacrificed at earlier time points of 1 to several days following the last drug injection. 5-

40 HT terminal axotomy was found throughout several forebrain regions including widespread 5-HT denervation of the cerebral cortex, basal forebrain with massive 5-HT reduction in the dorsal striatum, the intermediate lateral septum, hippocampal formation, amygdala, most of the midline and lateral thalamic nuclei and anterior and dorsomedial hypothalamus. These areas are highly innervated by the morphologically thin fine axons that ascend from the dorsal raphe. Thick axons with large beaded varicosities were spared in the ventral striatum, medial septum, paraventricular nucleus of the thalamus, and partially spared in the hippocampus. In addition, fibers of passage and terminal fibers in the lateral hypothalamus remained untouched. Raphe cell bodies and most fibers of passages remain intact, demonstrating that MDMA only affected 5-HT axon fibers.

Concurrently, Battaglia et al. (1988) examined the effects of 1-8 subcutaneous injections of 5, 10, or 20 mg/kg MDMA in SD rats 1 day to 1 year post injection.

Reductions of 5-HT and 5-hydroxyindolacetic acid 5-HTIAA were immediate and preceded changes in [SERT], indicating a metabolic change in 5-HT before onset of neurotoxicity. This is consistent with the pharmacological profile of MDMA. Reductions in SERT binding sites in the frontal cortex were seen in a dose-dependent manner with high multiple injections of the drug producing severe reductions in SERT. However, a partial recovery of SERT binding sites appeared six months to one year later. This return to baseline concentrations, however, may reflect upregulation of the protein rather than regeneration of axonal fibers (Battaglia et al., 1988). Rats with short survival periods exhibited both a loss of fibers and the existence of swollen, unhealthy, misshapen fibers, which disappeared following longer survival periods (Molliver, 1987).

41 Early primate studies conducted by Ricaurte et al. (1988) assessed multiple subcutaneous doses of 2.50-5 mg/kg MDMA in rhesus and squirrel monkeys. Reductions in both 5-HT and 5-HTIAA and loss of serotonergic axons were noted 2 weeks later in monkeys treated with 5.0 mg/kg dose regimens. No lasting changes in dopamine or norepinephrine were observed. In contrast to the rat, while there was no loss in the number of 5-HT immunoreactive cells within DR, some 5-HT cells were misshapen, with cytoplasmic inclusions reflecting the deformation of metabolic and morphological characteristics (Ricaurte et al., 1988; Ricaurte, 1989).

To compare routes of administration, doses of 5-10 mg/kg delivered orally or subcutaneously to rhesus macaques also produced reductions in 5-HT markers (Kleven et al., 1989). Likewise, Slikkler et al. (1989) administered MDMA orally at a multiple dose of 40-80 mg/kg in Sprague Dawley rats or 5-10 mg/kg in rhesus macaques; significant reductions in serotonin markers in the hippocampus, dorsal striatum, and frontal cortex were noted (Slikkler et al., 1988). Oral administration of MDMA in rats and monkeys does in fact produce serotonergic depletions similar to that of s.c. doses. Dose response studies found that a dose of 2.5-5.0 mg/kg s.c. MDMA in the monkey produced reductions in 5-HT near 50%, but in the rodent, doses of 10-20 s.c. mg/kg were needed to produce similar loss of 5-HT markers (Ricaurte, 1989). This early distinction in dose comparison lead to the conclusion that primates may be more sensitive to the drug, thereby establishing differences in interspecies dosing. While rodents have been noted to recover from serotonergic deficits one year following MDMA treatment, this recovery was not seen in monkeys. It is important to note the neurotoxicity of MDMA is not related to the compound itself but its metabolites. This was discovered by a number of

42 studies, which delivered MDMA intracerebrally by microinfusion directly into the raphe nuclei. These studies failed to produce neurotoxicity to 5-HT axon fibers, whereby peripheral administration did (Paris and Cunningham, 1990, 1992).

The majority of 5-HT forebrain fibers originate from the dorsal (DR) and median

(MR) raphe nuclei. These two brainstem structures send 5-HT projections that differ in both their morphology and anatomical projections (Abrams et al., 2004; Kosofsky and

Molliver, 1987; Lidov et al., 1980; Tork, 1990; Vertes and Linley, 2007, 2008). High doses of MDMA, PCA and MDA cause subsequent degeneration of thin axonal fibers, which primarily originate from DR. Moreover, the pattern of forebrain denervation following these high doses of MDMA reflects the neural structures receiving the majority of their 5-HT input from DR. Two types of 5-HT fibers were noted to remain in these cortical denervated structures: large swollen damaged fibers, and thick fibers with terminals with large, spherical varicosities (Brown and Molliver, 2000; Mamounas and

Molliver, 1988; Mamounas et al., 1991; Molliver, 1987; Wilson et al., 1989).

Previous studies have found no long-term damage of MDMA to other monoaminergic terminals. Several amphetamine analogues including methamphetamine, produce degeneration of dopaminergic, noradrenergic, and serotonergic terminals

(Armstrong and Noguchi, 2004; Brunswick et al., 1992; Quinton and Yamamoto, 2006;

Woolverton et al., 1989). However, dopamine and norepinpherine containing axons do not undergo neurotoxic changes following regimens of MDMA, MDA, fenfluramine, and

PCA (Green et al., 2003; O’Loinsigh et al., 2001). No differences in damage to 5-HT fibers were seen between 10 mg/kg and 20 mg/kg or higher doses of PCA, illustrating a

43 maximum neurotoxic effect for these drugs, despite slight individual differences in both the extent of 5-HT forebrain denervation as well as mortality (Mamounas et al., 1991).

Neurotoxicity can also be measured using other neuronal markers, including silver positive staining and glial cell hypertrophy, measured by examining increases in glial fibrilliary acidic protein (GFAP). Early studies found very high doses of MDMA

(40-80 mg kg/mg) in SD and Long Evans (LE) rats produced silver staining in specific cortical regions, however these doses were greater than 15 times relevant behavioral doses; smaller doses had no effect. Increases in GFAP were produced reliably after intracerebral injections of 5,7-DHT, however only extremely high doses of MDMA have produced responses of GFAP. Increases in GFAP do not directly correspond with 5-HT axon degeneration, as doses of 3 x 7.5mg-10mg MDMA produce significant loss of 5-HT fibers, but an absence of GFAP elevation in forebrain tissue (Rothman et al., 2001; Wang et al, 2004). There is incongruence between 5-HT fiber denevervation and other measures of neurotoxicity, suggesting axon damage may be a result of sizeable doses rather than the amphetamine analogue itself.

In addition to 5-HT neurotoxicity, high doses of MDMA and related compounds also disrupt other molecular and cellular processes. MDMA has been known to impact plastic and neurogenic factors. MDMA (8 x 5 mg/kg) hindered the survival of new granule cells in the dentate gyrus (Hernadez-Rabaza et al., 2006). MDMA intoxication decreased brain derived neurotrophic factor (BDNF) in the hippocampus (Martinez-

Turrillas et al., 2006). MDMA also produces a reduction in the protein calmodulin- dependent protein kinase II (Moyano et al., 2004, 2005).

44 Interspecies dose scaling relies on both the pharmacokinetics and pharmacodynamics of the drug. Classically, doses across animals and humans are calculated using allometric scaling, which use size and basal metabolic rate (BMR) to predict metabolic factors in the pharmacokinetics of the drug. Both size and metabolic rate have show that larger animals require smaller doses of a drug, while smaller animals with higher metabolic rate are expected to eliminate the drugs faster (Mager et al., 2009;

Sharma and McNeil, 2009). Basal metabolic rate and surface area can predict heart rate, circulation of drug, and subsequent elimination (Baumann et al., 2007). However dose scaling using size as a predictor has been inconsistent. Route of administration, distribution, receptor affinity, half-life, plasma concentration, and elimination/clearance of drugs should be factored (Mager et al., 2009; Sharma and McNeil, 2009).

In the 1990’s, less than 35% of an Ecstasy tablet contained MDMA, with the rest being fillers and other amphetamine derivatives. The purity of Ecstasy tablets has increased exponentially over the years (Cole et al., 2001). Now the amount of MDMA in

Ecstasy tablets in Europe and the United States contains on average between 60-100 mg of MDMA, which depending on subject weight, is a dose of 1-3 mg/kg (Baumann et al.,

2007; Cole et al., 2001; Schiffano, 2004; Vogels et al., 2009). Researchers have argued recreational Ecstasy intake in humans produces 5-HT neurotoxicity because MDMA animal doses used in studies are equivalent using allometric dose conversion. Using this algorithm, a dose of 1.7mg/kg is equivalent to a 5 mg/kg dose in monkeys and a 10 mg/kg dose in rats (Cole et al., 2001). However, others think 20 mg/kg in rats is equivalent to 20 mg/kg dose of MDMA in humans, or 20 total Ecstasy tablets (Green et al., 2009). 45 The interspecies dosing regimens from rats to primates used in most preclinical studies has been challenged because the allometric interspecies scaling, which takes into account size and basal metabolic rate of a mammal cannot been applied to MDMA pharmacokinetics. Both rat species vary in which the drug is metabolized, which is associated with heterogeneous nature of P450 liver enzymes, responsible in breaking down the drug (Baumann et al., 2007). MDMA can be metabolized via four different routes: demethylation, dealkylation, deamination, and conjugation. Of these, 8 different metabolites have been discovered, some of which are biologically active and known to be neurotoxic (Lim and Foltz, 1988, 1989). Metabolism in humans occurs through the enzmes cytochrome P450 2D6 (CYP2D6) and catechol-O-methyltransferase. Individual differences may be associated with genetic polymorphisms of these enzymes. In rats, cytochrome P450 2D1 is the analogous catalyst for CYP2D6 (Baumann et a., 2007;

Kumagai et al., 1994).

Baumann et al. (2007) has argued classic interspecies dosing for MDMA is inaccurate, and has applied the use of ‘effects scaling’ in lieu of allometric scaling to map out dose regimens of MDMA. Drugs that are extensively metabolized do not share the allometric equation across species. Using the ‘effects’ method, comparable physiological and behavioral effects of species suggest similar dose response curves between species.

The 5-HT substituted amphetamine fenfluramine demostrated species variability in plasma concentrations that do not account for the classic BMR to surface area relationship (Baumann et al., 2007). Cole et al. (2001) noted interspecies dose scaling used in most experiments “is contentious as: (i) the pharmacokinetics of MDMA are nonlinear, (ii) it does not take into account toxic metabolites, (iii) [Sprague Dawleys]

46 quoted in one such calculation is known to be extremely sensitive to MDMA-induced neurotoxicity and (iv) the effects of a single oral administration of 5 mg/kg in the primate are minimal.” MDMA, at a dose of 1-2 mg/kg in the rat (s.c) and human (taken orally,

PO) produced similar increases in [5-HT] and [DA], drug secretion, and neurohormonal response. In addition, the dose at which both rats and humans can discriminate MDMA is 1.5 mg /kg (as reviewed by Baumann et al., 2007). Given this information, Baumann et al. (2007) argued for a re-evaluation for the behavioral and neurotoxic effects of

MDMA in rodent species, using doses comparable to MDMA intake in humans.

Recreational Ecstasy users consume tablets that usually contain approximately 1.5 mg/kg MDMA. However, MDMA is not taken habitually but typically taken on weekends or over a span of a few days. Ecstasy is often consumed in a binge like manner.

That is, on one night or one occasion, individuals often ‘stack’ Ecstasy pills. Users will consume 1-2 pills every few hours over one night. In order to examine the effects of binge consumption in humans, a series of studies examined multiple daily injections of

MDMA in rats using doses related to human consumption. A binge dose of 3 x 1.5 mg/kg i.p. in SD rats did not produce a hyperthermic response or deplete [5-HT] in forebrain tissue. A larger binge dose of 3 x 7.5 mg/kg MDMA produced hyperthermia and subsequent long-term 5-HT neurotoxicity (Baumann et al., 2001). Similar results have been found in Dark Agouti rats (O’Shea et al., 1998). MDMA had little impact on markers which promote neuroplasticity, by comparison to other amphetamine derivatives.

Both PCA and METH produce reductions in the mRNA of both microtubule associated protein 2 (MAP2) and Arc gene, two compounds associated with synaptic plasticity.

47 Doses of 0.25 – 10 mg/kg MDMA have no effect on mRNA production of MAP2 and

Arc (Putzke et al., 2007).

The damage to the serotonin system following MDMA may not be related to the drug itself, but administration of improper doses. Doses of the SSRIs fluoxetine, sibutramine, and sertraline 10-100x their therapeutic dose produced swelling and abnormal changes in serotonergic fibers very similar to those seen with MDMA and fenfluramine (Kahlia et al., 2000). In addition, SSRI’s also share similar physiological properties with MDMA on the dopamine and serotonin systems. Both SSRIs and

MDMA inhibit vesicular reuptake of MDMA via VMAT2 (Yasumoto et al., 2009).

The U.S. Food and Drug Administration (FDA) is currently conducting clinical trials for the use of MDMA in post-traumatic stress disorder (PTSD) patients. PTSD is an anxiety disorder often triggered by a traumatic event. Triggers and memories often act as reminders to individuals. Mechanisms of fear extinction have been outlined in preclinical models, and new treatments for PTSD are focusing on extinction-based therapies rather than anxiolytic pharmacological intervention (Cukor et al., 2009).

MDMA produces subjective effects such as euphoria, empathy, and relaxation. MDMA- based psychotherapy is implemented to attempt to extinguish petrifying fear from the memory of trauma. Studies use a dose of 125 mg MDMA, which averages to be 1-2 mg/kg dependent on the weight of the participant. Preliminary studies have found promising results, with participants more open to recollection and working through fear memories during therapy while under the influence of MDMA (Johansen and Krebs,

2009; Sessa, 2005, 2007; Sessa and Nutt, 2007).

48 The majority of studies examining the neurotoxic effects of MDMA in rats have used Sprague Dawley strain (Brown and Molliver, 2000; Mamounas and Molliver, 1988;

Mamounas et al., 1991; O’Hearn et al., 1988; Schmued, 2003). Sex differences exist in

SD rats, with female rats having a lower concentration of the metabolite MDA than males, demonstrating differences in MDMA metabolism. Studies using Dark Agouti rats found that a single dose of 15 mg/kg produced a reduction in SERT mRNA and a substantial loss of SERT fiber immunoreactivity (Kirilly, 2010; Kovacs et al., 2007).

Female Dark Agouti rats display a deficiency in CYP2D, which is involved in the demethylenation of MDMA. Despite this, a dose dependent increase in the reduction of

[5-HT] was observed in SD rats in comparison to Dark Agouti rats (Chu et al., 1996).

Sex differences also exist in Dark Agouti rats, with females metabolizing debrisoquine slower than males. Females showed an increased hyperthermic response to MDMA, which correlated to a reduction in [5-HT] (Colado et al., 1995).

Zhou et al. (1996) compared the effects of PCA on different rat strains and found

Sprague Dawley rats also demonstrated a greater sensitivity to 5-HT related neurotoxic markers as compared to Wistar rats, which demonstrated these deficits only when a 4 mg/kg PCA dose was doubled (Zhou et al., 1996). Small doses of MDMA in Wistar rats can increase 5-HT synthesis, whereby larger doses (20-40 mg/kg) will produce a reduction (Muck-Seler et al., 1998). Doses of 8 x 20 mg/kg MDMA induced significant decreases in 5-HT binding using [3H] paroxetine autoradiography (O’Loisigh et al.,

2001). While behavioral MDMA studies have been conducted on Long Evans (LE) rats, there are few reports assessing the neurotoxicity of MDMA using this rat strain, and it is not known whether LE rats may show a greater or lesser sensitivity to 5-HT axonal

49 degeneration as a result of a large dose of the drug. Despite different sensitivities to the effects of MDMA, i.p., s.c., or p.o. administration of equivalent doses of MDMA across

SD, Dark Agouti, and Wistar rats result in similar plasma concentration of the drug and metabolites hours after receiving the dose (Green et al., 2009).

This study examined serotonergic axonal fibers in the rat forebrain after dose regimens of MDMA that are physiological and behaviorally associated with human intake using ‘effects’ scaling. Multiple groups of LE rats were given various high and low MDMA binge doses, a low chronic dose of MDMA, or physiological saline. After a survival period of one month, rats were perfused, their brains removed and series of sections were processed for immunohistochemistry using the antibody for the SERT to assess loss of serotonergic fibers. Catecholaminergic innervation was examined by processing a subsequent series of tissue using the antibody for tyrosine hydroxylase (TH).

This served as a control, to ensure changes in labeling of fibers were 5-HT-specific.

Serotonergic innervation and denervation throughout the entire forebrain is described in detail. Daily chronic or binge low doses of MDMA are expected to have no effect on the pattern of 5-HT fiber labeling in LE rats throughout the forebrain. Binge doses of 1.5 mg/kg did not produce hyperthermia nor significantly reduce [5-HT] in frontal or striatal tissue (Wang et al., 2004). Conversely, the binge high dose of MDMA should produce behavioral effects resembling the serotonin syndrome followed by a subsequent reduction in 5-HT axon terminals throughout the forebrain. Similar doses of MDMA resulted in hyperthermia and a reduction in [5-HT] forebrain tissue (Baumann et al., 2007). It was predicted that MDMA-induced neurotoxicity should be selective for 5-HT fibers, and not affect MDMA on forebrain catecholaminergic innervation of the forebrain was predicted.

50 MATERIALS AND METHODS

Subjects

39 Long Evans rats (Charles River, weighing 275-300 grams) housed in pairs.

Rats were randomly assigned to one of the following conditions: 1.) MDMA chronic low dose: MDMA (subchronic dose of 10 x 1.5mg/kg daily for 5 consecutive days across two weeks; n=10) 2.) MDMA low binge dose (4 x 1.5mg/kg every two hours for two consecutive days; n=10); 3.) MDMA binge high dose (2 x 7.5 mg/kg; n=9). 4.)

Physiological saline using either a binge or chronic administration pattern was used as controls (n=10).

Rats were originally housed in pairs until the onset of their injections schedules, then were moved to individual plastic cages and maintained on a 12hr light/dark cycle with lights on at 7 A.M. Rats had access to food and water ad libitium until the start of behavioral testing. All protocols were approved by the Florida Atlantic University

Institutional Animal Care and Use Committee and held in accordance to the National

Institutes of Health guidelines for the care and use of laboratory animals. Body weights were obtained regularly throughout the experiment. All injections were given i.p. These rats were behaviorally tested in the paradigm in experiment 2 and sacrificed directly following succession of testing. As such, rats were perfused one month (31 days) following their last treatment injection, their brains were removed and processed for immunohistochemistry.

51 Drugs

3,4-methylenedioxymethamphetamine (MDMA) HCl (gifted from the National

Institute of Drug Abuse, NIDA) was dissolved in physiological saline in a volume of 1 ml/kg.

Histology

On the 31st day following their last treatment injection, rats were deeply anaesthetized using isoflurane. The rats were first perfused transcardially with 30-50 ml of ice cold heparinized 0.1 M phosphate buffer saline (PBS) to exsanguinate the rat, followed by 200-300 ml chilled 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at a pH =7.4. Brains were removed and postfixed for 24-48 hrs in 4% paraformaldehdye in

0.1M PB. Brains were then placed in 30% sucrose in 0.1 M PB solution for another 48 hours. Following sucrose cryoprotection, 50 um sections were cut on a freezing sliding microtome. Sections were collected in a six well plate using 0.1 M PB as a storage solution, so that every sixth section was represented throughout the brain for each series of brain tissue sections. Sections were stored in 0.1 M PB in -4° C until tissue was prepared for immunohistochemistry.

SERT immunohistochemistry

For each rat, immunohistochemical analysis to detect serotonergic axons fibers was conducted with the antisera for the serotonin transporter (SERT) using an avidin biotin protein complex (ABC) protocol. A series of sections were moved from storage into crucibles and placed in pyrex crystallizing dishes rinsed with 0.1 M PB. Sections

were initially treated with a 30-min. 1% sodium borohydride (NaBH4) in 0.1 M PB

52 incubation to remove excess aldehydes. Following a copious amount of 0.1 M PB washes, sections underwent a second incubation for 1 hour (h) in 0.5% bovine serum albumin (BSA) in 0.1 M tris buffered saline (TBS; pH=7.6) containing 0.25% Triton X-

100.

Tissue was then incubated in the primary polyclonal antibody, rabbit anti-SERT

(Immunostar, Hudson, WI), in a diluent of 0.1% BSA TBS containing 0.25% Triton X-

100 at a concentration of 1:5,000 for at room temperature for a duration of 48-72 hours.

Cases were placed in scintillation vials during incubations for the primary, secondary, and tertiary antibody steps. Sections were then rinsed with several 0.1 M PB washes.

Sections were next placed in a secondary antibody, biotinylated goat anti-rabbit immunoglobulin (Vector Labs, Burlingame, CA) in the diluent at a 1:500 concentration for a 2 h incubation. This was followed by another series of PB washes. Sections were then placed in atwo-hour tertiary antibody incubation: biotinylated horse anti-goat immunoglobulin (Vector Labs, Burlingame, CA) in the diluent at a 1:500 concentration.

After washing the tissue in 0.1M PB, sections were incubated for 1 h in an avidin biotin complex (ABC) using the ABC Elite kit (Vector Labs, Burlingame, CA) with a diluent of

0.1% BSA in TBS containing 0.25% Triton X-100 at a 1:200 concentration. Following final 0.1 M PB washes, brown serotonin fibers expressing the SERT were visualized with the chromagen: 0.022% 3,3' diaminobenzidine (DAB) (Aldrich, Milwaukee, WI) and

0.003% hydrogen peroxide in TBS for approximately 4-6 minutes. The tissue was stored in 0.1 M PB in a -4°C freezer until they were mounted. Sections were mounted onto chrome-alum gelatin-coated slides, dehydrated using graded methanols and coverslipped with Permount. 53 Catecholaminergic Immunohistochemistry

Similar to the above protocol, immunohistochemical analysis to detect catecholaminergic axons fibers was conducted with the antisera for the enzyme tyrosine hydroxylase (TH), using an ABC protocol. The tissue was initially treated with a 30-

minute 1 % NaBH4 incubation in 0.1 M PB to remove excess aldehydes from fixation.

Following several PB washes, sections were then incubated for 1 h in 0.5% BSA in 0.1M

TBS (pH=7.6).

Sections were then incubated in the primary monoclonal antibody mouse anti tyrosine hydroxylase (Immunostar, Hudson, WI) in a diluent of 0.1% BSA TBS containing 0.25% Triton X-100 at a concentration of 1:5,000 for a duration of 24 hours at room temperature, followed by another 24-48 hours kept at -4 degrees Celsius. Cases were placed in scintillation vials during incubations for the primary, secondary, and tertiary antibody steps. Next, following copius 0.1 M PB washes, sections were placed in a secondary antibody, biotinylated goat anti-mouse immunoglobulin (Vector Labs,

Burlingame, CA) in the diluent at a 1:500 concentration for a 2 h incubation. This was followed by another PB wash. Sections were then placed in 2 h tertiary antibody incubation: biotinylated horse anti-goat immunoglobulin (Vector Labs, Burlingame, CA) in the diluent at a 1:500 concentration. After washing the tissue in 0.1M PB, sections were incubated in a one hour ABC Elite kit (Vector Labs, Burlingame, CA) with the diluent of 0.1% BSA at a 1:200 concentration. Following a final 0.1M PB wash, brown catecholaminergic fibers and cell bodies expressing the enzyme TH were visualized using

0.022% DAB (Aldrich, Milwaukee, WI) with 0.003% hydrogen peroxide in TBS for approximately 3-4 minutes. Sections were stored in 0.1M in a -4°C freezer until they 54 were mounted. Sections were mounted onto chrome-alum gelatin-coated slides, dehydrated using graded methanols and coverslipped with Permount.

Photomicroscopy and data analysis

The labeling for SERT and TH was analyzed for each case using light microscopy on a Nikon Eclipse E600 microscope. For each rat, the pattern and density of forebrain serotoninergic and catecholaminergic expression was noted across each forebrain cortical and subcortical structure. Subjects from each treatment condition were selected as a best representative case from their experimental group based on both the optimal quality of the tissue and the pattern and density of labeling. Interindividual variability across cases was minimal, and was attributed to differences in the signal to noise ratio of the reacted tissue and not individual differences in the pattern or intensity of fiber innervation.

Nomenclature and nuclear borders were defined using Swanson’s (2003) neuroanatomical rat atlas, except where denoted otherwise.

Lightfield photomicrographs at 100x magnification were taken throughout relevant regions across the forebrain for each case with a Q Imaging ICAM camera mounted on a Nikon Eclipse E600 microscope. Photomicrographs were captured and compiled using overlapping stitching at the 100x level using Nikon NS Elements imaging software. Photomicrographs were then saved and imported as tiff file images in Adobe

Photoshop for Creative Suite 4 for Mac (Mountain View, CA) where adjustments to brightness and contrast were made for illustrative purposes, and montages of images were created.

Density of serotonergic fibers was assessed qualitatively for each subject and across each treatment condition. Density was then averaged for each drug treatment

55 condition. Patterns of SERT and TH labeling were described as described as light (+), with light referring to only a few labeled fibers widely dispersed throughout a cortical or nuclear structure. The term moderate (++) labeling was used when a nucleus or cortical area exhibited a modest number of immunoreactive fibers dispersed throughout the forebrain structure. Dense (+++) labeling was noted as a profound concentration of labeled fibers generally occupying a significant portion of a nucleus or cortical area.

Very dense labeling (++++) was assigned to areas where fibers were heavily packed into a cortical or subcortical structure, which was more intense fiber labeling than dense structure. Very dense structures were innervated so strongly that most of the entire structure was covered with immunoreactive fibers. Absence (-) of fiber labeling was assigned to structures which exhibited no fiber expression, or only sparse fibers present within the entirety of the structure. The extent of changes in forebrain serotonergic and catecholaminergic fiber expression across several dose regimens as a result of MDMA dose regimens was examined in comparison with saline controls visually and qualitatively.

RESULTS

The results for serotonergic fiber expression across forebrain neural structures will be first described in control rats, followed by a comparison of any changes in the pattern or density of fiber labeling in the three MDMA experimental groups. The anatomical results of forebrain innervation below is organized by both regional and functional groupings. Cortical structures are discussed first, followed by the dopaminergic-targeted structures: the ventral and dorsal striatum and globus pallidus, the

56 basal forebrain, the hippocampal formation and amygdala, and lastly, the diencephalon.

As discussed in detail below, no differences in SERT fiber labeling was observed in

MDMA chronic low dose and MDMA binge low dose subjects. Where possible, all four treatment conditions were visualized with photomicrographs. However, to increase the size, quality, and detail of the histology, many of the figures below only illustrate differences in SERT innervation by displaying representative cases of the control and

MDMA binge high dose rat, where significant changes in fiber innervation were visualized.

Serotonergic innervation of the forebrain

Prefrontal Cortex

The nomenclature and prefrontal orbital medial subdivisions were taken from Van der Werd and Uylings (2008), which redefined the rostral frontal polar, orbital, and insular cortices in rodents as a result of common morphology. Table 1 lists the density of fibers expressing the SERT protein in the cortex across MDMA dosing conditions.

In control rats, at the very rostral tip of the frontal pole, the prefrontal cortex was uniformly moderately to densely labeled across all levels of the agranular medial (AGm), agranular lateral (AGl) cortex, and dorsal lateral orbital (DLO) cortex. A moderate to dense amount of SERT fibers were also observed in the lateral (LO), ventral lateral

(VLO), ventral (VO), and medial (MO) orbital cortices. 5-HT fibers in these orbital divisions were more densely packed in the 1st and inner (5/6) cortical layers. At this level, the prelimbic (PL) cortex expressed a dense amount of SERT immuoreactive fibers, which coursed throughout all cortical layers. This similar density and pattern of

57 SERT axon fiber labeling was noted in the MDMA chronic low dose and MDMA binge low dose groups.

Figure 1 is a brightfield photomontage of coronal sections through the rostral prefrontal cortex for a representative case in the control (A) and MDMA binge high dose

(B) treatment condition. Rostrally, MDMA binge high dose rats showed a substanial decrease in SERT fiber expression in VO, VLO, LO and MO, with only light to moderate remaining serotonergic fibers. PL, AGm, and DLO also showed decreases in serotonergic innervation, displaying only moderate fiber labeling throughout. By contrast, the high binge dose of MDMA had minimal effect on SERT expression at the rostral level of AGl.

The labeling of the orbital cortical subdivisions exhibited slight differences across their rostral caudal plane in both the pattern and intensity of SERT expression in control rats and the extent of fiber loss in MDMA binge high dose rats. At rostral levels, labeling was more pronounced in all the orbital frontal cortex (OFC) subdivisions, with the greatest concentration of SERT fibers witnessed in DLO and MO. Figure 2 is a high magnification brightfield photomontage of SERT fiber expression at the midlevel of the ventral orbital cortex in a representative case for the control (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA binge high dose (D) groups. At mid levels of the OFC, SERT labeled fibers remained densely expressed in layers 1,5, and 6 of the ventral, ventrolateral, and lateral orbital cortices. Labeling in layers 2 and 3, however, became noticeably less pronounced with only a light to moderate amount of immunostained fibers. This trend remains unchanged in the MDMA chronic low dose and MDMA binge low dose groups.

58 Forebrain sections from MDMA binge high dose rats showed a significant loss of

5-HT axons. Note the large loss of SERT immunopositive fibers throughout VO in the

MDMA binge high dose subject as compared with the other groups, however their laminar organization remained the same, with an increase in the density remaining with the first and inner layers of the cortical divisions, and only a sparse amount of fibers dispersed throughout layers 2/3. The largest loss of SERT immunoreactivity was observed in the caudal aspect of VLO, where the anterior forceps (fa) were well expressed.

The insular cortex includes an agranular, dysgranular (DI), and granular (GI) division. In control rats, the agranular dorsal (AId) and the agranular ventral (AIv) division showed dense SERT fiber expression at rostral to mid levels, with an increase in the number of fibers present in layers 1-3 as compared to the inner layers. At more caudal levels, layers 2/3 showed only light SERT labeling, while SERT axons were packed more heavily in the first and inner layers. This pattern of expression was unchanged in MDMA chronic low dose and MDMA binge low dose rats. There were variations in the change of fiber density in AId and AIv across the rostral caudal plane in

MDMA binge high dose rats. At the very rostral to mid expression of AId, only a light number of 5-HT fibers remained. AIv showed light to moderate number of remaining serotonin terminals, with moderate numbers more packed in layer 1 and meager fibers distributed throughout the remaining layers.

Pronounced SERT fiber labeling was observed in DI and GI in control rats than the agranular divisions. Figure 3 is a brightfield photomontage through levels of rostral

GI in each of the experimental groups. Overall, these structures received dense 5-HT

59 innervation, however layers 1 and 4 exhibited the very dense expression in GI, while layers 2,3,5, and 6 only had a moderate to dense SERT immunoreactive fibers. In

MDMA binge high dose rats, only minimal fiber loss was noted in GI. Layers 1-4 contained dense SERT immunoreactive fibers, while the inner layers only showed moderate innervation.

Also associated with the insular cortices, but caudal to the prefrontal cortex is the posterior agranular insular cortex (AIp), which first emerges at the levels of the rostral medial septum. In control, MDMA chronic low dose and MDMA binge low dose rats, serotonergic fibers were concentrated densely throughout the entire cortical division, with fibers more heavily packed in layers 1 and 5/6. A substantial loss of SERT immunoreactive fibers was noted in AIp in MDMA binge high dose rats, with only a light amount of fibers remaining throughout AIp, concentrated primarily in layer 1.

The medial prefrontal cortex (mPFC) is divided into a dorsal and ventral division.

The dorsal division includes the AGm, and the anterior cingulate (AC) cortex. The dorsal division of the anterior cingulate cortex (ACd) extends across over half of the forebrain.

The ventral division of (ACv) the anterior cingulate cortex emerges just rostral to the genu of the corpus callosum. The ventral mPFC includes the prelimbic (PL) cortex and infralimbic (IL) cortex. As mentioned, rostral PL contained a dense plexus of 5-HT fibers, which encompassed the entire cortical subdivision in control rats. This pattern of innervation persisted caudally in PL and until the emergence of the rostral aspect of IL.

At more caudal levels, very dense serotonergic fiber expression was present in layers 1,

5, and 6 in both PL and IL. A dense amount of SERT fiber labeling was recorded across layers 2/3, some of these fibers traversed mediolaterally across to the inner layers. There

60 was no difference in the density or pattern of SERT labeling in the MDMA chronic low dose or MDMA binge low dose rats. Figure 4 is a brightfield photomontage of the caudal ventral mPFC in a representative control (A) and MDMA binge high dose rat (B). Notice the heavy amounts of SERT axons throughout both PL and IL in the control rat, with a specific increase in density in layers 1 and 5/6. The MDMA binge high dose rat showed a strong reduction in the 5-HT axonal innervation. The greatest effect was observed in

PL, where only a light number of fibers labeling remain, while IL showed light to moderate number of SERT immunoreactive fibers intact.

Moderate to dense SERT fibers were noted in AGm and AC at their rostral extension, though fibers were more heavily concentrated in AC in control, MDMA chronic low dose, and MDMA binge low dose animals. The density of SERT labeled axons in AC increased at more caudal levels, with dense fibers concentrated in ACd but only moderate 5-HT input reaching ACv. Figure 5 is a brightfield photomontage through the caudal expression of AC for a representative subject in the control (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA binge high dose (D) condition. At caudal levels of the dorsal mPFC, there was an increase in the density of

SERT fiber expression through ACd, which remained constant across MDMA chronic low dose and MDMA binge low dose rats. A more extensive loss of SERT fiber expression in AC was noticed in comparison to rostral levels in MDMA binge high dose rats. These animals displayed only minute amount of fibers at the caudal aspect of ACd and ACv.

61 Table 1

Density of fibers expressing the serotonin transporter (SERT) protein in the cortex across MDMA dose conditions.

Figure 1: Brightfield photomontage showing the distribution of SERT immunopositive fibers through the rostral frontal cortex for a representative control (A) and MDMA binge high dose (B) subject. Note the dense labeling across the entire prefrontal cortex in the control subject. A binge high dose of MDMA produced a significant reduction in SERT labeled fibers throughout the orbitomedial pfrefrontal cortex: the anterior cingulate(AC), medial agranular cortex (AGm), prelimbic cortex (PL), and the dorsal lateral (DLO), lateral (LO), ventral lateral (VLO), ventral (VO), and medial (MO) cortex. SERT fiber density in the lateral agranular cortex remained consistent across the two conditions. Scale bar 500µm.

Figure 2: High magnification brightfield photomontage through the frontal cortex displaying SERT fiber expression through the midlevel of the ventral orbital (VO) cortex for a representative subject in the control (A), MDMA chronic low dose (B), MDMA binge low dose (C) and MDMA binge high dose rat (D). Note the dense plexus of fibers packed into layers I and V/VI in the control, MDMA chronic low dose and MDMA binge low dose rat. A significant reduction in SERT labeled fibers was observed in the MDMA binge high dose subject, where only light numbers of fibers were dispered throughout VO. Scale bar, 100µm.

Figure 3: Brightfield photomontage through the lateral frontal cortex displaying SERT fiber expression through the granular (GI) and dorsal agranular (AId) insular cortex for a representative control (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA binge high dose (D) subject. GI and AId contained dense SERT labeled fibers, with very dense fibers concentrated in the 1st and 4th granular layer in control and MDMA low dose rats. MDMA binge high dose rats showed a small reduction in SERT fiber expression, however layers 1 and 4 remained labeled intensely. Scale bar, 250 µm.

65 The taenia tecta (TT) lies on the ventral base of the frontal lobe and though not classically associated with the PFC, is expressed along the same anterior posterior plane.

TT is segregated into a dorsal (TTd) and ventral (TTv) division. In control rats, TTd received very dense SERT positive fibers through its very rostral expression. At the level of the anterior forceps, these SERT fiber terminals were concentrated primarily in layer

1. TTv also displayed dense numbers of SERT immunoreactive fibers, though comparably less in number than TTd. The pattern and intensity of innervation of the TT was unchanged in MDMA chronic low dose or MDMA binge low dose rats. Figure 6 is a photomontage of coronal sections through the rostral forebrain in a control (A) and

MDMA binge high dose (B) subject. MDMA binge high dose rats demonstrated substantial 5-HT fiber loss in both divisions of the taenia tecta, with only a light to moderate amount of SERT axons remaining in TTd and TTv.

Piriform Cortex, Endopiriform Nucleus, and Olfactory Tubercule

The piriform (PIR) cortex begins at the level of the very rostral frontal pole and extends virtually the entire length of the forebrain. In control rats, PIR received very dense serotonergic innervation, which persisted rostrally to caudally: SERT fiber labeling was more pronounced in layers 1 and 3. This pattern of innervation continued until anterior to mid thalamic levels, when a notable reduction in PIR SERT fibers was witnessed, with only moderate to dense number of fibers were noted. No change in the intensity or pattern of innervation was observed in the MDMA chronic low dose or

MDMA binge low dose groups. In MDMA binge high dose rats, there was a significant reduction in the density of serotonergic innervation. At rostral levels, these high dose rats showed only light to moderate amounts of 5-HT fibers in layers 1 and 3, with only a few

66 fibers remaining in layer 2. Caudal PIR, extending from mid striatal to mid to caudal thalamic levels, displayed only light serotonergic fiber expression, uniform across all layers.

The endopiriform (EPd) nucleus, also known as the deep 4th layer of PIR, lies just dorsalateral to PIR, and extends nearly the length of the forebrain. In the control,

MDMA chronic low dose, and MDMA binge low dose groups, EPd received a very dense plexus of SERT fibers, uniformly distributed throughout the structure, persisting across the entire telencephalon. At mid to caudal levels of the thalamus, a slight decrease in fiber density with EPd was observed. The MDMA binge high dose group showed a small reduction in SERT fiber expression across EPd. At rostral levels, moderate to dense SERT labeled fibers were visualized, while at more caudal septal and diencephalic levels, EPd only received a moderate amount of SERT immunoreactive fibers.

The olfactory tubercule (OT) received very heavy serotonergic input. Very dense

SERT immunopositive axons were observed homogenously distributed across all layers throughout its entire expression. MDMA chronic low dose and MDMA binge low dose rats showed no overall changes in the pattern or intensity of OT labelling. A substantial reduction in SERT labelled fibers was witnessed in OT following a binge high dose of

MDMA; dense SERT fiber expression was still seen throughout OT. A greater reduction in the density of SERT fibers was witnessed at more caudal expressions of OT, beginning at the level of the septum, where only a moderate number of fibers remained.

Figure 4: Brightfield photomontage of SERT fiber expression in the caudal ventral medial prefrontal cortex in a representative control (A) and MDMA binge high dose (B) subject. Note the dense number of SERT axons throughout both the prelimbic (PL) and infralimbic (IL) cortex in the control rat, with an increase in density in layers 1 and 5/6. The MDMA binge high dose rat showed a strong reduction in the SERT labeled fibers. The largest reduction in fibers was observed in PL, where only a light amount of fibers labeling remaining, while IL showed light to moderate number of SERT immunoreactive fibers. Scale bar, 250 µm.

Figure 5: Brightfield photomontage of SERT fiber expression in the anterior cingulate (AC) cortex in a control (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA binge high dose (D) subject. Note the dense number of fibers labeled in the dorsal anterior cingulated (ACd), and more moderate SERT fiber expression across the ventral division (ACv) in control rats which remained similar in the MDMA chronic low dose and MDMA binge low dose condition. MDMA binge high dose rats exhibited a strong reduction in SERT immunopositive fibers across AC. Scale bar, 500 µm.

Figure 6: Brightfield photomontage of the pattern of SERT fiber expression in the piriform cortex (PIR), endopiriform nucleus (EPd), and olfactory tubercule (OT) in a representative control (A) and MDMA binge high dose (B) subject. Note the very dense SERT fiber expression in PIR, EPd, and OT in the control rat, and heavy. MDMA binge high dose rats showed a large reduction in SERT immunoreactivity in PIR, with only a sparse number of fibers remaining in PIR. Dense SERT immunopositive axons remained throughout EPd and OT, despite the significant reduction in axonal processes. See list for abbreviations. Scale bar, 500 µm.

70 Figure 6 is a brightfield photomontage taken through a coronal section of the caudal PFC, anterior to the genu of the corpus callosum (ccg), displaying the rostral OT,

PIR, and EPd for a representative case of a control (A) and MDMA binge high dose subject (B). Note the very dense uniform SERT fiber expression in EPd and OT in the control rat, and heavy SERT fiber labelling in layers 1 and 3 of PIR. MDMA binge high dose rats showed a significant reduction in SERT immunoreactivity, with only a sparse number of fibers remaining in PIR. Dense numbers of SERT immunopositive axons spread throughout EPd and OT, despite the large reduction in axonal processes following the binge high dose of MDMA.

Remaining Cortical Mantle

Serotonergic neurons send dense projections throughout the entire neocortex, with a progressive decrease in the number of immunoreactive fibers moving posteriorly, and an increase in density moving ventral laterally. Moderate to dense SERT immunopositive fibers were exhibited in the primary motor cortex (MOp) in control rats, with heavy numbers of fibers packed in the first and inner layers. This pattern of intensity continued until the emergence of the rostral lateral septum, where only moderate numbers of SERT positive fibers terminated homogenously across the cortex. At its rostral extension, moderate to dense SERT positive fibers were distributed evenly across cortical layers of the secondary motor (MOs) cortex. At the level of the septal fimbrial nucleus, the number of SERT fibers reduced to moderate amounts, which were seen until the posterior end of MOs. There was no change in the density of serotonergic innervation in MDMA chronic low dose and MDMA binge low dose rats. MDMA binge high dose rats displayed a significant reduction in SERT innervation of the motor cortices. At

71 rostral levels, only moderate number of SERT immunoreactive fibers remained in both

MOp and MOs. Only light numbers of fibers were dispersed throughout MOp and moderate fibers remaining in MOs at caudal levels.

The somatosensory cortices (primary, SSp and secondary, SSs) received strong serotonergic input in control rats. Extremely dense SERT positive fibers were visualized in layers 1 and granular layer 4. Dense 5-HT input also targeted the inner layers, while layers 2/3 only received a moderate amount of SERT fiber terminations. The posterior division of SSp and SSs showed a decrease in density; beginning at levels of the rostral diencephalon, where moderate fiber labeling was seen consistent throughout both cortical divisions. Figure 7 is a high magnification brightfield photomontage of micrographs through a coronal section of the midrostral diencephalon illustrating the outer layers of

SSp for a representative case of each of the four treatment conditions. Note the absence of change in the pattern of dense SERT fiber labeling in the control, MDMA chronic low dose, and MDMA binge low dose rat. The MDMA binge high dose rat displayed a significant reduction in SERT fiber expression, demonstrating only a light remaining labeled fibers dispersed across the SSp. Layer 4, however, by contrast, still contained moderate to dense fibers. Overall, there was a progressive increase in serotonergic denervation in the posterior plane across SSp and SSs in MDMA binge high dose rats.

The auditory (AUD) and visual (VIS) cortices received substantial 5-HT innervation in control rats. AUD (dorsal, posterior, and ventral divisions), contained a moderate number of fibers, dispersed evenly throughout their layers across the anterior posterior plane. This pattern of innervation was unchanged in MDMA chronic low dose and MDMA binge low dose rats. Dense SERT labeled terminals were dispersed

72 throughout the rostral divisions of VIS, including the primary (VISp), anterolateral

(VISal), anteromedial (VISam), and rostrolateral (VISrl) divisions. At more posterior levels, only moderate SERT immunoreactivity was visualized in the posteromedial

(VISpm), VISal, and laterolateral (VISll), interomedial lateral (VISi), and mediolateral

(VISml) divisions. Widespread neocortical serotonergic denervation was observed in the

MDMA binge high dose condition, with only a light number of fibers dispersed throughout the entire visual and auditory cortical subdivisions.

The retrosplenial (RSP) cortex emerges at levels of the midrostral thalamus at the termination of AC, and includes a ventral (RSPv), dorsal (RSPd), and agranular (RSPagl) division. In control rats, RSPv received only moderate serotonergic input, with dense

SERT positive fibers concentrated in layer 1, and only light fibers found in the remaining layers. Moderate to dense numbers of SERT labeled fibers were dispersed more evenly throughout RSPd and the RSPagl. The posterior parietal (PTLp) cortex, lateral to RSP, showed a homogenous plexus of dense 5-HT terminal fibers dispersed throughout the cortical division in control rats. This remained unchanged in MDMA chronic low dose and MDMA binge low dose MDMA rats. Figure 8 is a brightfield photomontage taken through coronal sections illustrating RSP and PTLp for a representative subject in the control (A) and MDMA binge high dose (B) condition. Note the scarcity of fibers remaining in RSPv and only a light amount of SERT immunoreactive fibers dispersed throughout both RSPd and PTLp in the MDMA binge high dose subject. This pattern of fiber labeling remained consistent throughout the entire expression of the cortical divisions, with only a light number of remaining serotonergic fibers also seen in RSPagl.

73 The entorhinal (ENT) and the perirhinal (PERI) cortices exhibited very dense fiber labeling, with the concentration of SERT immunopositive fibers the most intense in the first and inner layers of PERI. The ectorhinal (ECT) cortex also contained dense numbers of SERT fibers, as did the visceral (VISC) and temporal (TEv) cortical divisions. There was no change in serotonergic innervation of these cortical areas in

MDMA chronic low dose and MDMA binge low dose rats. MDMA binge high dose rats exhibited substantial SERT fiber denervation in VISC, TEv, ECT, and PERI cortices, with only a light amount of SERT positive axons diffused across the remaining cortex. A drastic reduction in SERT fiber expression was observed in the lateral entorhinal cortex

(ENTl), however the medial (ENTm) division showed little change. Figure 9 is a lightfield photomontage through coronal sections displaying the rhinal cortices in a representative control (A) and MDMA binge high dose (B) subject, illustrating SERT fiber density in PERI, ECT, and the lateral entorhinal (ENTl) cortex. Note the very dense plexus of SERT immunopositive terminals through PERI, ENTl, and ECT. Stark differences in the MDMA binge high dose subject were visualized, which showed widespread loss of these axons across all layers of each of the rhinal cortices.

Dorsal and Ventral Striatum, and Globus Pallidus

Dense SERT labeled axons in the dorsal striatum/ caudate putamen (CP) were revealed in control rats, however differences were noted both the anterior-posterior, dorsal-ventral and mediolateral planes. The rostral pole of CP was uniformly packed with dense SERT fibers. At rostral and midrostral levels of the dorsal striatum, the dorsal lateral CP exhibited very dense SERT fiber expression, whereby the medial and ventral aspects were less dense. This pattern of serotonergic striatal innervation continued until

74 the fusion of the septum, where the dorsal lateral and ventral CP displayed dense fiber expression, while other striatal areas only showed moderate to dense numbers of axonal terminations. At the level of the septofimbrial nucleus, the greatest amounts of SERT labeled fibers were observed in CP, with very dense fibers witnessed in the ventromedial

CP. At the very caudal aspect of CP, expressed at the level of the diencephalon, density in 5-HT innervation was reduced, with only a moderate to dense number of fibers visualized.

Figure 10 is a high magnification brightfield photomontage through a coronal section at the midrostral level of the dorsolateral CP for a representative control (A),

MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA binge high dose

(D) rat. Note the strong intensity and pattern of labeling in CP, which was similar across control and low dose MDMA groups. MDMA binge high dose rats were noted to have widespread 5-HT axonal denervation across CP. There was a near obliteration of 5-HT axons in the rostral CP. Only a few fibers remained sparsely scattered from the rostral

CP pole to midlevels of expression. At the fusion of the septum, fiber loss was not as severe; light to moderate SERT labeled fibers remained in the ventral and ventromedial

CP, while light fiber labeling enveloped the dorsal CP. Light numbers of 5-HT terminals remained dispersed throughout the structure.

Nomenclature and anatomical borders for both the accumbens nucleus (ACC; ventral striatum) and ventral pallidum were derived from Paxinos and Watson (1998), which delineated the core (ACCc) and shell (ACCs) divisions of ACC. Dense SERT immunoreactive axonal processes were observed in ACC in control rats. Fibers were packed more densely in the ACCc than the surrounding ACCs. Overall, the rostral and

75 midlevel ACC was more intensely innervated than the caudal aspect, where ACCs only expressed a moderate number of SERT labeled fibers. Serotonergic innervation of ACC was unchanged in MDMA chronic low dose and MDMA binge low dose rats. MDMA binge high dose rats exhibited a significant reduction in SERT fiber expression throughout ACC. The core division showed the greatest fiber loss, with only minimal to light numbers of 5-HT fibers remaining. The shell division also showed a reduction in

SERT density, however light to moderate number of fibers sustained the high dose of

MDMA. Figure 11 is a lightfield photomontage through a midlevel section of ACC for a representative control (A) and MDMA binge high dose (B) rat. Note the minute number of SERT immunoreactive axons throughout ACC in the MDMA binge high dose subject, in comparison to the control, where dense fibers spread throughout ACC.

The globus pallidus (GP) contained very dense SERT labeled fibers, more intense than CP. This intensity of innervation persisted across the anterior posterior plane, and there was relativity little difference in SERT expression across the medial and lateral divisions. The pattern and density of SERT innervation was unchanged in the MDMA chronic low dose, MDMA binge low dose, and the MDMA binge high dose condition.

Figure 12 is a brightfield photomontage through coronal sections of GP at the level of the rostral thalamus, displaying GP for a representative control (A) and MDMA binge high dose (B) case. Note the absence of change in the intensity of SERT labeled fibers throughout GP, indicating a high binge dose of MDMA did not produce axonal denervation in GP.

Figure 7: High magnification brightfield photomontage for a representative case in the control (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA binge high dose (D) illustrating the pattern of SERT fiber expression in the outer layers of the primary somatosensory cortex (SSp). Note the absence of change in the pattern of dense SERT fiber labeling in control, MDMA chronic low dose, and MDMA binge low dose rats. MDMA binge high dose rats displayed a significant reduction in SERT fiber expression, with only a light amount of remaining labeled fibers dispersed across the SSp. Scale bar, 100 µm.

Figure 8: Brightfield photomontage of the pattern of SERT fiber expression in the retrosplenial (RSP) and posterior parietal (PTLp) cortex in a representative control (A) and MDMA binge high dose (B) rat. Dense SERT positive fibers were observed in layer 1 of the ventral retrosplenial (RSPv) cortex with only light amounts of fibers found in the remaining layers. Moderate to dense SERT labeled fibers were dispersed more evenly throughout the dorsal retrosplenial (RSPd) cortex and PTLp. Note the scarcity of fibers remaining in RSPv and only a light amount of SERT immunoreactive fibers dispersed throughout both RSPd and PTLp in the MDMA binge high dose subject. See list for abbreviations. Scale bar, 500 µm.

Figure 9: Brightfield photomontage of the pattern of SERT fiber expression through the rhinal cortices in a representative control (A) and MDMA binge high dose (B) subject. PERI, ECT, and the lateral entorhinal (ENTl) cortex. Note the very dense plexus of SERT immunopositive terminals through the perirhinal (PERI), lateral entorhinal (ENTl), and ectorhinal (ECT) cortices in the control rat. Widespread loss of SERT labeled fibers was observed across the rhinal cortices in the MDMA binge high dose subject. See list for abbreviations. Scale bar, 500 µm.

79 Table 2 Density of fibers expressing the serotonin transporter (SERT) protein in the basal forebrain in the control, MDMA chronic low dose, MDMA binge low dose, and MDMA binge high dose conditions.

Figure 10: High magnification brightfield photomontage of the pattern of SERT fiber innervation across a midrostral level of the dorsolateral caudate putamen (CP) in a control (A), MDMA chronic low dose (B), MDMA binge low dose (C) and MDMA binge high dose (D) rat. Note the dense SERT labeling in CP in control animals, and similar pattern of intensity in control and low dose MDMA rats. A significant widespread reduction in SERT fibers was observed in MDMA binge high dose rats throughout CP. Scale bar, 100 µm.

Figure 11: Bightfield photomontage of the pattern of SERT fiber expression through the a midlevel section of the nucleus accumbens (ACC) in a representative control (A) and MDMA binge high dose (B) rat. Dense SERT immunoreactive axonal processes were observed in ACC in control rats. Fibers were packed more densely in the accumbens core (ACCc) than the surrounding accumbens shell (ACCs). Note the small amount of SERT immunoreactive fibers remaining in ACCc and ACCs in MDMA binge high dose subject. See list for abbreviations. Scale bar, 500 µm.

82 Basal Forebrain and Claustrum

Table 2 displays the density of SERT fiber expression in the basal forebrain and claustrum. The claustrum (CLA) first emerges in the frontal cortex, rostral to the anterior forceps and extends almost the entire length of the forebrain. In controls, the entire claustrum receives a homogenous plexus of dense 5-HT innervation, which only slightly degrades in intensity as the structure progresses posteriorly. This remained unchanged in the MDMA chronic low dose and MDMA binge low dose rats. MDMA binge high dose rats exhibited a reduction in SERT fiber density, with moderate amounts of SERT positive fibers remaining in the rostral claustrum, and moderate to dense numbers of fibers at while more midlevel sections. A decrease in the density of fibers was seen in caudal CLA.

The ventral pallidum (VP) lies in the basal forebrain, ventral to ACC, first emerging at the anterior posterior plane of the rostral unfused septum and extends caudally to rostral levels of the diencephalon. In the control, MDMA chronic low dose, and MDMA binge low dose condition, the anterior VP exhibited very dense SERT labeling, more so than the adjacent ACC. Further caudally, VP fiber expression decreased in intensity, though a strong number of serotonergic fibers were still present.

Only moderate SERT immunoreactive fibers were observed in the MDMA binge high dose group.

Figure 12: Bightfield photomontage lightfield of the pattern of SERT fiber expression through coronal sections of the globus pallidus (GP) at the level of the rostral thalamus, in a representative control (A) and MDMA binge high dose (B) rat. Note the absence of change in the very dense SERT labeled fibers throughout GP following a high binge dose of MDMA. See list for abbreviations. Scale bar, 250 µm.

84 The septal nuclei are targeted heavily by serotonergic input. The lateral septum

(LS), rostral division (LSr) received very dense 5-HT fibers in control rats at levels of the caudal ventral mPFC. Very dense 5-HT fibers were visualized throughout the medial septum (MS), with the exception of the very caudal MS, as the septal fimbrial nucleus began its expression, where only dense fibers coursed through and targeted the nucleus.

Dense SERT labeled fibers were also found in the lateral septum, intermediate division

(LSi), while the lateral division of the lateral septum (LSl) only contained a moderate number of labeled axonal processes. There were no changes in the density or pattern of innervation of MS or the LSl in MDMA chronic low dose and MDMA binge low dose rats, and only minute differences were seen in MDMA binge high dose rats. There were also no changes in serotonergic innervation of the lateral septum following chronic or binge low doses of MDMA.

There was only a small decrease in the number of SERT immunoreactive fibers at the very rostral aspect of LSr in MDMA binge high dose rats, but 5-HT fibers remained packed densely. Progressing posteriorly, loss of SERT labeled fibers denervation of was substantial in LSi, with only a light amount of immunoreactive fibers remaining. Figure

13 is a brightfield photomontage through the septum for a representative control (A) and

MDMA binge high dose subject (B). Note the large reduction in intensity of SERT reactive axons in LSi in the rat pretreated with a high binge dose of MDMA, while the pattern of innervation in MS, LSl, and the nucleus of the diagonal band (NDB) remained relatively unchanged in comparison to control.

Control, MDMA chronic low dose and MDMA binge low dose rats contained very dense serotonergic fiber labeling throughout the NDB. MDMA binge high dose rats

85 exhibited only minimal decreases in serotonergic fiber expression in NDB, where moderate fiber labeling number of SERT immunoreactive fibers was observed at the rostral levels, with an increase in density seen at the fusion of the septum. Very dense

SERT immunoreactive fibers were also dispersed throughout the magnocellular (MA) nucleus, and the islands of Calleja (isl), which was unaffected by MDMA pretreatment regardless of experimental group. Both low and high doses of MDMA also had no impact on serotonergic denervation of the substantia innominata (SI) and median preoptic nucleus, (MePO) which were densely packed with SERT positive fibers in all treatment conditions. Figure 6 shows a similar pattern and intensity of SERT fiber expression in

SI, just ventral to GP, in control and MDMA binge high dose rats.

The lateral preoptic area (LPO), anteroventral (AVP), anterodorsal preoptic nuclei

(ADP), and medial preoptic (MPO) nucleus contained a moderate density of SERT fiber labeling, which varied across the anterior posterior plane from light to moderate and moderate to dense in some areas. This pattern and density of SERT fiber expression was not altered in MDMA chronic low dose or MDMA binge low dose rats. The MDMA binge high dose group showed small reductions in SERT fiber expression across preoptic nuclei.

The bed nucleus of the stria terminalis (BST) received moderate to dense 5-HT fiber innervation in control rats, which varied in intensity across the anterior posterior plane. At its very rostral expression, at levels of the fusion of the septum, the anterior dorsal (BSTad) division displayed moderate fiber labeling. At mid-caudal levels of

BSTad, SERT fiber expression was augmented. BST received mostly moderate serotonergic input throughout the remainder of its anterior posterior expression with the

86 exception of the dorsolateral posterior division (BSTdlpr), which contained dense SERT positive terminal fibers. The pattern and density of fiber expression remained unchanged in the MDMA chronic low dose and MDMA binge low dose groups. MDMA binge high dose rats exhibited a reduction in SERT terminals, with light to moderate number SERT fibers persisting across all BST divisions.

Hippocampal formation and amygdala

Table 3 is a density chart of SERT fiber expression in the amygdala (AMY) and hippocampus (HPC). The amygdaloid complex received very heavy serotonergic input, as revealed by an abundance of SERT immunoreactive fibers in control rats. With the exception of the central nucleus of the amygdala (CEA), which only contained a light blanketing of serotonergic axons, dense innervation was observed across the AMY. The basal lateral amygdala (BLA) and lateral amygdala (LA) contained very dense SERT labeled fiber terminations. The rostral basal medial amygdala (BMA) also displayed very dense SERT labeling but this density decreased slightly progressing posteriorly. Dense

SERT fiber expression was also noted across the anterior amygaloid area (AAA), intercalated nuclei (IA), medial amygdala (MEA), and the posterior nucleus of the amygdala. Both the cortical amygdala (COA) and the piriform amygdalar area (PAA) were also densely innervated. This pattern and density of fiber expression did not vary in

MDMA chronic low dose and MDMA binge low dose rats.

MDMA binge high dose rats displayed substantial SERT fiber loss across the amygdala, but several nuclei continued to contain strong SERT expression. Figure 14 is a lightfield photomontage through a coronal section of the amygdala for a representative control (A) and MDMA binge high dose (B) case. While a reduction in SERT

87 immunoreactive fibers was witnessed across the entire AMY, dense numbers of axons were still visualized in BLA and LA. Greater serotonergic fiber loss was observed in

MDMA binge high dose rats across AAA, COA, IA, PA, and PAA, which exhibited only a light SERT immunoreactivity. Moderate to dense serotonin terminals were observed in

MEA and BMA.

SERT axonal labeling throughout the hippocampal formation was extensive, but heterogeneous across layers and divisions. Figure 15 displays brightfield photomontage of coronal sections through the dorsal hippocampus for a representative case of each of the four treatment groups. In control, MDMA chronic low dose and MDMA binge low dose rats, the dorsal hippocampus contained a dense plexus of SERT immunoreactive axons, which demonstrated a common laminar organization across Ammon’s horn: both the stratum oriens (so), stratum radiatum (sr), and stratum lucidum (slu) contained very dense SERT positive terminal fibers in the CA3 field of Ammon’s horn (CA3), while the middle pyramidal layer only received light to moderate SERT innervation. While dense numbers of fibers were also visualized in so and sr of the CA1 field of Ammon’s horn

(CA1), it was notably less intense than CA3. The stratum lacunosum moleculare (slm) of

CA1 received the strongest innervation, with very dense SERT immunoreactive fibers noted. CA2 field of Ammon’s horn (CA2) exhibited dense number of fibers diffused across laminar divisions. The intensity and pattern of SERT fiber innervation of

Ammon’s horn remained fairly consistent across the dorsal and ventral hippocampus.

The dentate gyrus (DG), by contrast, only received a light to moderate SERT fiber expression distributed across its molecular and polymorphic layers in the dorsal HPC. A notable increase in the amount of fiber distribution was found in the posterior sections of

88 DG, at levels of the subiculum. MDMA binge high dose rats exhibited a significant loss of hippocampal SERT fiber expression. Moderate amounts of SERT axons persisted in the so, sr, and slu layers of CA3 and in slm, while so, sp, sr of CA1, sp of CA3, and DG remained lightly labeled. There was only a small increase in the amount of SERT fiber expression in the ventral hippocampus, with dense SERT terminals remaining in so of

CA3 and slm.

The subiculum, including the dorsal and ventral subiculum was densely innervated with serotonergic fibers in control rats. The density of SERT fiber expression was increased in both the presubiculum (PRE) and postsubiculum (POST). This pattern and intensity of innervation remained unchanged in MDMA chronic low dose and

MDMA binge low dose rats. Figure 16 is a brightfield photomontage through coronal sections showing the subiculum for a representative control (A) and MDMA binge high dose (B) rat. Note the paucity of remaining fibers in the MDMA binge high dose subject and the minute number of SERT axons in TEV. SERT were fibers packed densely in the subiculum and associated areas in the control rat.

Thalamus

Descriptive anatomy for experimental conditions for the thalamus is organized by groups, which are anatomically and functionally related. Table 4 is a chart with the overall relative density measurements of SERT fiber innervation for each thalamic and hypothalamic nuclei across each of the four treatment conditions: control, MDMA chronic low dose, MDMA binge low dose, and MDMA binge high dose rats.

Figure 13: Brightfield photomontage of SERT fiber expression through the septum in a representative control (A) and MDMA binge high dose subject (B). Note the large reduction in intensity of SERT reactive axons in the intermediate lateral septum (LSi) in the rat pretreated with a high binge dose of MDMA. Dense fibers dispersed throughout the medial septum (MS), lateral division of the lateral septum (LSl), and the nucleus of the diagonal band (NDB) did not differ between the experimental groups. See list for abbreviations. Scale bar, 500 µm.

90 Table 3 Density of fibers expressing the serotonin transporter (SERT) protein in the hippocampus and amygdala in the control, MDMA chronic low dose, MDMA binge low dose, and MDMA binge high dose conditions.

Figure 14: Brightfield photomontage of the pattern of SERT fiber expression through the amygdala (AMY) in a representative control (A) and MDMA binge high dose (B) rat. Very dense SERT labeled fibers were packed in the basal lateral (BLA) and lateral (LA) amygdala in control rats. Dense fiber expression was noted across the basal medial (BMA) and cortical (COA) amygdala. Moderate amounts of fibers were observed in the medial (MEA) amygdala, and only a light sheath of fibers were contained the central nucleus of the amygdala (CEA). A significant reduction in the amount of SERT labeled fibers was observed across the AMY in MDMA binge high dose rats, however dense fiber expression was still observed in BLA and LA. See list for abbreviations. Scale bar, 500 µm. 92

Figure 15: Brightfield photomontage of the pattern of SERT fiber expression across the dorsal hippocampus in a representative control (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA binge high dose (D) subject. In control, MDMA chronic low dose and MDMA binge low dose rats, the dorsal hippocampus contained a dense plexus of SERT immunoreactive axons, which demonstrated a common laminar organization:: both stratum oriens (so), stratum radiatum (sr), and stratum lucidum (slu) in the CA3 field of Ammon’s horn (CA3), and the stratum lacunosum moleculare (slm) of CA1 field of Ammon’s horn (CA1) contained very dense SERT positive terminal fibers Dense fibers were observed in so and sr of CA1 and throughout the CA2 field of Ammon’s horn (CA2), and moderate to dense fiber labeling was present in the dentate gyrus (DG). MDMA binge high dose rats exhibited significant reductions in SERT immunoreactivity, with only light fiber labeling remaining across the hippocampus, with the exception of CA3, which contained moderate SERT axon fibers. Scale bar 500 µm. 93

Figure 16: Brightfield photomontage of the pattern of SERT fiber expression through the subiculum (SUB) in a representative control (A) and MDMA binge high dose (B) rat. SUB was densely innervated with SERT labeled fibers in control rats. This density was increased in the presubiculum (PRE) and postsubiculum (POST). Note the paucity of remaining fibers in SUB, PRE, and dentate gyrus (DG) in the MDMA binge high dose subject. A large reduction in SERT fiber density was also observed in POST. See list for abbreviations. Scale bar, 500 µm.

94 The anterior nuclei include the anterodorsal (AD), anteromedial (AM), anteroventral (AV), and interoanterodorsal nuclei (IAD), and are expressed at the very rostral plane of the thalamus. In control rats, AD received the lightest SERT immunoreactive labeling of all the anterior groups. AM, IAD, and IAM displayed a dense number of SERT labeled fibers. AV received the most desnse 5-HT innervation, with very dense SERT expression. There was no change in the pattern or intensity of fiber expression in the anterior thalamus in response to the MDMA chronic or MDMA binge low dose conditions. Figure 17 displays a brightfield photomontage through the anterior thalamus for a representative control (A) and MDMA binge high dose (B) subject. The MDMA binge high dose regimen produced significant loss of SERT fiber expression in each of the anterior nuclei. AM and IAD demonstrated only light 5-HT labeling. A sparse amount of fibers remained throughout the extent of AD. Moderate to dense SERT labeled fibers were observed in AV. IAM, which lies on the midline and sometimes associated with midline nuclei showed a variation in intensity of fiber labeling along the anterior posterior plane, with only a light amount of 5-HT fibers seen at very rostral levels, and more moderate SERT fiber labeling through midrostral thalamic sections.

The midline thalamic nuclei include the paraventricular (PV), paratenial (PT), reuniens (RE), and rhomboid (RH) nuclei. PV and RE extend across two thirds of the diencephalon, and rostral caudal differences in intensity of fiber labeling were exhibited in control rats. RH, RE, and PV expressed very dense SERT labeling throughout the extent of the thalamus. Rostral PV exhibited more intense fiber labeling than the posterior division. The very rostral aspect of PT showed a moderate number of SERT

95 immunopositive fibers, however the remainder of the nucleus only exhibited light labeling. There were also differences across the medial lateral plane in RE, with a very dense plexus of fibers in the center core, and moderate to dense SERT expression across the lateral wings. In MDMA chronic low dose or MDMA chronic binge dose rats, no changes in the pattern or intensity of labeling in comparison to controls was observed.

Figure 18 is a brightfield photomontage illustrating the rostral midline thalamus in a representative control (A) and MDMA binge high dose (B) subject. Note the very dense plexus of SERT fibers throughout the dorsal and ventral midline. In MDMA binge high dose rats, the greatest amount of fiber loss in the thalamus was found along the midline, including the central medial (CM), IAM, and RE nuclei. After the high dose of

MDMA, serotonergic input to PT was virtually eliminated, with only a few fibers remaining. At the very rostral thalamus, both PT and RE showed substantial SERT fiber reduction, with only moderate amount of fibers remaining in these very dense 5-HT targets. Moderate to dense SERT imnunoreactivity was noted in the anterior portion of

PV (PVa) at rostral to mid levels of the thalamus in MDMA binge high dose rats. The posterior portion of the paraventricular nucleus (PVp) only exhibited a moderate number of SERT fibers. Fiber loss was consistent along the anterior posterior plane, with RE and

RH having only moderate number of 5-HT fibers.

The intralaminar nuclei include the central lateral (CL), paracentral (PCN), and

CM nuclei rostrally, and the parafasicular (PF) nucleus at the caudal end of the diencephalic mesencephalic junction. Dense serotonergic fibers innervate both CM and

CL heterogeneously across their anterior posterior plane, while PCN receives moderate to dense number of 5-HT fibers in control rats. PF was immersed with dense SERT fibers

96 throughout its nucleus, with fibers of passage witnessed within PF and the adjacent fasciculus retroflexus. The density and pattern of innervation of the three nuclei in the IL central complex does not differ in MDMA chronic low and MDMA binge low dose rats.

In the MDMA binge high dose condition, CM exhibited only a sparse number of SERT immunoreactive fibers throughout at anterior to mid level expression. At caudal levels of the thalamus, where CM lies above the third ventricle, more moderate numbers of serotonergic fibers remained. PCN also demonstrated a virtual absence of fiber expression following the high dose MDMA treatment. Light to moderate SERT labeled fibers were observed consistently throughout the expression of CL. Reductions in SERT fiber expression across PF in the MDMA binge high dose group were small and insignificant.

The interomedial dorsal (IMD) nucleus of the thalamus expressed a dense amount of SERT fibers in control rats; a moderate number of SERT labeled fibers was observed in MD. MD showed heterogeneity in the density of 5-HT immunolabeled fibers across its subdivisions, with more fibers noted in the lateral (MDl) and central (MDc) divisions than the medial (MDm) division. There was no significant change in SERT expression across IMD and MD in MDMA chronic low dose and MDMA low binge dose rats. In

MDMA high binge dose rats, only light SERT fibers were dispersed evenly throughout all subdivisions of MD and rostral IMD, whereby, while more moderate numbers were observed at the caudal aspect of IMD.

The lateral thalamic nuclei include the lateral dorsal (LD) nucleus rostrally, and lateral posterior (LP) nucleus, caudally. LD displayed very dense expression of SERT fibers in controls. Moderate to dense numbers of SERT immunoreactive fibers were

97 visualized in LP. MDMA chronic low dose and MDMA binge low dose rats exhibited no difference in this pattern or intensity of SERT innervation. Figure 19 is a brightfield photomontage of coronal sections through LD for a representative case of each of the 4 experimental groups. Note the very dense plexus of SERT labeled fibers packed into LD in the control, MDMA chronic low dose, and MDMA binge low dose rat. MDMA binge high dose rats exhibited significant reduction in SERT fibers, with only light to moderate fibers dispersed throughout the structure. Light 5-HT fiber labeling was also noted in LP in the MDMA binge high dose group.

The submedial nucleus (SM) is located in the ventral thalamus, with RE and RH positioned medially and the ventromedial (VM) nucleus laterally. In control rats, SM is moderate labeling is observed in rostral SM, however, further caudally, fibers are scattered lightly throughout the nucleus. SERT expression did not change in the pattern or intensity of labeling in MDMA chronic low dose or MDMA binge low dose rats. A small number of fibers was dispersed across the entire expanse of the nucleus following the binge high dose. The ventral thalamus includes the ventral anterior lateral (VAL) nucleus, VM, nucleus, the ventrobasal (VB) complex, which includes the ventral posterior medial and ventral posterior lateral nuclei, and the posterior (PO) nucleus of the thalamus. Overall, serotonin only lightly innervated the ventral thalamus, with the exception of VM. Moderate to dense 5-HT fibers were observed throughout the rostral to midlevel expression of VM, however, progressing further caudally, only a light number of fibers were visualized. MDMA binge high dose rats exhibited light SERT fiber expression across VM, however there was a quiescence of labeling in VAL, VB,

98 and PO, where only a few SERT labeled fibers were scattered throughout the expansive nuclei.

The reticular nucleus in the rat emerges at the very rostral diencephalon, extends across two thirds of the thalamus, and lies on the lateral aspects of the diencephalon, adjacent to the internal capsule. Overall, control rats only exhibited a light number of

SERT immunoreactive fibers throughout RT with the exception of the very rostral aspect of RT, which showed a moderate fiber labeling. There was no difference in the pattern or density of SERT innervation of RT in rats given chronic or binge low doses of MDMA.

Moderate numbers of SERT immunoreactive fibers were still detected in very anterior thalamic sections of RT in MDMA binge high dose rats. Further caudally, only sparse fibers were noted.

The lateral and medial geniculate nucleus is expressed at the diencephalic mesencephalic border. In control rats, the lateral geniculate (LGN) nucleus expressed the highest density of fibers witnessed across the entire thalamus with an increase in density in the ventral division as compared to the dorsal division. This same intensity in fiber expression was also seen in the intergeniculate leaflet (IGL). MGN displayed only a light amount of 5-HT fibers dispersed evenly across its subdivisions. The pattern and density of fiber expression was unchanged in MDMA chronic low dose and MDMA binge low dose rats. Figure 20 is a brightfield photomontage of coronal sections through the LGN complex for a representative control (A) and MDMA binge high dose (B) subject. MDMA binge high dose rats exhibited a significant reduction in SERT fiber expression in the LGN and IGL; a light blanket of SERT positive fibers remained in

99 LGNd and IGL. A moderate amount of SERT immunoreactive fibers were observed in the LGNv.

The habenular nuclear complex, consisting of the medial habenula (MH) and lateral habenula (LH) comprises the epithalamus. MH exhibited moderate SERT fiber expression homogenously throughout the extent of the nucleus, while LH only contained a light number of SERT positive fibers in control animals. The pattern and density of fiber expression was unaffected following pretreatment in the MDMA chronic low dose and MDMA binge low condition. In MDMA binge high dose rats, a light number of

SERT positive fibers was noted across MH, while LH demonstrated a virtual absence of

SERT immunolabellng. SERT immunoreactive fibers are packed very densely throughout the entire extent of the zona incertia (ZI), many of which are fibers of passage. The intensity of labeling in ZI did not change in MDMA chronic low dose and

MDMA binge low dose rats. MDMA binge high dose rats showed only a small reduction in SERT: dense SERT fibers were still observed through most of ZI.

100 Table 4 Density of fibers expressing the serotonin transporter (SERT) protein in the diencephalon across dose conditions.

101

Figure 17: Brightfield photomontage of the pattern of SERT fiber expression through the anterior thalamus in a representative case of control (A) and MDMA binge high dose (B) subject. Very dense SERT fiber expression was observed in the anteroventral (AV) nucleus in control rats. The anteromedial (AM) and interoanterodorsal (IAD), and interanteromedial (IAM) nuclei contained dense numbers of labeled axons, while the anterodorsal (AD) nucleus received only a light sheath of fibers. MDMA binge high dose rats demonstrated significant loss of SERT fiber expression across all of the anterior nuclei, with only light fiber labeling observed in AM, IAM, and IAD, moderate fiber labeling witnessed in AV, and a paucity of fibers remained in AD. See list for abbreviations. Scale bar, 250 µm. 102

Figure 18: Brightfield photomontage of SERT fiber expression across the rostral midline thalamus in a representative control (A) and MDMA binge high dose (B) rat. Note the very dense plexus of SERT fibers throughout the paraventricular (PV), rhomboid (RH) and reuniens (RE) nuclei in control rats. Dense fiber expression was also noted in the central medial (CM) and interanteromedial (IAM) nuclei. Light numbers of fibers were dispersed throughout the parataenial (PT) nucleus. There was a large reduction in SERT immunoreactivity in the midline thalamus in MDMA binge high dose rats: RE and RH contained only moderate SERT labeled fibers, while sparse fibers were scattered through CM, IAM, and PT. There was only a minimal loss of SERT fiber expression in PV. Scale bar, 500 µm.

103

Figure 19: Brightfield photomontage of the pattern of SERT fiber expression through the lateral dorsal (LD) nucleus of the thalamus in a representative control (A), MDMA chronic low dose (B), MDMA binge low dose (C) and MDMA binge high dose (D) rat. Note the very dense plexus of SERT labeled fibers packed into LD in the control, MDMA chronic low dose, and MDMA binge low dose rat. MDMA binge high dose rats exhibited significant SERT fiber denervation, with only light to moderate SERT fibers were dispersed throughout the structure. Scale bar, 250 µm.

104

Figure 20: Brightfield photomontage of SERT fiber expression through the lateral geniculate nucleus (LGN) complex in a representative control (A) and MDMA binge high dose (B) case. The lateral geniculate nucleus, dorsal (LGNd) and ventral (LGNv) divisions and the intergeniculate leaflet (IGL) contained very dense numbers of SERT labeled fibers, with an increase in intensity noted in IGL and LGNv. MDMA binge high dose rats exhibited a significant reduction in SERT fiber expression in the LGN and IGL, with only a light blanket of SERT positive fibers remaining. A moderate amount of SERT immunoreactive fibers was observed in the LGNv. See list for abbreviations. Scale bar, 250 µm.

105 Hypothalamus

With the exception of the dorsomedial hypothalamic (DMH) nucleus, where only a light amount of SERT immunoreactive fibers throughout the rostral caudal expression, other hypothalamic nuclei received strong terminals and fibers of passage. The lateral hypothalamus (LHy) received very dense serotonergic input. Dense terminal fibers as well as passing axonal projections coursed through LHy. The pattern of SERT immunolabeled fiber expression did not change in the MDMA chronic low dose or

MDMA binge low dose groups. There was also no long-term effect of MDMA on SERT expression across the anterior (AHN), tuberal (TUB), and arcuate (ARH) hypothalamic nuclei, which all contained a dense amount of fiber labeling following low doses.

MDMA binge high dose rats showed a reduction in the number of SERT labeled fibers in the PV and VMH nuclei, with only moderate numbers of immunopositive fibers remaining throughout.

MDMA binge high dose rats also displayed large reductions in SERT axonal fibers across the caudal hypothalamus. MDMA binge high dose rats only expressed light amounts of SERT immunoreactive fibers across the posterior (PH), dorsal (PMd) and ventral (PMv) premmamillary, supramammillary (SUM), and tuberomammillary (TMN) hypothalamic nuclei, all of which exhibited a dense amount of serotonergic input in control, MDMA chronic low dose, and MDMA binge low dose rats. Only a sparse amount of 5-HT immunoreactive fibers was dispersed throughout the medial (MM) and lateral (LM) mammillary bodies: MM displayed only a moderate amount of 5-HT axons in control subjects, while LM displayed a dense amount of SERT labeled axonal processes.

106 Lastly, the visual and circadian hypothalamic nuclei including the suprachiasmatic (SCN), retrochiasmatic (RCN), and supraoptic (SO) nuclei contained a very dense amount of SERT immunoreactive fibers, which remained unchanged following pretreatment with a chronic low dose or binge low dose of MDMA. Figure 22 is a brightfield photomontage through the SCN for a representative control and MDMA binge high dose treated subject. Notice the very intense numbers of serotonergic fibers packed into the SCN in both the control and MDMA pretreated animal, with a significant reduction of SERTlabeled fibers in the surrounding PVH in the MDMA binge high dose rat.

Catecholaminergic Immunohistochemistry

No notable differences in TH immunoreactivity were observed between the control, MDMA chronic low dose, MDMA binge low dose, and MDMA binge high dose conditions, indicating no long-term effect of MDMA on catecholaminergic activity.

Figure 22 is a brightfield photomontage of coronal sections of the frontal cortex, displaying TH labeled fibers at two levels of the mPFC for a representative control and

MDMA binge high dose rat. Note the similarity in the pattern and intensity of TH immunoreactivity in both rostral and caudal PL. TH fibers were moderately dispersed throughout the prelimbic (PL) and medial orbital (MO) cortices. Progressing caudally, the prelimbic cortex and infralimbic cortex also showed dense fiber labeling in layers 5/6.

Light numbers of DA fibers were present across the other layers.

The ventral orbital, ventral lateral orbital, lateral orbital, and dorsal lateral orbital cortices exhibited comparable patterns of moderate TH fiber labeling. At the frontal pole, moderate numbers of immunoreacted fibers were observed at the rostral aspect of

107 VO. Light to moderate TH fibers were observed distributed fibers through layer 1-5 in

VO, VLO, LO, and DLO, with a moderate amount of TH fibers present in deep layer 6.

The most intense TH immunoreactivity was noted in the dorsal and ventral striatum and the olfactory tubercule. Figure 23 depicts photomicrographs of coronal sections demonstrating TH immunoreactivity across the rostral nucleus accumbens

(ventral, striatum, ACC) for a representative case in each of the four treatment conditions.

ACC exhibited very dense TH labeled fibers throughout the nucleus. The core showed a slight increase in the intensity of fiber labeling in comparison to the shell division across rostral levels, however the shell division of ACC caudally at the level of the fusion of the septum contained the most intense TH labeling across the entire forebrain. OT was very densely packed with TH positive fibers, and this density persisted throughout the rostral caudal extension of the nucleus. MDMA chronic low dose, MDMA binge low dose, and the MDMA binge high dose groups showed no differences in the pattern or density of innervation in these areas.

The dorsal striatum (caudate putamen, CP) also exhibited very dense TH immunopositive fibers. Figure 24 is a brightfield photomontage of a representative case for each of the four treatment conditions through CP at the level of the fused septum.

Note the intense TH labeling across all treatment conditions. At this level, dense TH fibers were also observed in the intermediate and lateral divisions of the lateral septum.

The very rostral expression of the rostral lateral septum (LSr) contained a dense number of TH immunopositive fibers, however at more rostral levels, both LSr, MS, and the diagonal band nucleus only displayed a light amount of TH fiber expression. The caudal

108 aspect of the intermediate division of the lateral septum (LSi) showed an increase, with a moderate amount of TH labeled fibers.

No differences in catecholaminergic fiber expression across the treatment groups indicated a selective neurotoxic effect on serotonergic terminals as witnessed in the

MDMA binge high dose when processed with SERT immunohistochemistry. All subjects displayed very dense TH immunoreactivity across ACC, CP, the globus pallidus, ventral pallidum, and the arcuate nucleus and lateral hypothalamus. Dense fiber expression was noted in the paraventricular hypothalamus nucleus (PVH), where TH positive neurons were not only densely packed in the A11 and A13 cell groups, but scattered throughout the entirety of PVH. As mentioned, dense TH fibers were located throughout the prefrontal cortex, and lateral septum. The majority of the thalamus in the rat was devoid of TH positive axons in rats. The paraventricular (PV) thalamic nucleus displayed moderate to dense amounts of TH immunopositive fibers, which increased in intensity in the posterior paraventricular nucleus. The lateral habenula also displayed a moderate amount of TH positive axons, as did the parafasicular nucleus (PF).

Moderate amounts of TH activity were also noted in the amygdala including the anterior amygdaloid area (AAA), central nuclei, lateral, basal lateral, and basal medial amygdala, and cortical amygdala in control rats. The medial amygdala only displayed a light number of TH positive axons. A moderate amount of TH positive fibers was detected in the magnocellular preoptic nucleus, claustrum, visual cortex, and perirhinal cortex, and insular cortices. The remaining cortical mantle only displayed a light blanketing of TH labeled fibers.

109

Figure 21: Brightfield photomontage through the suprachiasmatic nucleus (SCN) in the hypothalamus for a representative control and MDMA binge high dose treated subject. Notice the very intense numbers of serotonergic fibers packed into the SCN serotonergic input to SCN in both the control and MDMA pretreated animal, with a significant loss of SERT labeled fibers in the surrounding paraventricular hypothalamic nucleus (PVH) in the MDMA binge high dose rat. Scale bar, 100 µm.

110

Figure 22: Brightfield photomontage of TH immunoreactivity across the accumbens (ventral, striatum, ACC) nucleus and olfactory tubercule (OT) in a representative control (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA binge high dose (D) rat. Very dense fiber labeling was observed in both the core (ACCc) and shell (ACCs) division. OT was very densely packed with TH positive fibers. MDMA chronic low dose, No differences in the pattern and intensity of fiber labeling was noted in the MDMA binge low dose, and the MDMA binge high dose group. See list for abbreviations. Scale bar, 250 µm.

111

Figure 23: Brightfield photomontage of TH immunoreactivity across the accumbens (ventral, striatum, ACC) nucleus in a representative control (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA binge high dose (D) rat. Very dense fiber labeling was observed in both the core (ACCc) and shell (ACCs) division. No differences in the pattern and intensity of fiber labeling were noted in the MDMA chronic low dose, MDMA binge low dose, and the MDMA binge high dose group. See list for abbreviations. Scale bar, 250 µm.

112

Figure 24: Brightfield photomontage of TH fiber expression through the dorsal striatum (caudate putamen, CP) in a representative control (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA binge high dose (D) rat. Note the very intense TH labeling across all treatment conditions. At this level, dense TH fibers were also observed in the intermediate and lateral divisions of the lateral septum. See list for abbreviations. Scale bar, 250 µm.

113 There was a paucity of TH fiber labeling throughout CA3, CA1, and DG in all rats. A virtual absence of TH activity was found throughout the dorsal and ventral hippocampus. Only a few fibers are also seen across the subiculum, however both the presubiculum and postsubiculum show an increase in TH immunoreactivity, with light to moderate numbers of labeled fibers present in each division. There was also a moderate number of fibers present in the entorhinal (ENT) cortex, with an increase in density witnessed in the lateral ENT as compared to the medial division. The anteroventral, ventrobasal, ventral anterior lateral, ventral medial, and reticular nuclei also displayed a light number of fibers dispersed throughout the nuclei.

DISCUSSION

Rats treated with a high binge dose (2 x 7.5 mg/kg) of MDMA showed significant serotonergic forebrain axonal denervation one month later, but no impairments in TH immunoreactivity. A chronic low dose (10 x 1.5 mg/kg) and binge low dose (2 days of 4 x 1.5 mg/kg spaced 2 hours apart) of MDMA did not produce deleterious long-term effects on serotonergic or catecholaminergic axonal innervation of the forebrain. Long

Evans rats given these dose regimens exhibited SERT fiber density patterns similar to controls. MDMA binge high dose rats showed a substantial reduction in SERT axon fibers. However the pattern and extent of serotonergic damage was heterogeneous across specific regions and structures rather than global. The prefrontal cortex, including the prelimbic, infralimbic, anterior cingulate, orbital cortical divisions, and insular cortex was very negatively impacted by the high binge dose of MDMA. The dense SERT fiber expression seen in controls and rats treated with low doses was reduced to only light to

114 moderate amounts of remaining fibers. This same loss of SERT labeled terminals was observed across the entire neocortical mantle. Both the dorsal and ventral striatum were also highly sensitive to the effects of a high binge dose of MDMA. CP was one of the most vulnerable forebrain areas to the neurotoxic effects of the drug. SERT positive fibers were nearly annihilated, with only a few axons remaining. Despite this, there was no region, which was completely denervated of thin 5-HT axon fibers in response to the

MDMA binge high dose.

In MDMA binge high dose rats, widespread serotonergic denervation occurred across the hippocampal formation including CA1, CA2, and CA3, the subiculum, and the entorhinal cortex, specifically the lateral enthorhinal cortex. The dentate gyrus and medial entorhinal cortex only showed minor reduction in SERT immunoreactivity. A large loss of SERT fibers was also observed in the piriform cortex, olfactory tubercule, and lateral septum, and the anterior amygdaloid area, and the intercalated nuclei, piriform amygdalar area, and the cortical and posterior amygdala. There were only more subtle changes in SERT fiber expression in the nucleus of the bed stria terminalis, endopiriform nucleus, ventral pallidum, and claustrum, In spite of these regional changes; none of the neural structures were completely devoid of 5-HT input. Further caudally, the midline thalamic nuclei including RE, RH, CM, IAD, and IAM showed considerable decreases in

SERT fiber expression. The anteroventral and anteromedial thalamic nuclei also displayed reductions in the density of SERT labeled fibers. The lateral dorsal, lateral geniculate complex and intergeniculate nuclei also displayed a large loss of serotonergic terminations following a binge high dose of MDMA. There were also changes in the intensity of SERT fiber expression across several thalamic nuclei including the

115 paraventricular, interomedial dorsal nucleus, central lateral, and paracentral nuclei, and the subthalamic zona incertia, though less severe than midline and lateral thalamic nuclei.

Despite widespread loss, 5-HT input to certain neural structures remained unaffected by the binge high dose of MDMA. This included most of the hypothalamus, with the exception of the paraventricular, posterior, mammillary, and supramammillary nuclei, medial septum, cholinergic basal forebrain groups, preoptic nuclei, and globus pallidus. As expected, variable low and high doses of MDMA had no long term impact on dopaminergic innervation of the forebrain, as measured by TH immunohistochemistry.

There were no large differences in the pattern or intensity of TH fiber labeling between

MDMA chronic low dose, MDMA binge low dose, and MDMA binge high dose rats in comparison to controls. This has been well documented in previous studies, and demonstrates the selectivity of neurotoxicity to serotonergic fiber terminals, whilst leaving other monoaminergic systems in tact (McKenna and Petrouka, 1990; O’Hearn et al., 1988; Stone et al., 1986)

There is a paucity of data on the serotonergic neurotoxicity of MDMA in Long

Evans (LE) rats using immunohistochemical methods. Previous immunocytochemical studies which looked at tryptophan hydroxylase, serotonin, or the serotonin transporter used Sprague Dawley (SD), Wistar, or Dark Agouti rats (Kovacs et al., 2007; Kirilly,

2010; O’Hearn et al., 1998; Mamounas et al., 1991; Molliver et al., 1989). Early studies using very high doses of MDMA found patterns of serotonergic neurotoxicity similar to those described here; however, the degree of axonal degeneration was more severe.

MDMA at total doses of 20 mg/kg to over 200 mg/kg in SD rats over short periods of time produced near total denervation of several forebrain structures including the frontal

116 cortex, neocortex, and striatum (O’Hearn et al., 1988; Mamounas and Molliver, 1988;

Molliver et al., 1990; Mamounas et al., 1991). Likewise, Xie et al. (2006) replicated earlier findings in SD rats: a total dose of 45 mg/kg given orally across 4.5 hours nearly destroyed all SERT positive axons in the parietal cortex, which is densely labeled in control cases. The results in this experiment show that 2 x 7.5 mg/kg MDMA produced substantial serotonergic damage to forebrain input, however, all targets still had light to moderate SERT immunoreactive axons.

There are two factors which could account for the differences in the intensity of damage between this experiment and other studies. First, the dose used in this experiment was not as large as those used in previous experiments. Behaviorally, LE rats in the current study demonstrated the behavioral and physiological signs of serotonin toxicity following administration of i.p. MDMA injections. This dose was selected to induce serotonin syndrome, without producing lethal effects. Even so, a dose of 2 x 7.5 mg/kg produced death hours following the injections rats in both experiment 2 and 3.

Conversely, little difference in the extent of forebrain denervation was seen across escalating doses of PCA, MDMA, and MDA, indicating a ceiling effect for the drug. For example, previous studies of this laboratory (Hughes et al., 2007; Vertes et al., 2007) examined variable doses of PCA (2 x 5mg/kg PCA or 2 x 10 mg/kg PCA) in Sprague

Dawley rats. The findings of these studies concluded there was no difference in the amount of 5-HT denervation of the forebrain between the two doses. Both doses produced widespread destruction of SERT labeled fibers, leaving some structures with only a minimal number of fibers. While the loss of 5-HT fibers as a result of PCA was significantly more injurious than the histological results of the current MDMA group, in a

117 similar manner, doses of 2 x 7.5 mg/kg or 3 x 15 mg/kg may produce similar physiological and subsequent neurotoxic effects.

Likewise, Skelton et al. (2008) found no behavioral differences in rats given one dose regimen of 4 x 15 mg/kg s.c. or rats given five weekly regimens of the same dose.

Differences in sensitivity to MDMA across rat strains may account for differences in the intensity of axonal degradation. SD rats have shown a heightened sensitivity to the behavioral and neurotoxic effects of MDMA in comparison to both Wistar and Dark

Agouti rats (Chu et al., 1996; Muck-Seler et al., 1998; O’Loisigh et al., 2001; Zhou et al.,1996). This same difference in sensitivity may exist between LE rats and SD rats, however there are no current studies examining a direct comparison of doses in the two rat strains.

Early tracing studies have delineated two classes of 5-HT axons, based on their origin: DR neurons have 5-HT axons with thin fibers and small minute varicosities whilst

MR axons are thick with large beaded varicosities (Kosofky and Molliver, 1987,

Molliver, 1987). Amphetamine analogues show a selective destruction of thin fibers. 5-

HT axonal denervation is morphologically specific, whereby 5-HT fine axon fibers are selectively vulnerable to the deleterious effects of MDMA and related compounds, and the larger beaded terminals with thick varicosities remain immune to the degrading effects of the drugs. The few remaining fibers in neural structures which showed significant loss of serotonergic input were thick, and displayed large varicosities

(Molliver 1987; Mamounas et al., 1991). However, it is very possible the origin of these vulnerable fine 5-HT axonal fibers may be associated with other raphe nuclei.

118 MDMA binge high dose rats displayed similar patterns of reductions in SERT labeled fibers. Structures targeted heavily by DR showed substantial loss of SERT positive fibers. The majority of structures targeted primarily by MR showed little to no change in SERT immunoreactivity. By contrast, both fine and beaded remaining axons were visualized in these cases. This distinction can be best visualized by examining the effect of MDMA on neural structures which receive their serotonergic input exclusively from DR or MR. The subdivisions of the septal nuclei display a distinct segregation of raphe input. The DR projects to the intermediate lateral septum, whereby the medial septum and lateral division of the lateral septum receive their 5-HT projections from MR.

This pattern of fiber labeling found in the MDMA binge high dose group: a significant reduction in SERT fiber labeling, is seen throughout the intermediate lateral septum, with the density of fibers in the medial septum remaining unscathed in comparison to controls.

In the visual circadian system, MR projects solely to the SCN whereby serotonergic input to the LGN and IGL originate from DR (Meyer-Bernstein and Morin,

1996; Vertes et al., 2007; Vertes and Linley, 2008; Vertes et al., 2009). Similarly, a very significant loss of 5-HT fibers was present across the LGN complex. In comparison, there was no loss of density of SERT fibers in the SCN in the MDMA binge high dose group. Though McCann and Ricaurte (2007) did find a reduction of 5-HT immunoreactivity in SCN following MDMA, our laboratory has found no evidence of decreased 5-HT and SERT expression in the SCN following administration of the related amphetamine analogue PCA (Vertes et al., 2007).

There were some regional inconsistencies in the present study regarding the hypothesis of variable susceptibility of the neurotoxicity in DR and MR following

119 MDMA. For instance, there was a widespread and significant loss of 5-HT fibers throughout the hippocampus. Serotonergic innervation of the hippocampus is supplied primarily by the MR. DR does send a significant number of fibers to the hippocampal formation, however DR mainly targets the dorsal hippocampus. Mamounas et al. (1991) described a specific loss of 5-HT fine axon fibers originating from DR in the dorsal hippocampus. Here, a reduction in SERT immunoreactive fibers was observed across the entire dorsal and ventral hippocampus, with only a slight increase in fibers present in the ventral HPC in high dose rats. By contrast the posterior hypothalamus and mammillary nuclei, which are thought to receive large amount of serotonergic input from MR, showed a substantial loss of SERT immunoreactivity in MDMA binge high dose rats.

Cholinergic groups, such as the substantia innominata, which receive large 5-HT projections from DR, showed little changes in serotonergic density following a high binge dose of MDMA. The differences in susceptibility of MDMA neurotoxicity in morphological 5-HT axons may encompass structures beyond DR and MR. For instance, both B9, and the caudal linear nucleus contain 5-HT neurons sending input to the forebrain, and caudal raphe nuclei have displayed ascending projections to forebrain structures in tracing studies (Bowker, 1986; Loiser and Semba, 1993; Parent et al., 1981;

Vertes and Crane, 1997). The morphology of these axons may also be vulnerable to

MDMA neurotoxicity.

Despite previous studies that support the delineation of MDMA neurotoxicity across the two types of fibers, there is little evidence as to why these thin axons show preferential axonal degradation. Brown and Molliver (2000) postulated that differences of 5-HT fibers in vulnerability to neurotoxicity may be related to the expression of the 120 serotonin transporter protein. DR sends heavy projections to the core of the nucleus accumbens, while MR sends input to the shell division. 5-HT fibers located in the core of the nucleus accumbens heavily express the SERT protein, where as 5-HT fibers in the shell area had low [SERT]. MDMA and methamphetamine produced a large denervation of the core, but not the shell (Brown and Molliver, 2000). No studies have replicated this finding, and we have found no differences between the expression of 5-HT and

SERT in our immunohistochemical studies, indicating differences in expression may in fact be differences in the density of innervation of the two subregions of the ventral striatum (Vertes et al., 2007).

Injection schedules in the MDMA chronic low dose and MDMA binge low dose groups were chosen to assess MDMA’s impact on the serotonin system when given in doses which may better reflect human intake by using effect scaling. The previous aforementioned studies justify using doses of MDMA 200 x or greater than those consumed by humans based on allometric scaling: an algorithm taking the small body mass and increased basal metabolic rate of rats results in an equivalency of a 10 mg/kg dose of the drug in rats to 1.75 mg/kg in humans (Baumann et al., 2007; Savage et al.,

2008). Allometric scaling is a common method in determining initial human dosages in preclinical drug development studies. Many pharmacokinetic factors have been found that are also involved in the determination of interspecies dosing, including the route of administration, distribution and binding sites of the drugs, and liver enzymes and metabolic routes (Sharma and McNeill, 2009). To try and account for these differences

“effects scaling” was proposed as an alternate interspecies dose scale for MDMA across rodents and primates. MDMA, at a dose of 1-2 mg/kg in the rat (s.c.) and human (taken

121 orally, PO) produced similar increases in [5-HT] and [DA] and metabolites in plasma, drug secretion, and neurohormonal response. This dose also has reinforcing properties in rats using self-administration and operant conditioning paradigms. (as reviewed by

Baumann et al., 2007). Based on these findings, it has been postulated that effects scaling is a better predictor of interspecies dose scaling, as compared to using an allometric equation.

Low doses of MDMA given in either a daily or binge regimen produced no long term detrimental effects on the serotonin system when assessed immunohistochemically.

These findings are congruent with reports, which have found similar results using other behavioral and histological methods. While high doses of MDMA produce hyperthermia, this effect is not seen in the low binge regimen of 1.5 mg/kg MDMA.

MDMA induced 5-HT neurotoxicity is known to be dependent on hyperthermia.

Prevention of hyperthermia attenuates subsequent serotonergic damage. Dopamine is crucial to the hyperthermic effects of the amphetamine compounds and pharmacological intervention which blocks MDMA’s effects on the dopamine system also prevents hyperthermia and subsequent 5-HT neurotoxicity (Cadet et al., 2007; Green et al., 2003,

2004; Lyles and Cadet, 2003).

In the current study, while rectal temperatures were not obtained directly, MDMA chronic low dose and MDMA binge low dose rats did not exhibit physical signs of hyperthermia or serotonin syndrome while under the influence of the drug. A more detailed description of the behavioral effects of MDMA is discussed in experiments 2 and

3. Multiple binge injections of a low dose of MDMA has been previously ineffective in reducing [5-HT] in the cortex or striatum using HPLC methods, while higher doses show

122 a significant reduction in [5-HT] neural tissue (Baumann et al., 2001; O’Shea et al.,

1998). Based on these findings, Baumann and colleagues (2007) have now ascribed the term behavioral dose and noxious dose to describe their injection regimens, rather than low and high doses.

There were no differences in serotonin forebrain innervation between MDMA chronic low dose and MDMA binge low dose groups. That is, 1.5 mg/kg injected 4 times daily every two hours across two consecutive days did not have a long-term effect on 5-

HT, demonstrating no additive effects of binge dosing. However, as mentioned, there are differences in the MDMA metabolic pathway across rats and humans. It has been argued that humans display an autoinhibition of MDMA metabolism, so that 1-hour after ingestion, if more is consumed, blood plasma concentrations increase. This inhibition persists for at least 24 hours, and some researchers have speculated self inhibition of

MDMA metabolism may last for days. Farre et al. (2004) administered 2 x 100 mg

MDMA to healthy participants in a controlled setting spaced one day apart, and found the second dose produced an increase in drug plasma concentrations and augmented somatic and behavioral effects when compared with the first dose. This led to speculation that binge consumption cannot be modeled in rats, and does not reflect binge Ecstasy intake in recreational users (Green et al., 2009). However, a recent study found separate metabolic pathways both in vitro rat liver microsomes and human liver cells which fuelled autoinhibition of the compound (Antolino-Lobo et al., 2010). This suggests behavioral and physiological effects in binge regimens across species to be comparable.

123

EXPERIMENT 2

INTRODUCTION

In humans, deficits in attention and executive functioning, working memory, and episodic and verbal memory have been found in current and former Ecstasy users (Fisk et al., 2005; Fox et al., 2001, 2002; Montgomery and Fisk, 2007; Quednow et al., 2004,

2005, 2007; Wareing et al., 2004; Soar et al., 2004; Nulsen et al., 2010; Ward et al., 2006;

Zakzanis and Campbell, 2006). While other studies report no lasting impairments directly related to Ecstasy use, polydrug use that included THC and amphetamines was correlated with memory deficits (Golding et al., 2007; Gouzoulis-Mayfrank et al., 2005;

Groth-Marnat et al., 2007; Jager et al., 2009; Hanson et al., 2008; Hanson and Luciana,

2010; Hoshi et al., 2007; Lamers et al., 2006; Roiser et al., 2007; Smith et al., 2006). The amount of drug, duration of use, length of abstinence, and demographics of subjects included in these studies contribute to these conflicting results. Though long-term memory deficits have been strongly associated with verbal tasks (Laws and Kokkalis,

2007; Schlit et al., 2007; Wareing et al., 2000, 2004). A more definitive way to extrapolate the impact of MDMA on cognition and memory is the utilization of animal models to eliminate confounds.

124 Subsets of memory systems are governed by different neural systems. Working memory is known to be reliant on the prefrontal cortex, in accordance with several other neural systems, whilst long-term declarative memory is thought to rely on structures within the medial temporal lobe (including the hippocampal formation, amygdala, and rhinal cortices) and maintained in cerebral cortical structures. These memory systems are also involved in the transformation of the acquisition and encoding of latent memories in short term memory, their consolidation into a stable permanent state, and the reactivation of these memories, where reconsolidation or degradation can occur (Squire, 1987; Squire and Kandel, 1999; Eichenbaum, 2002). Developing paradigms that can measure memory directly in rodents in a language independent manner that is translational across mammalian species is difficult. Spatial learning is one such paradigm that may allow evaluation of episodic memory that is language independent. Physical attributes of the surrounding environment such as distance, direction, depth of water, and other physical dimensions are thought to be represented topographically in the hippocampus (O’Keefe and Nadel, 1978; Eichenbaum, 2002).

In rodents, one type of memory which is associated with episodic memory is spatial memory and navigation. These tasks may require “episodic” recall of a location in space, a cognitive map, or a “route to destination.” Both idiothetic and environmental cues in their environment are used to remember the place or locations. Versions of the

Morris water maze can be used to test long term and working memory as well as prefrontal functioning. Over a series of trials, rodents use allothetic and idiothetic cues to swim and navigate to the platform to escape the water. Repetitive trials allow rodents to theoretically “remember” the location of platform. This task was originally developed to

125 determine if hippocampal lesions impaired acquisition of place location of the submerged platform (Morris et al., 1982).

Typically, the submerged hidden platform is placed in one of four quadrants, divided into equally spaces across the circular arena (pool). Initially, naïve rats will spend longer times swimming to find the hidden platform. After several trials, this time is greatly reduced and after several training sessions, an asymptotic level of performance is attained where rats swim directly to the escape platform, despite their release point in the maze. Shorter latencies to find the platform location presumably indicate the utilization of the surrounding spatial cues to navigate. After reaching asymptotic escape latency, rats undergo a probe test, where the platform is removed from the maze. The rat’s location in the maze is tracked and recorded with a video camera. Rats who recall the platform’s location will spend an above chance amount of time searching in the quadrant where the platform was located during training (Vorhees and Williams, 2006).

The water maze paradigm has been used extensively to test the functions of different neural structures, and the effects of pharmacological agents on memory.

Subsequent modification of variables in the water maze have allowed additional memory systems to be evaluated. Evaluation of behavioural flexibility has been conducted using a spatial reversal test. Boulougouris and Robbins (2010) stated, “by definition, reversal learning presupposes retention of a previously acquired discrimination.” The previously learned but currently irrelevant information must be applied to adapt behavior to respond to a new correct stimulus. Reversal learning, often used interchangeably with cognitive or behavioral flexibility, is an example where previously learned information is used in a flexible manner to modify behavior to make a correct selection.

126 One way to test behavioral flexibility in rats is through spatial reversal learning using a variation of the Morris water maze. The prefrontal cortex was shown to be critical to complete the reversal task (De Bruin et al., 1994, 1997, 2001). This variation involved first learning the location of the hidden submerged platform. The platform was then moved to the opposite quadrant (reverse position). During this spatial reversal learning phase, the rats undergo training to learn the new platform location. Behavioral flexibility is examined by determining if rats can disregard the previously learned place information to navigate to the new location. Perseveration of the prior location would be apparent if increased latencies to find the platform are found or if significantly more time is spent searching the previously correct quadrant rather than the new correct reversal location during a probe trial.

The prefrontal cortex is intricately involved in executive functions; including decision-making, behavioral strategies, behavioral inhibition, and behavioral flexibility

(Chikazoe, 2010; Chudasama and Robbins, 2006; Dalley et al., 2004, 2008; Holmes and

Wellman, 2009; Kehagia et al., 2010; Robbins, 2007; Robbins and Arnsten, 2009). Rats with lesions of the medial prefrontal cortex did not show impairments during the acquisition and retention phases of a water maze task, but were impaired on the reversal task. (De Bruin et al., 1994). Medial prefrontal cortical lesions also impair egocentric response learning in the water maze, leaving allocentric place unaffected (De Bruin et al.,

1997, 2001; Lacroix et al., 2002; Jo et al., 2007). Furthermore, the integrity of the orbitofrontal cortex is necessary for optimal acquisition, consolidation, and recall of spatial learning with a water maze paradigm (Vafaei and Rashidy-Pour, 2004).

127 Previous research examining the behavioral and mnemonic effects of MDMA have focused primarily on the Sprague Dawley (SD), Wistar, and Dark Agouti strains of rat (Able et al., 2006; Adori et al., 2010; Chipana et al., 2008; Kurling et al., 2008;

Morini et al., 2010; Schaefer et al., 2008; Shioda et al., 2008; Skleton et al., 2006, 2008;

Thompson et al., 2009). Studies have found that non-spatial hippocampal dependent tasks such as novel object recognition, were not affected by acute high doses of MDMA, despite the presence of neurotoxic serotonergic denervation (Able et al., 2006; Ludwig et al., 2008; Meyer et al., 2008; Piper and Meyer, 2004).

The effects of MDMA on spatial memory have been contradictory, and the results of studies weigh heavily on age of rats, dose of the drug, and time of delivery (Able et al.,

2006; Arias-Cavieres et al., 2010; Camarasa et al., 2008, 2010; Cunningham et al., 2009;

Skelton et al., 2008; Sprague et al., 2003). Sprague et al. (2003) administered a total dose of 40 mg/kg (2 x 20 mg/kg) MDMA s.c. to adult Sprague Dawley rats and began MWM testing one week later. MDMA treated rats showed no impairments in acquisition of platform location during training however a deficit in retention of the platform location was witnessed during the probe trial. Recall deficits corresponded with a reduction in [5-

HT] in hippocampal tissue (Sprague et al. 2003).

Several experiments tested the effects of some amphetamine analogues on spatial memory in Sprague Dawley rats using the Morris and Cincinnati water maze (Able et al.,

2006; Schaefer et al., 2008; Skelton et al., 2008; Vorhees and Williams, 2006). The

Cincinnati water maze (CWM) which is a “labyrinth” maze with opaque dark borders eliminates all distal cues. This test measures path integration, where rats must navigate from start to finish solely using their ideothetic cues. The Morris water maze (MWM),

128 by contrast, tests place learning, whereby rats rely on distal cues and egocentric learning to navigate to the submerged platform (Vorhees and Williams, 2006; Skelton et al.,

2008). These studies examined the 1.) deficits in reference memory and path integration after MDMA administration during crucial postnatal periods and 2.) whether or not these deficits persist into adulthood, and 3.) behavioral impairments are associated with monoaminergic dysfunction as measured by HPLC (Williams et al., 2003; Vorhees et al.,

2003; Vorhees et al., 2004; Cohen et al., 2005; Skelton et al., 2006; Skelton et al., 2008).

Able et al. (2006) tested the effects of MDMA in adult SD rats and replicated results similar to Sprague et al. 2003. Rats were tested on the CWM and MWM following a dose regimen of 4 x 15 mg/kg MDMA s.c. There were deficits in path integration in the CWM and recall in the MWM. Pretraining in the CWM did not attenuate impairments in the MWM (Able et al. 2006). In a follow up study, Skelton et al. (2008) found results that conflicted with both studies. Following a single dose regimen of either 4 x 15 mg/kg MDMA s.c or 5 weekly treatments of 4 x 15 mg/kg s.c.

MDMA, rats were tested on a novel object recognition task, the CWM, MWM, and a

MWM reversal. Both the single binge regimen of 4 x15 mg/kg or weekly binge regimen produced significant reductions of [5-HT] in neuronal tissue, though the five week group did exhibit some recovery of loss. MDMA treated rats showed pronounced deficits in path integration in the CWM; however, there were no deficits in the acquisition, recall or reversal stages of the MWM. However, when platform size was reduced to ¾ of its original size in a double reversal phase, recall of the platform was impaired.

Pretraining during the Cincinnati water maze may account for the differences between these studies. However, Able et al. (2006) found MWM retention impairments

129 despite pretraining using the “dead reckoning” task. The experiments conducted by

Vorhees and colleagues found a significant loss of serotonin in forebrain tissue as measured by HPLC (Able et al., 2006; Skelton et al., 2008). As discussed previously, the doses employed in these studies were neurotoxic, but do not necessarily reflect

MDMA/Ecstasy intake in humans (Baumann et al., 2007, De La Garza et al., 2007;

Easton and Marsden, 2006; Schenk, 2009).

Adolescence is a crucial time of neurohormonal and morphological development of the central nervous system, and a critical time when recreational drug use and substance abuse begins. To model, researchers test rats during their periadolescent window, which starts approximately around postnatal day 28 (Smith, 2003). The effects of more moderate doses of MDMA, similar to human recreational Ecstasy intake, were tested using place learning in the MWM in periadolescent rats. SD rats were given 0.2 or

2.0 mg/kg MDMA s.c. one hour prior to training sessions in the MW. MDMA impaired acquisition of the platform location. There was no difference in the recall of the platform location during the probe trial. These moderate doses of MDMA produced no reductions in [5-HT], however, in post-mortem in vitro hippocampal slices, long term potentiation

(LTP) induction was impaired (Arias-Cavieres et al., 2010). This study is important to the literature on Ecstasy use because it is one of the first animal studies to illustrate that non-neurotoxic doses of MDMA, similar to human intake, can disrupt mnemonic functioning while under the influence of MDMA that subsequently may affect synaptic plasticity.

Cunningham et al. (2009) also found a difference in recall of platform location in the MWM during the probe session when Sprague Dawley rats were pretreated with 4 x

130 7.5 mg/kg i.p. MDMA, but acquisition and reversal was left unaffected. In addition, chronic unpredictable stress (CUS) was coupled with MDMA, as chronic stress is a common among drug users. Chronic unpredictable stress alone did not impact performance in the MWM but exacerbated the deficits of MDMA (Cunningham et al.,

2009).

In the current study, administration of variable doses of MDMA on place and spatial reversal learning was tested using Long Evans (LE) rats. Very few studies have assessed the behavioral, physiological, and neurotoxic effects of MDMA in the Long

Evans (LE) rat strain. Ethanol potentiates the physiological and behavioral effects of the

MDMA in LE rats (Cassel et al., 2004, 2006; Ben Hamida et al., 2007; Hamida et al.,

2008, 2009; Riegert et al., 2008; Jones et al., 2010). In periadolescent Long Evans’ rats after a binge regimen of MDMA (3 x 10 mg/kg i.p.); LE male rats showed greater hyperlocomotor responses, and had a greater mortality rate related to lethal hyperthermia than female rats. However, there were no differences in neurotoxicity (Koenig et al.,

2005). Subcutaneous injections of 15 mg/kg MDMA produced memory impairments in both a novel object recognition task and the MWM. These deficits were rescued when memantine, a nicotinic and NMDA receptor antagonist, was coadministered. MDMA does not have an impact on object recognition in the SD rats, however LE rats exhibited significant impairments. MDMA pretreated LE rats also show deficits in retention of platform location in the MWM (Camarasa et al., 2008).

This study examined the effect of MDMA on place and spatial reversal learning in the MWM using adult Long Evans rats using interspecies “effects” scaling discussed in Experiment 1 to model human Ecstasy intake. To test the long term effects of both low

131 dose and high dose regimens of MDMA, rats were given a dose regimen of MDMA and tested with a water maze protocol similar to those used in prior studies (Vorhees and

Williams, 2006). LE rats were given one of the following MDMA treatment regimens: a chronic low dose of 10 daily injections of 1.5 mg/kg spaced across two weeks; a low dose binge regimen (2 daily regimens of 4 x 1.5 mg/kg with injections spaced two hours apart, or two injections of 7.5 mg/kg spaced two hours apart. These groups were compared to two control groups given saline injections in either a chronic or binge injection schedule. Following two weeks of recovery, rats were trained on the Morris water maze and given a retention probe test. Rats then underwent training and were tested on a reversal task. Lastly, a final serial reversal test, or “double reversal” task was conducted (see Vorhees and Williams, 2006). During this probe trial, MDMA pretreated rats received a drug challenge of low dose (2.0 mg/kg) MDMA to test the effects of retention and search strategies while under the effect of the drug.

It is expected the binge high dose of MDMA should produce deficits in recall of the platform location during the retention probe, however acquisition or the reversal task should not be impacted. Several studies have found specific deficits in the retention probe trial during MWM tests (Able et al., 2006; Camarasa et al., 2008; Sprague et al.,

2003). Reference memory, as measured in a radial arm maze task was also hindered following high doses of MDMA (Kay et al., 2009, 2011). Low dose regimens of

MDMA, which do not produce 5-HT neurotoxicity, are not expected to impair performance on the acquisition, retention, or reversal MWM tasks. Previous studies have found no effect of MDMA on the acquisition and retention of spatial reversal in the

MWM (Able et al., 2006; Cunningham et al., 2009; Skleton et al., 2008). While the

132 acquisition of reversal and subsequent serial reversal phases is predicted to be unaffected, inter-trial analysis for the probe tracks were evaluated to determine if MDMA produced more subtle effect, such as perseverative inter-probe search strategies. Lastly, it is hypothesized that low doses of MDMA administered immediately before the final serial reversal probe trial may impair recall of the platform location in the MDMA chronic low dose, MDMA binge low dose, and MDMA binge high dose conditions. Low doses of

MDMA given immediately prior to MWM trials impaired acquisition and recall (Arias-

Cavieres et al., 2010).

MATERIALS AND METHOD

Subjects

Long Evan rats (n=39; Charles Rivers, Wilmington, VA) weighed 275-300 grams at the start of the experiment. Rats were originally housed in pairs until the onset of their injections schedules, then were moved to individual plastic cages and maintained on a

12hr light/dark cycle with lights on at 7 A.M. Rats had access to food and water ad libitium until the start of behavioral testing. All protocols were approved by the Florida

Atlantic University Institutional Animal Care and Use Committee and held in accordance to the National Institutes of Health guidelines for the care and use of laboratory animals.

Body weights were obtained regularly throughout the experiment.

Dose Regimen

Rats received one of the following treatment conditions: MDMA chronic low dose (10 x 1.5 mg/kg daily for 5 consecutive days over two weeks); MDMA binge low dose (4 x 1.5 mg/kg every two hours for two consecutive days); MDMA binge high dose

133 (2 x 7.5 mg/kg); saline administered in a chronic (10 daily) or binge (2 x 1 day) injection schedule. Rats were randomly assigned to one of the five groups prior to the start of the experiment. All injections were given i.p.

Drugs

3,4-methylenedioxymethamphetamine (MDMA) was gifted from the National

Institute of Drug Abuse (NIDA), and dissolved in physiological saline and injected at a volume of 1ml/kg.

All rats were given two days of room habituation, followed by two days of saline injections to familiarize them with the environment and reduce stress. Next, the respective treatment regimens began. Rats were wheeled into the injection room 30 minutes prior to the start of their daily session and were left in the test area a maximum of

8 hours after their last injection to ensure that they would return to the vivarium without being under the influence of MDMA.

Maximum onset of the drug occurred 20-30 minutes after the injection. The half- life of i.p. MDMA in rats is less than 1 hr, and biologically active metabolites’ half-life is approximately 2 hr (Baumann et al., 2009). Rats were monitored every 15 min and assessed for signs of serotonin toxicity: rearings, increased locomotive movement, head shakes, body tremors, increased salivation, piloerection, forepaw treading, penile ejaculate, defecations, hind limb splaying, hyperthermia, and straub tail. In addition, rats that showed signs of hyperthermia were rescued in a cool water bath (n=3).

Apparatus

Water maze: a white polyethylene circular tank measuring 70 inches in diameter and 30 inches in height. The tank was filled with tap water to 22 inches and kept at a 134 temperature of 21-23 degrees Celsius using two electric aquarium heaters. The water was made opaque by using a nontoxic Tempra latex white paint so that the submerged platform was visually hidden. The square platform measured 10 cm, and was attached to a polyethylene circular base (20 inches in height). The maze was surrounded by large distal cues. Black curtains partitioned the maze from the rest of the room. A monochromatic camera mounted in the ceiling above the maze was attached to a computer tracking system (Ethovision 3.1; Noldus Information Technology, US) to record swimming trials. Immediately following each trial, rats were placed in a Plexiglas holding cage with a suspended heat lamp and fan to prevent hypothermia. Figure 25 is an illustrative drawing of the water maze arena displaying the release points, quadrants, and platform locations used in the experiment.

Procedure

Spatial Memory

Figure 26 is a flow chart outlining the timeline and procedure for the water maze protocol used in this experiment. Training began fourteen days after the last drug injection. Four days prior to the first training session, rats were given two days of room habituation, followed by two days of pool habituation. During pool habituation, the platform was placed in the center of the maze. Rats were placed into the maze in one of 4 release points, named to its cardinal direction: Northwest (NW), Northeast (NE),

Southwest (SW), or Southeast (SE). Release point was pseudorandomly generated for trials, but fixed across rats. Rats were allowed to explore the arena for 90 seconds before being guided to the center platform. The rat was left on the platform for 60 seconds before being removed from the maze.

135

Figure 25: Scaled illustrative drawing of the water maze apparatus used in experiment 2. Quadrants represented in this diagram are labeled respective to their cardinal directions and the experimental design (start, target, adjacent, and opposite) used in the retention probe. Shown above is the platform location. The center and surround are referred to as the target search zone = 2 x radius of the platform.

136

Figure 26: Outline and timeline of the behavioral paradigms used in Experiment 2.

137 Rats were trained across 5 daily sessions. During the daily training session, a series of 4 trials were given. During each session, rats were released from each of the four release points at the cardinal directions of North (N), South (S), East (E), and West (W) for each trial and allowed to swim and locate the submerged hidden platform (NW quadrant). The order of quadrants for each training session was pseudorandomized but remained constant for all rats. During each training trial, if the platform was not reached after 90 seconds, the rat was guided to the platform. Once the rat reached the platform, it remained there for 60 seconds to become familiar with the allothetic cues before being removed from the maze. With the exception of two groups (n=2; n=3), rats were tested in groups of 4 during each daily session. This allowed the rats to have inter-trial intervals

(ITI) spaced in between each of the 4 trials. During the ITI, rats were placed under the heating lamp and fan to prevent hypothermia.

On the sixth day, rats were given a probe test. The platform was removed from the maze. Each rat was released from a novel start point and allowed to search and swim for the platform for 60 seconds. The start release point for the retention probe test was the

SW quad is referred to as the start quadrant. For each probe test, the start quadrant was randomly selected, but remained consistent for all rats. The target quadrant refers to the quadrant in which the platform was located for during the training sessions (NW quadrant). The opposite quadrant was positioned in the opposite diagonal space of the maze (SE for the retention probe). The adjacent quadrant to the target quadrant was the

NE quad for the retention probe.

138 Spatial Reversal Task

Following the probe test, rats underwent three daily training sessions for the reversal phase. During this training, the platform was placed in the opposite quadrant

(SE). Each reversal session consisted of 4 trials. The order of release points for each trial was again randomized but fixed for all rats in a session. One day after the last reversal training session, rats received another probe test to assess learning of the new location of the platform. The start quadrant was selected pseudorandomly (NE quadrant).

As stated, the target quadrant was now the SE quadrant, the adjacent quadrant was the

SW quadrant, and the opposite quadrant was the NW quadrant, the target quadrant from the retention probe.

Serial Reversal and Drug Challenge

A rest period of three days was given after the second reversal probe test. Three daily training sessions were conducted with the platform returned to its original location, the NW quadrant. This was followed 24 hours later by a serial reversal drug challenge probe test. In the drug challenge probe, all rats that had previously received a MDMA dose regimen were given 2.0 mg/kg MDMA thirty minutes prior to the 60 sec probe trial to test place recall under the influence of the drug. Controls received injections of saline.

For the probe trial, the platform was removed; rats were released into the pool from the

NE quadrant (start quadrant) and allowed to explore the water maze for 60 sec before being removed. After the final probe test, rats were immediately perfused and their tissue was processed for immunohistochemistry.

139 Histological Analysis

Rats were deeply anesthetized with isoflurane, intracardially perfused with 30-

50ml of ice cold heparinized phosphate buffered saline (PBS) to exsanguinate the rat, then fixed with 150-200 ml of ice cold 4% paraformaldehyde in 0.1 M phosphate buffer

(PB). Brains were removed, post fixed in 4% paraformaldehyde for 24-48 hours and then placed in 30% sucrose for at least 48 hours. Following sucrose cryoprotection, 50um coronal sections were cut throughout the forebrain up to the rostral raphe nuclei on a freezing sliding microtome and collected in 0.1 M PB and prepared for SERT immunohistochemistry. Loss of 5-HT fibers was visualized using an antisera for the serotonin transporter protein (SERT) as described in detail in Experiment 1.

Data Analysis

Ethovision 3.1 (Noldus Information Technology, US) recorded individual training and probe trials were recorded via video tracking software. The mean escape latency to reach platform (seconds), cumulative distance to platform (CDT, centimeters), and mean velocity (swim speed, centimeters per second) were extrapolated and analyzed for each block of daily trials (mean average of 4 trials for each training day) for the acquisition of spatial memory, reversal, and serial reversal training sessions. The platform distance can be used as an accurate measure of the route a rat takes to reach its target location, and can be more accurate if motor deficits or swim speed impacts mean latency scores.

Each measure was analyzed using a repeated measures analysis of variance

(ANOVA) with daily session as the within-subjects factor and drug treatment condition as the between-subjects factor. Posthoc analysis was calculated using the Tukey HSD test, when equal covariance was assumed, and the Tamahane’s T2‘s test when covariance

140 was not equal among treatment groups. Both univariate and multivariate ANOVA were calculated for mean velocities across daily sessions for training phases. Mauchley’s test of sphericity tested significance differences in covariance. If a significant difference was present, the Greenhouse Geisser correction was used to determine repeated ANOVA results, and is denoted by the change in degrees of freedom.

For each probe trial, repeated measures multivariate analysis was conducted on percent time spent across each of the 4 quadrants. To examine search extinction and search strategies, the total search duration in each quadrant was analyzed across 15- second increments from start to finish of the 60 sec probe trial by conducting a repeated

ANOVA with total search time in quadrant being the dependent variable, time interval as the within subjects factor (0-15, 15-30, 30-45, and 45-60 seconds) with a between subjects factor of treatment condition.

Univariate analysis of variance tests were conducted on swim speed, first latency of occurrence to the target quadrant, first latency of occurrence in the target search zone, and mean distance traveled to the center of the target platform. Individual probe trials recorded in Ethovision were converted to tiff files and imported into Adobe Photoshop.

Individual probe tracks were analyzed for each subject in every treatment condition to assess differences in search strategy and to eliminate any indiscernible search patterns or thigmotaxis. Representative subject for each group were selected by overlaying individual tracks over each other in photoshop and selecting the case which displayed the most universal swimpath.

Circular zones were created in the NW and SE quadrants surrounding the platform location within the target quadrants. These small (center) and large (surround, 141 twice the circumference of the platform) zones of NW or center SE was known as the target search zone and analyzed. Measures such as number of crossings and time spent in target search zone were analyzed. Percent of time spent in each of the 4 quadrants was calculated using the following equation: total duration (s) in Quad X / by 60 seconds

(total duration of probe trial) x 100 = % Time spent in Quad X. Estimates of effect size

2 were calculated using partial eta squared (np ). All measures calculated used an alpha level of .05 to reach statistical significance. All statistics were run in SPSS 170.0 and

18.0 for Mac and PC.

RESULTS

Physiological Effects of MDMA

MDMA chronic low dose rats (10 x 1.5 mg/kg) behaved similar to controls.

There was a brief increase in psychomotor behavior, including increased rearings and locomotor activity, along with sensitivity to noises. These rats returned to a normal behavioral state soon thereafter. MDMA binge low dose rats (2 days of 4 x 1.5 mg/kg) showed motor patterns very similar to the MDMA low chronic group following their first daily injection. However, after subsequent injections across the hours, they displayed active psychomotor profiles that included increased rearings, locomotor activity, defecations, and piloerection.

MDMA binge high dose rats (2 x 7.5 mg/kg) showed signs of serotonin syndrome by 20 minutes following the first injection. These included increased psychomotor effects, defecations, piloerection, ejaculation, head weaving and bobbing, forepaw treading, hind limb splaying, and increased body temperature. These symptoms 142 persisted through the first and second injections and continued for 4-6 hours. One rat expired following the first high dose MDMA regimen. A second rat in the binge low dose MDMA group expired within two minutes following the 2 mg/kg dose of MDMA given before the serial reversal probe test.

Body weights were recorded throughout the experiment. Though the MDMA high binge dose lost an average of 15-20 grams one day post injection, this was quickly recovered one week later with no long lasting effects of MDMA on body weight.

Behavioral Testing

Acquisition and Retention

The mean latency, distance to platform, and velocity were averaged for each rat across the four trials, and mean daily values were used in statistical analysis. In the tables and figures for the results, the following labels were used to define the treatment groups:

Control Binge: 2 saline injections in one day across two hours; Control Chronic: 10 x daily saline injections; MDMA Chronic Low Dose: 10 x 1.5 mg/g MDMA daily; MDMA

Binge Low Dose: 4 x 1.5 mg/kg spaced two hours apart given on two consecutive days;

MDMA Binge High Dose: 2 x 7.5 mg/kg, spaced two hours apart.

The mean latency to the platform for each daily session for all treatment groups across the five training sessions was calculated. A significant within-factor effect for

2 mean escape latency across treatment days, F (2.248, 89.907) =75.769, p < .001, np =

.654, was noted. There was no significant between-group effect of drug treatment regimens for mean escape latency to find the platform across any of the five daily

2 training sessions, F (4, 40) =1.067, p > .05, np = .054. These results indicate a significant reduction in latency to reach platform across training sessions for all rats. A Bonferroni

143 pairwise comparison found there were significant reductions in mean escape latency of all rats from day 1 to 2, day 2 to 3, and day 3 to 5 (p < .01). There was no significant difference in escape latency between days 3 and 4 or day 4 and 5, indicative of an asymptotic plateau in performance around day 4 of training. No interactions between drug treatment conditions and escape latency to platform across training session indicated all rats acquired the location the platform at the same rate. Figure 27A is a line graph displaying the decrease in escape latency for all treatment groups during the acquisition phase. The means and standard deviations for mean escape latency during the training acquisition phase are summarized in Table 5.

The means and standard deviations for cumulative distance to platform (CDT) during the acquisition training sessions are summarized in Table 5. Figure 27B depicts a line graph illustrating mean CDT for each experimental group. No significant within- factor effects were revealed across the five daily training sessions, F(1.01, 40.036)= .906,

2 p > 0.05, np = .022. While there were no overall significant within-subjects differences across training days, Bonferroni pairwise comparisons looking at mean difference between each of the daily sessions found a significant difference for day one and day 2

(M difference=7583.816), day 4 (M difference= 11261.321cm; p < .001) and day 5 (M difference= 11871.568cm; p < .001) and between day 2 and day 5 (M difference=

4287.751; p= .05).

There was no significant between-subjects effect or interaction of drug regimen on mean CDT to the target platform across training sessions, F (4, 40)= 1.067, p > 0.05,

2 np = .096. Levene’s test of homogeneity revealed significant differences in the variance between treatment groups for cumulative distance to the target platform on day 2 [F(4,

144 40)=3.182, p <.05] ,day 3 [F(4. 40)=5.190, p < .01] and day 5 [F(4, 40)=2.821, p < .05].

Large differences in variance seen within groups as well as large variances illustrated in standard deviations throughout each training day (see Table 3) may be responsible for the lack of significance in the reduction of distance traveled to the platform across training sessions. There was a distinguishable trend in the reduction of distance traveled to reach the platform for all groups as the training session progressed (see Figure 27B).

To assess if MDMA produced any gross motor deficits between treatment groups, an ANOVA was completed on the mean velocity/swim speed (cm/s) for each daily training session. Means and standard deviations for velocity during the training acquisition phase are summarized in Table 5. No significant differences in velocity between drug treatment conditions were noted (data not shown here). However, equality of variance could not be assumed for mean velocity across treatment groups using

Levene’s test during day 1, F(4, 40)=3.815, p= .01, and day 2, F(4, 40)= 4.320, p < .01, of training. No significant changes in swim speed across daily training sessions, F (4,

2 160) =.755. p > .05, np = .019. This indicated that there was no difference in swim speed during the acquisition phase of the experiments, indicating no persistent effects of

MDMA, given at any of the selected dose regimens, on psychomotor behavior. In summary, MDMA, given at a low dose, low binge dosing, or high dose, produced no significant effects on spatial navigation in the acquisition of location of a submerged platform in the MWM.

To test retention of the platform location, a 60 second probe test was administered

24 hours after the last training session. Figure 25 is a graphic illustration of the water maze, outlining the labeled quadrants and zones, which were used to assess the search

145 patterns of rats during the retention probe test. A significant within-measures effect was

2 found, F (1.714, 68.576) = 120.961, p < .001, np = .751 for percent of time spent across the 4 quadrants. There was no between subjects effect percent in quadrants interaction, F

2 (4, 40) = 1.221, p > .05, np = .109, indicating while all rats illustrated a preference for the target location, where the platform had been in previous training trials, there was no effect of drug treatment on retention.

Figure 28 shows the percent of time spent searching in each of the four quadrants during the probe trial. All experimental groups spent significantly more time in the target

(NW) quadrant than the start (Mean difference 22.405, p < .001), adjacent (Mean difference=40.720, p < .001), and opposite quadrants (Mean difference= 41.860, p <

.001). All rats spent significantly more time in the start quadrant than the adjacent (Mean difference= 18.314, p < .001) and opposite quadrants (Mean difference 19.455, p < .001).

To analyze whether the MDMA dose regimens resulted in different search strategies, a separate repeated measures ANOVA analyzed the total duration spent in each quadrant with 15 sec intervals of the 60 sec probe trial (0-15 seconds, 15-30 seconds, 30-45 sec, and 45-60 seconds) as the within factor and treatment condition as the between factor. Table 6 displays the means and standard deviations for total duration of time spent in each quadrant across the four 15-sec time intervals. There was a significant decrease in time spent searching in the target NW quadrant from the start of the probe (0-15 sec) to the end (45-60 sec) of the probe trial, F (1.801, 70.236) = 10.066,

2 p < .001, np = .205. There was no significant between-subjects effect in search time in

2 the NW quadrant across 15 sec time intervals, F(4, 39) = .737, p < .573, np = .70. Rats

146 spent significantly more time searching in the target quadrant during the first 0-15 sec interval than the other 45 sec of the probe trial.

There was no significant difference across time intervals from start to finish of the probe trial for total duration spent in the start (SW) quadrant, F(1.97, 78.784) = 2.643, p

2 2 > .05, np = .062, and no between-subjects effect, F(4, 40) = 1.44, p > .05, np = .126.

In contrast to the target quadrant, there was a significant increase in the duration

2 of time (sec) spent searching in the adjacent NE, F(3, 120) = 3.144, p < .05, np = .073 and

2 opposite SE, F (1.815, 782.620) = 9.728, p < .001, np = .196, from the start of the probe trial across 15 sec time intervals to the end of the 60 second probe trial. However, there

2 was no significant between-subjects’ effect for the adjacent, F(4,40) = .298, p > .05, np =

2 .029 or opposite, F(4,40) = .846, p > .05, np = .078, quadrants. This reflects the reduction of time searching in the target location, indicating after searching for the platform in the target location, rats’ shifted their behavior and explored the pool to locate the escape platform in another location. Likewise, opposite to the target quadrant, all rats spent significantly more time in the opposite SE quadrant as well as the adjacent NE quadrant during the last 15 sec (45-60 sec) interval than the first 45 sec of the probe trial.

147 Table 5 Daily Mean (Standard Deviations) latencies to reach platform (seconds, s), cumulative distance traveled to platform (cm), and velocity/swim speed of each trial for each drug treatment during the daily sessions for the acquisition phase.

148

Figure 27: A: Daily mean escape latency to platform (seconds=s; y axis) for each of the drug treatment groups during the acquisition phase training sessions (x axis). There was 2 a significant within-subjects effect, F (2.248, 89.907) =75.769, p > .001, np = .654; all treatment conditions showed significant reduction in escape latency to platform across treatment days. No between-subjects interaction for treatment groups were present. B: Mean daily values for cumulative distance to platform measured in centimeters (cm; y axis). A trend in a decrease in cm to platform across daily sessions, F (4, 40) =1.067, p > 2 .05, np = .054, did not reach significance, and no between treatment conditions effect, 2 F(1.01, 40.036)= .906, p > 0.05, np = .022.

149

Figure 28: Percent of search time (y axis) in each of the four quadrants (x axis) during the retention probe trial. Repeated measures analysis of variance detected a significant 2 within-factor effect, F (1.714, 68.576) = 120.961, p < .001, np = .751. There was no 2 between-subjects X percent in quadrants interaction, F (4, 40) = 1.221, p > .05, np = .109. All rats displayed a search preference for the target platform, spending significantly more time in the NW quadrant than the other three locations. Rats also spent significant more time in the start quadrant in comparison to the adjacent and opposite quadrants.

150 Individual tracks of each rat during the probe trial were analyzed to determine if there were any visible differences in distinct search strategies between treatment conditions. An example retention probe track for each drug MDMA treatment condition and a control group is displayed in Figure 29. Due to the limited space, only one control group was illustrated for each probe trial. Control groups, which had separate injection schedules, were matched in the study. Probe tracks revealed subjects in each treatment condition showed pronounced search strategies in the target quadrant, with specific interest around the target search zone.

In addition, univariate statistics were used to determine if there was a drug treatment effect on other discrete measures of the probe test. There was no effect of drug treatment on latency of first occurrence to the target quadrant, F(4, 40)= 1.409, p > .05,

2 np = .123 or for latency of first occurrence into the target search zone, F(4, 39)= 1.002, p

2 > .05, np = .093. The mean distance to center of target NW platform for each of the drug condition measured in centimeters was as followed: Control Saline (M=59.39,

SD=7.547); Control Chronic (M=49.075, SD=8.103); MDMA Chronic Low Dose

(M=55.913, SD+ 11.58); MDMA Binge Low Dose (M= 51.241, SD= 9.226); MDMA

Binge High Dose (M=51.908, SD= 7.443). There was no significant difference in the

2 mean distance to the target platform, F(4, 40)= 1.066, p > .05, np = .386.

Finally, the mean velocity/swim speed for each rat was calculated during the 60 sec probe retention trial. Figure 30 displays the mean velocity (cm/s) across the 60- second trial for each of the three probe sessions across drug treatment condition. No significant difference between drug treatment conditions in swim speed during the

2 retention probe trial was found, F(4, 40)= 1.089, p > .05, np = .098. 151 Table 6 Mean (Standard Deviations) search duration (seconds, sec) during each of four 15 second intervals (0-15, 15-30, 30-45, and 45-60 sec) in the 60 second retention probe trial listed for each treatment condition.

152

Figure 29: Schematic illustration of individual probe tracks, displaying the search strategies during the entire 60 sec. retention probe trial for a representative case for each of the drug treatment conditions: control binge (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA high binge dose (D). The location of the platform location during training trials is indicated by the black dot in the target (NW) quadrant in Figure 38A. For control cases, one track was selected from both the binge and saline groups for spacing purposes. Drug treatment had no overall visible effect on search strategy. Rats showed a distinct and purposeful search, where the target quadrant remained the focal point of the search.

153 Reversal Task

Figure 31A depicts a line graph of mean escape latency across the three reversal training sessions for each of the treatment groups. Table 7 lists the means and standard deviations of mean escape latencies during the reversal stage. There was a significant within-factor effect for latency to reach platform across the three daily training sessions,

2 . F(1.542, 80)= 54.273, p < .001, np =.576 , but no significant between-subjects effect of

2 treatment on escape latencies, was found F(4, 40)= 1.223, p > .05, np = .109. All rats showed a significant reduction in escape latencies to the platform from Day 1 to Day 3.

Rats disregarded the previously correct location and navigated to the new correct placement of the platform at the same rate, reaching an asymptotic level of escape latency equal to the acquisition phase at training session day 3 equivalent to day 4 in the acquisition phase.

Figure 31B is a line graph which displays the cumulative distance to the platform for all drug treatments. There was a significant reduction in the cumulative distance

2 traveled to the platform across training days 1 to 3, F(1.371, 80)= 66.009, p < .001, np =

.623. There was no between-subjects effect of drug treatment condition on cumulative

2 distance to platform during the reversal phase, F (4, 40) = .850, p > .05, np =. 078.

Means and standard deviations for cumulative distance to target platform are listed in

Table 7. No between-subjects differences were found for swim speed (cm/s) during the

2 reversal phase, F (4,40)= 1.208, p> .05, np = .108.

Figure 32 is a bar graph illustrating percent of time spent in each quadrant across all treatment manipulations. A significant within-subjects effect was found, F(1.996,

2 79.833)= 36.549, p < .001, np = .477. No between-subjects effects were present. 154 Pairwise comparisons of the within-subjects factor using the Bonferroni test found all rats spent significantly more time in the SE target quadrant than the start (M difference=

19.696, p < .001), opposite (M difference = 19.569, p < .001) and adjacent (M difference

= 17.818, p < .001), quadrants of the pool. There were no significant differences, F(4,

2 40)= 1.479, p > .05, np = .129, in time spent searching between the start, adjacent, and opposite quadrants

Means and standard deviations for duration of search time (seconds) for each time interval spent in each of the 4 quadrants for all treatment conditions (0-15 sec, 15-30 sec,

30-45 sec, and 45-60 sec) are displayed in Table 8. There was a significant within- factor difference in the mean duration of search time across 15 second time intervals in

2 the target SE quadrant, F(3, 120)= 49.202, p < .001, np = .552. There was however, no significant between-subjects interaction for 15 sec time intervals X treatment condition,

2 F(4, 40)= .527, p > .05, np = .05, for the target SE quadrant. All rats spent a significantly more time searching in the quadrant target for the first half (0-30 sec) of the probe trial than the second half of the reversal probe trial (30-60 sec).

No significant within-subjects effects were noted, F (2.671, 106.825)= 1.021, p >

2 .05, np = .025, in search duration across 15 sec time intervals from start to finish of the probe trial and no significant between-subjects treatment effect, F(4, 40)= 1.861, p > .05,

2 np = .157, in the NE start quadrant, where rats were released. All rats spent relatively similar amounts of time searching in the start quadrant across all 60 sec of the probe trial.

Lack of significant effects in the start quadrant for the retention and reversal probe may be related to an initial increase in the duration spent in the first 15 sec due to the release of the animal in the quadrant at the start of the trial.

155 2 There was a significant within-factor effect, F(3, 111) = 12.438, p < .001, np =

.252, but no significant within-subjects factor X treatment condition interaction, F(12,

2 111) = .957, p > .05, np = .094 for search duration for rats in the adjacent SW quadrant.

Pairwise comparisons using the Bonferroni test indicated that all rats spent significantly less time searching for the escape platform during the first 30 sec. of the trial (0-30 sec) than the latter half (30-60 sec.) of the reversal probe test. Repeated measures analysis of variance did reveal a significant within-factor effect, F(2.423, 94.515)= 22.160, p < .001,

2 np = .362, of duration of time spent searching in the opposite NW quadrant across 15 sec. time intervals. There was no between-subjects treatment effect, F(4, 39)= 1.001, p > .05,

2 np = .093. Rats spent significantly less time searching in the opposite quadrant during the first 15 sec compared to the last 30 sec of the probe trial. Moreover, all rats spent significantly more time searching the opposite NW quadrant during the 30-45 sec timeframe than the other 45 sec of the probe trial (data not shown). This may reflect the predilection of search patterns due to the learned NW platform location during the acquisition phase.

Individual tracks of the reversal probe trial for all subjects in each treatment group were analyzed to look for unique search strategies. Figure 33 displays a representative track of the reversal probe test for each treatment condition. For the reversal probe, an example from the chronic saline control condition represented all control subjects. All treatment groups showed a distinct and deliberate search pattern focused around both the target quadrant and searching within the target search zone.

156

Figure 30: Mean velocity (cm/seconds; y axis.) across each of the three probe trials (retention, reversal, and serial reversal; x axis) for each of the treatment conditions. A 2 significant within-subjects effect, F(42, 78) = 5.044, p < .01, np = .115, of probe trial was found but no significant trial X treatment condition was observed, F(8, 78) = 1.03, p > 2 .05, np = .096. Rats had a slower swim speed during the retention probe (Mean difference= 1.482 cm/s) as compared to the reversal probe regardless of MDMA dose regimen.

157 Table 7

Daily mean (standard deviation) latency to reach platform (seconds, s), cumulative distance traveled to platform (centimeters, cm), and velocity during the daily reversal training sessions.

158

Figure 31: A: Daily mean escape latency to platform in seconds (y axis) for each of the drug treatment groups across the reversal phase training sessions (x axis). There was a significant within-factor effect for latency to reach platform across the three daily training 2 . sessions, F(1.542, 80)= 54.273, p < .001, np =.576 , but no significant between-subjects 2 effect, F(4, 40)= 1.223, p > .05, np = .109. B: Mean cumulative distant to the platform traveled (centimeter, cm; y axis) across daily sessions (x axis). There was a significant reduction in the cumulative distance traveled to the platform across training days 1 to 3, 2 F(1.371, 80)= 66.009, p < .001, np = .623, but no between-subjects effect of drug 2 treatment condition, F (4, 40) = .850, p > .05, np =. 078.

159

Figure 32: Percent of total search time (y axis) in each of the four quadrants (x axis) during the reversal probe trial. A significant within-subjects effect, F(1.996, 79.833)= 2 36.549, p < .001, np = .477, was found, but no between-subjects effect of treatment condition were noted. All rats spent significantly more time in the SE target quadrant than the start, adjacent, and opposite quadrants.

160 Table 8 Mean (Standard Deviation) search duration (seconds, sec) across each of four 15 second intervals (0-15, 15-30, 30-45, and 45-60 sec) in the 60 second reversal probe trial listed for each treatment condition.

161 There was no significant difference in the mean latency to reach the target search

2 zone during the reversal probe, F(4, 36) = .743, p < .05, np = ..076 (group mean= 6.8704 seconds; SD= 9.944) or latency to reach the opposite NW target during the reversal

2 probe, F(4, 40) = .609, p < .05, np = .057, with a group mean latency of first occurrence=

23.79 seconds; SD=15.987. There was no significant difference in mean velocity (cm/s) between each of the drug treatment condition during the reversal probe trial, F(4, 40) =

2 .556, p < .05, np = .071. Means and standard deviations of swim speed for all treatment conditions are displayed in Figure 30. All subjects reached an asymptotic level of escape latency and cumulative distance to the platform by Day 3 of training in the reversal phase, which was analogous to Day 4/5 of the acquisition phase. Rats spent more time exploring the target SE quadrant during the reversal probe trial than the other three quadrants. No subjects exhibited perseverative search strategies in the opposite and previously correct NW quadrant, nor did they exhibit perseveration of search duration in the SE quadrant during the reversal probe test. MDMA had no effect on acquisition or retention of the new reversal platform location in the reversal phase of the experiment.

Serial Reversal Drug Challenge

The serial reversal refers to subsequent reversal of platform placement to the original NW quadrant. As with the reversal phase, rats were given three days of training sessions, each day consisting of four trials. Twenty-four hours following the last training day, a 60 second drug challenge probe test was conducted. MDMA pretreated rats were given an injection of 2.0 mg/kg MDMA 30 minutes prior to the probe trial, with control animals receiving an injection of sterile physiological saline.

162 Means and standard deviations for these serial reversal measures are displayed in

Table 9. Figure 34A is a line graph, depicting the mean escape latency across each daily training session for the MDMA drug treatment conditions. There was a significant

2 within-subjects effect, F(2, 80) = .20.151 p > .001, np = .335, for mean escape latency to platform across daily training sessions, but there was no significant interaction of

2 treatment condition X daily training sessions, F(8, 80) = 1.632, p < .05, np = .14.

Pairwise comparisons using the Bonferroni test found a significant difference between each of the three daily training sessions, with a longer mean escape latency to platform of all subjects for Day 1 in contrast to day 2 (M difference seconds= 4.792, p < .01) and day

3 (M difference = 8.331 seconds, p < .001).

Repeated multivariate analysis yielded a significant within-subjects effect of daily training sessions on cumulative distance to the target platform, F(2, 80) = 15.616, p <

2 .001 np = .281. There was no significant between-subjects effect of treatment condition,

2 F(4, 40) = 1.823, p < .05, np = .154. Figure 34B displays the average cumulative distance to the platform for each of the treatment conditions across the three daily sessions in a line graph. Cumulative distance to the platform was significantly greater for day 1 in comparison to days 2 (M difference = 1698.723 cm, p < .05) and 3 (M difference =

2876.51 cm, p < .001), but there was no significant difference between Days 2 and 3 (M difference = 1177.787, p < .001).

163

Figure 33: Schematic illustration of individual probe tracks demonstrating search strategies for a representative case in each of the treatment groups: control chronic (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA high binge dose (D) during the 60 second reversal probe trial. Only one representative case was visualized for the control groups for spacing purposes. Drug treatment had no effect on search strategy. Rats showed a distinct and purposeful search, using the target (SE) quadrant as the focal point of the search.

164 Table 9

Daily mean (Standard Deviation) latency to reach platform (s), cumulative distance traveled to platform (cm), and velocity/swim speed of each trial for each drug treatment during the three daily sessions in the serial reversal phase.

165

There was a significant difference in mean velocity of rats across daily sessions,

2 F(2, 39) = 8.726, p < .001, np = .309 but no significant velocity X treatment group

2 interaction, F(8, 80) = .882, p < .05, np = .081. Pairwise comparisons using the

Bonferroni test found a significant difference in mean velocity of training trials between day 1 and day 3 (M difference = 2.168, p < .001) but no differences in mean velocity between days 2 and 3. The swim speed for all rats during day 1 (total mean velocity =

23.427, SD= 3.352) was significantly slower than day 3 (total mean velocity = 25.653,

SD= 4.168), with a total mean difference greater than 2 cm/s, Means and standard deviations for drug treatment groups are displayed in Table 9.

Repeated measures multivariate analysis was conducted for the percent of total duration spent searching in each quadrant during the serial reversal drug challenge probe trial, with treatment condition as the between-subjects factor and the 4 quadrants as the within subjects factor: Target (NW), Start (NE), Adjacent (SW), and Opposite (SE).

Figure 35 is a bar graph illustrating the percent of dwell time spent in each of the four quadrants for each of the treatment conditions. A significant within-subjects effect of

2 quadrant, F(2.150, 83.868) = 120.342, p < .001, np = .755, was noted, but there was no

2 between-subject effect of treatment condition, F(4, 39) = 1.357, p > .05, np = .122. All rats spent a significantly greater amount of time searching in the target NW quadrant than the NE start, (M difference= 32.03%, p < .001) SW adjacent (M difference= 29.208, p <

.001), and SE opposite quadrant (M difference= 34.68, p < .001). There were no significant differences between % dwell across the other three quadrants.

166 The means and standard deviations of total duration (s) during each 15 sec time interval for the four quadrant in each of the drug treatment conditions is listed in Table

10. Results found a significant within-subjects effect of time interval for the NW target

2 quadrant, F(3, 117) = 5.277, p < .01, np = .119, however, no between-subjects effect of

2 treatment condition was present, F(4, 39) = 1.594, p > .05, np = .14. Pairwise comparisons using the Bonferroni test illuminated all subjects spent a significantly more time searching the target quadrant during the first 15 seconds of the trial than the last 45 seconds (15-30, 30-45, and 45-60 seconds). No significant differences in search duration were found between any other time intervals.

There was a significant within-subjects effect across time interval, F(3, 111) =

2 12.944, p < .001, np = .259, for total search duration in the SW adjacent quadrant, however there was no time interval X treatment condition interaction, F(12, 111) = .774,

2 p > .05, np = .077 Rats spent significantly greater search duration in the adjacent target in the last 45 seconds of the trial in comparison to the first 15 seconds. There was no effect

2 on time interval, F(3, 117) = 1.461, p > .05, np = .036 or treatment condition, F(4, 39) =

2 .846, p > .05, np = .08 for total search duration of the NE start quadrant. There was also

2 no significant within-subjects effect of time interval, F(3, 117) = .192, p > .05, np = .04,

2 or time interval X treatment condition interaction, F(12, 117) = 1.132, p > .05, np = .104, for the opposite SE (reversal) quadrant.

167

Figure 34: A: Daily mean escape latency to platform (seconds= s; y axis) for each of the drug treatment groups for the serial reversal phase. There was a significant within- 2 subjects effect, F(2, 80) = .20.151 p > .001, np = .335, across daily training sessions, but there was no significant interaction of treatment condition X daily training sessions, F(8, 2 80) = 1.632, p < .05, np = .14. B: Daily averaged cumulative distance to the platform (CDT) for each daily session during the serial reversal phase. A significant within- subjects effect of daily training sessions on CDT was noted, F(2, 80) = 15.616, p < .001 2 np = .281, but there was no significant between-subjects effect, F(4, 40) = 1.823, p < .05, 2 np = .154. CDT was significantly greater for day 1 in comparison to days 2 (M difference = 1698.723 cm, p < .05) and 3 (M difference = 2876.51 cm, p < .001), with no significant difference between Days 2 and 3. All rats, independent of treatment condition, illustrated acquisition to the original platform location.

168

Figure 35: Percent of total search time (y axis) spent in each of the four quadrants (x axis) during the serial reversal drug challenge probe trial. A significant within-subjects 2 effect, F(2.150, 83.868) = 120.342, p < .001, np = .755, was present, but there was no 2 between-subjects effect, F(4, 39) = 1.357, p > .05, np = .122. All rats spent significantly longer duration in the NW target quadrant than the start, adjacent, and opposite quadrants.

169 Figure 36 depicts a sample track from the best representative subject for each of the MDMA drug treatment conditions and the control binge saline group. As illustrated, all treatment conditions showed a search strategy, which indicated a preference for the target location. Univariate analysis of variance revealed no significant differences between drug treatment groups in the latency of first occurrence (seconds) in the NW

2 target search zone, F(4, 39) = .779, p > .05, np = .074 or the latency of first occurrence to the search zone of the previous correct SE reversal (opposite) target, F(4, 33) = .783, p >

2 .05, np = .087 (data not shown). There were no significant between-subjects effect in

2 mean distance to the center point of the target platform, F(4, 39) = 1.159, p > .05, np =

.106, or mean distance to the center point of the SE reverse (opposite) quadrant , F(4, 39)

2 = 2.368, p > .05, np = .195 (data not shown).

Results indicated there was no significant difference between treatment groups,

2 F(4, 39) = .39, p > .05, np = .038, on mean velocity during the probe trial. Figure 30 displays the mean velocities for each of the three probe trials in a bar graph. A repeated measures ANOVA was conducted to see if there was any difference in mean velocity across the three probe tests. There was a significant within-subjects effect, F(42, 78) =

2 5.044, p < .01, np = .115, of probe trial for mean velocity (cm/s) but no significant trial X

2 treatment condition was present, F(8, 78) = 1.03, p > .05, np = .096. All rats had a swim speed that was slower during the retention probe (total mean= 27.0277, SD= 2.6105) than the reversal probe (total mean= 27.465, SD= 3.097), but no effects were found across the drug challenge probe. The mean difference between the two trials was 1.482 cm/s. All rats swam at the relatively same speed, providing inter-probe trials with minimal variance

170 (see standard errors in Figure 39). Therefore the significant but small difference in velocity across these two trials was most likely due to artifact.

Immunohistochemistry

The results of the histological analysis was described in detail in Experiment 1 and reviewed briefly here. There were no overt visually discriminative differences in the serotonergic fiber expression detected using the antisera for SERT between in the

MDMA chronic low and MDMA binge low group as compared to controls. All rats showed a strong yet heterogeneous expression of SERT immunoreactive fibers throughout the forebrain to the mesencephalon. The only distinguishable differences in

5-HT forebrain innervation were noted in the MDMA high binge dose. Rats given 2 x

7.5 mg/kg MDMA exhibited a significant decrease in SERT immunoreactive fibers in several cortical and subcortical structures across the entire forebrain, including several limbic structures known to mediate spatial navigation in rats.

DISCUSSION

MDMA, administered at low or high doses, did not produce any significant effects of acquisition or retention of the spatial location of a submerged platform, nor were deficits seen in the acquisition or retention of reversal or serial reversal.

Pretreatment of MDMA did not produce perseveration in navigating to the learned location of the platform in previous training sessions, nor did it produce perseverative search strategies as a result of failure of extinction to search for the platform in the target quadrant during the during probe trials. A low dose of MDMA given immediately prior to the probe trial to MDMA pretreated rats did not impair the retention of the spatial

171 location or of the hidden platform or result in perseverative search strategies in the serial reversal drug challenge probe.

It was hypothesized that Long Evans (LE) rats given high binge doses (2 x 7.5 mg/kg), would exhibit impairments in the retention of the location of a submerged platform during the retention probe trial, but there would be no measurable difference in acquisition of the spatial location of the platform. This would correspond with a loss of

5-HT innervation of the forebrain. The conservative lower doses of MDMA (chronic and binge low dose regimens), which are more representative of recreational MDMA use in humans that produce no measurable 5-HT axonal denervation, should have performed similar to controls in both acquisition and retention of the platform location during training and probe test. However, in contrast to predicted results, all rats acquired the spatial location of the platform at similar rates and showed a preference for the target quadrant during the retention probe, indicating accurate recall of the platform location.

These findings are in contrast to reports, which have found specific recall deficits in a MWM protocol following multiple s.c. injections of 15-20 mg/kg MDMA (Able et al., 2006; Cunningham et al., 2009; Sprague et al., 2003). Skelton et al. (2008) failed to find any changes in behavioral performance on a MWM task following high binge doses of MDMA. Rats in this study underwent a battery of behavioral testing, and pretraining on other spatial paradigms immediate prior to MWM may account for the attenuation of deficits.

172 Table 10 Mean (Standard Deviation) total search duration (seconds, sec) in each of four 15- second intervals (0-15, 15-30, 30-45, and 45-60 sec) across the 60 second serial reversal drug challenge probe trial listed for each treatment condition.

173

Figure 36: Schematic illustration of individual probe tracks illustrating search strategies during the serial reversal drug challenge probe test for a representative case of each of the treatment groups: control binge (A), MDMA chronic low dose (B), MDMA binge low dose (C), and MDMA high binge dose (D). Only one representative case was visualized for the control groups for spacing purposes. The platform location for the serial reversal probe was moved to the original target quadrant (NW) and indicated with a black dot in Figure 45A. No effect of treatment condition was observed. Rats showed a distinct and purposeful search, using the target (NW) quadrant as the focal point of the search.

174 Camarasa et al. (2008) found deficits in the recall of the platform location in LE rats one week after receiving 8 x 15 mg/kg MDMA, indicating strain may not account for the differences across experiments. In the current study, the cumulative dose of 15 mg/kg MDMA had no effect on retention when training began two weeks following the last dose regimen. It may be possible that higher doses than 15 mg/kg MDMA is needed to disrupt platform retention in the MWM. Recovery period following MDMA treatment may facilitate behavioral recovery. Retention deficits in the MWM following large binge doses of MDMA have been observed when training commenced 7 days after the last treatment injection (Sprague et al., 2003). Two contrasting studies, which found opposing conclusions on the retention probe began training on MDMA, pretreated rats longer than two weeks after their lat dose regimens. However, these rats were trained and tested on several other behavioral paradigms in the interim (Able et al., 2005; Skleton et al., 2008). The recovery period of two weeks was selected so rats began training on both behavioral paradigms (see Experiment 3) at the same time point post injection schedules.

Other spatial paradigms have also found retention deficits in MDMA treated rats.

MDMA produced significant increases in reference working memory errors on a version of the 8 arm radial maze (Kay et al., 2009, 2011). While the neurotoxic effects on the serotonergic system using 2 x 7.5 mg/kg i.p. in LE rats were extensive, a substantial amount of SERT forebrain innervation persisted following the binge high dose of

MDMA. There may be other morphological, neurochemical, and electrophysiological changes produced by larger doses of MDMA which reflect the behavioral differences found in other studies.

175 The reversal phase examined the role of MDMA and serotonergic lesions on spatial reversal learning. Reversal learning can be tested via a number of different paradigms to examine attentional and executive functioning in rats, primates, and humans

(Boulougouris and Robbins, 2010). Reversal learning requires the proper acquisition of a rule or place response. Boulougouris et al. (2008) stated “Efficient reversal learning calls upon specific operations such as 1.) detection of the shift in contingency, 2.) inhibition of a prepotent learned response, 3.) overcoming ‘learned irrelevance,’ and 4.) new associative learning.” There are several different reversal learning paradigms, some of which utilize appetitive learning under visuospatial contingencies or intersensory discriminative paradigms, and others which involve aversive directional and place learning.

While serotonergic input to regions of the prefrontal cortex are critical to behavioral flexibility tested in discriminative reversal learning paradigms, central 5-HT depletion via MDMA did not impact spatial reversal learning in a water maze in LE rats.

This is consistent with results which found MDMA lesioned SD rats did not show any perseveration towards the previously correct quadrant during reversal tests (Able et al.,

2006; Skleton et al., 2008).

The results of spatial reversal learning are not surprising when compared to previous studies, but there is uncertainty as to why spatial reversal learning in the water maze remains unaffected by MDMA neurotoxicity in SD or LE rats. Kay et al. (2011) discovered a spatial reversal learning deficit in MDMA treated rats using an 8 radial arm maze. Rats were first trained on a 4 baited arm delayed match to sample task. Following acquistion, the rules of the task changed so that rats had to learn that the 4 arms

176 previously unbaited now contained the food rewards. MDMA treated rats committed significantly more working and reference memory errors during this reversal phase in comparison to controls. Over all, however, rats committed a greater number of reference memory errors (Kay et al., 2011). High doses of methamphetamine (METH) produced significant longer latencies during training in the reversal phase of the MWM in periadolescent SD rats, but adult rats performed similarly to MDMA treated subjects

(Vorhees et al., 2005). Neurotoxic methamphetamine also given to SD rats impaired spatial reversal learning in a T-maze (Daberkow et al., 2008). However, METH is neurotoxic to all monoaminergic axonal fibers, so any deficits could be linked to dysregulated catecholaminergic systems.

It is possible there are separate chemical and structural systems that mediate different forms of reversal learning. Excitotoxic lesions of the medial prefrontal cortex were involved in impaired spatial reversal learning in the MWM in rats (De Bruin et al.,

1994, 2001; Lacroix et al., 2002). Nonspatial forms of reversal learning, which involve the “reversal” of rules or behavioral contingencies reliant upon visual, odor or tactile sensory discrimination, have been found to be influenced by homeostatic 5-HT input and modulated specifically by 5-HT innervation of the orbitofrontal cortex (McAlonan and

Brown, 2003; Clark et al., 2004; Clarke et al., 2004, 2005, 2007).

5-HT has also been known to modulate spatial reversal (and serial) reversal learning in an operant delay match to sample lever press paradigm, known to be dependent on the orbital frontal cortex (Boulougouris and Robbins, 2010). In this paradigm, rats learn a fixed ratio of response to a lever on one side of the chamber to receive a reward. Following acquisition, the location of a lever is reversed to the

177 opposite side, followed by a serial reversal. Lights discharge within trials to serve as an

irrelevant stimulus. 5-HT2A antagonists impair reversal learning and increase perseverative responding, whilst 5-HT2C antagonists facilitate reversal learning in this paradigm (Boulougouris et al., 2008). Further investigation found 5-HT2C antagonism specifically in the orbital frontal cortex facilitates spatial reversal learning (Boulougouris and Robbins, 2010). As mentioned above, spatial reversal learning in the MWM is not mirrored contingency reversal learning. Therefore, it is likely this example of spatial reversal learning in the MWM may be governed by the mPFC, and is independent of the visuospatial discriminative reversal learning paradigms controlled by serotonergic influence in the orbital frontal cortex (OFC).

Probe trials can be deconstructed to measure whether or not the subjects can perform extinction during the inter-probe trial. After searching for the platform in its target platform, they explore the remainder of the maze in search of a new escape location. Behavioral extinction in fear conditioning is regulated by the ventral medial prefrontal (prelimic and infralimbic) cortex in rodents (Akirav and Maroun, 2007; Peters et al., 2009; Sortes-Bayon et al., 2006). The perseveration deficits of spatial reversal learning in the MWM witnessed in medial prefrontal cortical lesions may in fact, be an example of a deficit in behavioral acquisition of extinction rather than deficit in behavioral flexibility.

For the probe trials, extinction was examined by analyzing the total search duration in each quadrant across 15-second time intervals across the 60-second trial. The rat will initially spend more time in the target quadrant when it accurately recalls the location of the platform, as compared to the other areas. Following this, rats will spend

178 an increased amount of time in the other quadrants in search to locate the escape platform. During this time, the search duration in the target quadrant significantly decreases in comparison to the surrounding quadrant locations. Rats should spend significantly greater amounts of search duration in the other quadrants during the latter half of the trial in comparison to the target quadrant. If rats are impaired in extinction, they may persist in searching for the hidden platform in the target quadrant throughout the trial, displaying a perseverative search response.

Previous water maze studies have not examined if MDMA impacts extinction behavior in the MWM during the probe trials. Results from the retention and reversal probe trials indicated there was no long-term impact of MDMA on extinction or search strategy during the probe tracks, on search strategy. All rats preferred the target quadrant, and displayed significantly longer search durations across the first 15 to 30 seconds of the probe trial in comparison to the last 30 sec. An opposing increase in the amount of search time occurred in the other quadrants during the last 30 and 45 sec. intervals.

The serial reversal drug challenge probe was designed to test the recall and search strategy for platform in the serial reversal (original) location in MDMA pretreated rats under the influence of MDMA. To minimize the psychomotor effects of the drug, a low dose of MDMA (2.0 mg/kg) was selected to mirror a recreational dose of Ecstasy, but also so not to induce serotonin syndrome in the rats. Serotonin toxicity produces psychomotor behaviors such as head weaving, forepaw treading, tremor, and hind limb splaying, making the rats unable to swim and may produce thigmotaxsis or rotational behavior. Similar to the acquisition/retention and reversal phases, MDMA pretreated rats

179 showed no impairments in acquisition of the serial reversal in comparison to control subjects. All treatment conditions displayed a sharp learning curve for escape latency and distance to platform which reached an asymptotic level by day 3, mirroring that of reversal training.

A low dose of MDMA did not impact the swim speed during the serial reversal probe, nor did it produce gross motor deficits such as thigmotaxis, stereotypies, or ataxic behavior. Individual probe tracks for each subject were studied, and no evidence of thigmotaxic or rotational behavior was found. This is similar to data collected by Arias-

Cavieres et al. (2010), which found no motor impairments in rats given doses of .2 to 2.0 mg/kg MDMA before water maze trials. This ensured the data gathered from the probe trial was related to higher cognitive behaviors rather than psychomotor performance.

During the serial reversal drug challenge probe, pretreated rats received low dose of 2.0 mg/kg MDMA 30 minutes prior to the trial, so the search for the platform would occur under peak actions of the drug. MDMA did not affect recall of the platform location, search strategies, or extinction during the probe trial for any of the pretreatment dose regimens.

It was hypothesized that MDMA administration would impair recall of the probe location during the drug challenge test. But in the case, retention was unaffected, it was predicted pretreated rats under the influence of MDMA may show deficits in extinction and may show a perseverative search response in the target quadrant. The acute effects of MDMA have been demonstrated to impede working memory in other spatial and nonspatial appetitive tasks (Harper et al., 2005,2006; LeSage et al., 1993; Young et al.,

2005). Kay et al. (2001) recently tested the effects of subacute effects of MDMA after

180 being trained on a radial arm maze, which used a baited arm protocol. A dose of 4.0 mg/kg MDMA negatively affected performance in both rats treated with a high binge dose of MDMA and saline controls. Increases in both working and reference memory errors were noted.

Arias-Cavieres et al. (2010) gave low doses of MDMA immediately prior to training and probe sessions using the Morris water maze on periadolescent Sprague

Dawley rats. They found MDMA significantly increased escape latencies to reach a submerged hidden platform during training trials. Retention of the probe location in this study was measured by comparing the search time in the target quadrant previous to training to the amount of the time in the quadrant after the training session rather than comparing search time across all quadrants. Low doses of MDMA, which did not produce a significant reduction of serotonin in hippocampal tissue, impaired LTP in postmortem in vitro slices (Arias-Cavieres et al., 2010). In the absence of 5-HT neurotoxicity or behavioral deficits, there is the possibility MDMA is morphologically and physiologically impacting the CNS in measures not obtained here.

All rats in the aforementioned study exhibited a learned preference for the target quadrant, however control rats spent significantly more time in the target quadrant than

MDMA treated rats, showing a drug effect (Arias-Cavieres et al., 2010). Differences in the results may be reliant on several factors, including the difference in age and rat strain.

It is possible that rats are more sensitive to the effects of MDMA during the periadolescent development period, though anecdotal evidence gathered from pilot studies in this laboratory suggested increases in age and weight in rats resulted in a greater sensitivity to the hyperthermic effects of the drug, and had a higher attrition rate.

181 MDMA was not administered during the training trials in the current study, but only prior to the retention probe.

MDMA also had no effect on search strategies or extinction in pretreated rats during the serial reversal probe. Similar to the retention and reversal probe trials, search strategies during the serial reversal drug challenge for MDMA pretreated rats did not differ from controls. Rats showed a significant preference of search for the target quadrant during the first half of the probe trial and spent an increasingly greater amount of time exploring the other three quadrants during the latter half, demonstrating no perseverative search response.

Previous work on serotonin systems has indicated that depleting 5-HT produces deficits in reversal learning using discriminative paradigms (Clarke et al., 2004, 2005,

2007). However, a low dose of MDMA enhances serotonergic activity, which conversely, could facilitate reversal learning, or moreover may attenuate deficits in lesioned rats. Facilitation and impairment of reversal learning, attention, and decision making has been documented by pharmacological manipulation of 5-HT receptor profiles

(Bondi et al., 2008; Boulougouris et al., 2008; Boulougouris and Robbins, 2010;

Burnham et al., 2010; Danet et al., 2010; Hatcher et al., 2005; Nikiforuk et al., 2010;

Passetti et al., 2003; Seibell et al.,2003; van der Plasse et al., 2007; Zeeb et al., 2009).

There is no literature on the acute effects of MDMA on reversal learning, but it stands to reason that acute 5-HT release in critical prefrontal areas may promote behavioral flexibility, or in fact, attenuate deficits, boosting 5-HT activity and ameliorating dysregulation caused with pretreated neurotoxic doses. Neither MDMA-induced facilitation of extinction or attenuation of deficits of spatial reversal learning was

182 observed in the current study due to the absence of any measurable performance differences between treatment conditions. Deficits in fear extinction are thought to be pronounced in post traumatic stress disorder (PTSD) and extinction may be critical to recovery and the alleviation of negative symptoms (Liberzon and Sripada, 2008; Milad et al.,, 2006; Milad and Rauch, 2007; Quirk et al., 2006). Low doses of MDMA may manifest positive effects on extinction behaviors and behavioral flexibility. This may suggest one mechanism of action related to the efficacy of MDMA in current clinical trials assessing MDMA-associated psychotherapy in PTSD patients (see Experiment 1 -

Bouso et al., 2008; Cukor et al., 2009; Johansen and Krebs, 2009).

It has been speculated that MDMA may facilitate learning and memory. In this light, low doses of MDMA might facilitate recall rather than impair it in LE rats. D- amphetamine and related compounds have been associated with facilitation of learning and memory (de Jongh et al., 2008). Adderall is a cognitive enhancer, with improvements in attention and focus has been recorded in sample populations (Farah et al., 2009). Modafinil improved both place learning in the water maze and cued fear conditioning in the rat and cognitive enhancing effects have been seen in humans

(Beracochea et al., 2001; Shuman et al., 2009; Repantis et al., 2010). Chronic treatment of L-amphetamine in aged rats improved performance on the water maze task, to levels matched to young controls (Gelowitz et al., 1994). D-amphetamine facilitated both acquisition and recall of platform location during the probe trial in the MWM (Brown et al., 2000). Amphetamine has also been known to enhance attention and vigilance in rats in visuopatial tasks and has been widely studied as a cognitive enhancer for psychiatric

183 conditions such as schizophrenia (Evenden et al., 1993; Turner, et al., 2004; Barch and

Carter, 2005; Silber et al., 2006).

Despite a large body of literature that promotes low dose amphetamine as a cognitive mnemonic enhancer, there have been reports of negative attributes of executive processes as well. Moderate doses of the drug impaired reversal learning, increased perseverative responses, and impaired behavioral inhibition (Ridley et al., 1981; Fillmore et al., 2005; Idris et al., 2009). Comparatively, it is not a far stretch that low to moderate doses of the amphetamine analogue, MDMA, may have a dual nature in its influence of executive functions and memory processes, similar to its analogue, d-amphetamine. Thus far,, low doses of the drug have been known to acutely impair acquisition of place learning, and working memory in a delayed match to sample paradigm in rodents, which suggest a negative influence on cognition (Camarasa et al., 2008; Cunningham et al.,

2009; Harper et al., 2005, 2006). Studies have failed to find any lasting behavioral deficits as a resultant of MDMA in primates (Taffe et al., 2001, 2002, 2003). The inconsistency in long-term deficits is shadowed by a scarce amount of literature on the subacute effects of the drug on behavior.

The lack of deficits in the face of neurotoxic insult on the serotonergic system may be related to an inverse relationship between 5-HT and spatial learning. While

SSRIs are potent mood enhancers and have cognitive enhancing properties in human patients, chronic administration of fluoxetine and other selective serotonin reuptake inhibitors impaired acquisition and performance in a water maze task (Majlessi and

Naghdi, 2002; Chow et al., 2007). Conversely, Valluzzi and Chan (2007) found chronic fluoxetine administration to rats had little impact on a version of the water maze task but

184 impaired rats’ performance on a nonspatial object recognition task. Keith et al. (2007) also found chronic fluoxetine had no impact on MWM performance. This indicates tonic enhancement of serotonin efflux does not facilitate learning and memory, and 5-HT may not be involved in place learning in the rat. If this stands to be correct, loss of serotonergic function would have little impact, if not an opposite effect, as seen in this study.

This inconsistency in effect on spatial learning has also been witnessed with other serotonergic agents. For example 5-HT depletion with p-chlorophenylalanine (PCPA) produced no deficits in the MWM, while scopolamine produced small deficits. The combination of the two drugs produced drastic impairments in the MWM (Dringenberg and Zalan, 1999). Several studies have found similar bidirectional relationship between

5-HT and ACH in the maintenance of memory processes (Santucci et al., 1995;

Ruotsalainen et al., 1998). 5-HT may not work solely on modulation of behaviors but synergistically with ACH, which may remain intact in amphetamine analogue induced neurotoxicity.

Studies examining the effects of Ecstasy in human recreational users have found deficits in several tasks known to be dependent upon hippocampal function (Daumann et al., 2005; Fox et al., 2001, 2002; Gouzloulis-Mayfrank et al., 2003; Jacobsen et al.,

2004). These studies are plagued with confounds and the influence of polydrug use and premorbid effects of psychiatric and addiction conditions cannot be ignored. Kuypers and Ramaekers (2006) tested the effects of MDMA in humans in a controlled laboratory setting and reported similar results as discovered in the current study. Previous or current

Ecstasy users were given 75mg/kg and then tested t on two visuospatial and spatial

185 working memory tasks during intoxication and one day following the drug administration. While weights were not reported, subjects were stated to be on “healthy body weights” which would make the dose of MDMA given to participants approximately 1-1.5 mg/kg. Results of the task found performance deficits/increase in errors while under the influence of MDMA however these deficits were absent one day later, indicating MDMA did not produce long lasting deficits in visuospatial memory

(Kuypers and Ramaekers, 2006).

Low and high dose regimens of MDMA did not impact acquisition, retention, reversal, and serial reversal place learning in LE rats. Despite this, MDMA binge high dose rats exhibited significant and long lasting serotonergic axonal denervation throughout the forebrain as measured by SERT immunohistochemistry. The decision to use the Long Evans strain rather than the albino SD rat was two fold. First, little data has been gathered on the behavioral and neurotoxic effects of MDMA on Long Evans rat.

Second, unlike other albino strains, pigmented Long Evans’ rats visual acuity is sharper compared to other strains (Harker and Whishaw, 2002; Prusky et al., 2002).

The absence of behavioral deficits in the current study may be attributed to the LE strain. Long Evans’ rats increased visual acuity may have enhanced their learning and recall abilities. LE rats are more sensitive to certain variables of the water maze task.

For instance, Vorhees and Willliams (2006) point out LE rats are sensitive to the size of the tank and will show immediate learning without a slope for escape latency in a small tank due to swimming a fixed distance from the wall. Here, the tank was an adequate size, and the acquisition of place learning reflected those common in water maze literature, with asymptotic levels being reached at days 4 and 5. Tonkiss et al. (1992)

186 found enhanced performance on the MWM in LE rats may be independent of differences in visual acuity. LE and SD rats exhibited no differences in a visual cued platform task in the MWM despite better perfomance of LE rats during hidden platform trials.

Brown et al., (2010) recently proposed that the large differences in the rodent and human neuropsychological literature relating to MDMA’s effect on cognition is a matter of the complexity of the tasks. They used verbal memory tasks which varied incrementally from levels that were ‘simple’ to ‘deep’ and found a positive correlation between Ecstasy related deficits and the complexity of the task. Moreover they found tasks reliant upon the prefrontal cortex produced more severe deficits in Ecstasy users than those which did not. Brown et al. (2010) summated the effects best stating “tasks with higher cognitive complexity might be more vulnerable to Ecstasy-related damage than tasks with low cognitive complexity.” Neurotoxic doses of MDMA may impact place learning in LE rats, but water maze paradigms are not complex enough to discriminate the multifarious behavioral traits.

In sum, chronic and stacked ‘binge’ regimens of the drug did not impair behavioral performance in the water maze or produce 5-HT axonal denervation of the forebrain. Conversely, high doses of MDMA given to LE rats produced significant 5-HT neurotoxicity in the forebrain, but left place learning unaffected. A low dose of MDMA did not impact retention or search strategy in a serial reversal MWM probe trial in pretreated rats. These results indicate the short-term and long-term effects of MDMA on spatial navigation and place learning using the MWM in Long Evans rats are negligible.

187

EXPERIMENT 3

INTRODUCTION

Executive functioning is the term given to a group of attentional and cognitive abilities involved in the regulation, selection and operations of behavior. It includes selective attention, shifting attentional sets, decision making, thought organization, behavioral inhibition, behavioral flexibility, and working memory (Dalley et al., 2004;

Robbins, 1996). Over the past several decades, numerous studies have outlined the neurochemical and anatomical control of these behaviors. Each of these executive processes is governed by its own set of neuroanatomical structures, which involve distinct subdivisions of the prefrontal cortex and interconnected structures.

Neuromodulation by specific neurochemical input to each of these regions is unique across these traits (Chase et al., 2008; Kim and Ragozzino, 2005; Ragozzino, 2007;

Ragozzino et al., 1999, 2002; Vertes, 2006). Serotonin is involved in the regulation of separate executive processes, which depend on 5-HT’s discrete input to specific prefrontal cortical divisions (Boulougouris and Tsaltas, 2009; Chudasama and Robbins,

2006; Clark et al., 2004; Dalley et al., 2004, 2008; Robbins, 1996; Robbins and Arnsten,

2009; Robbins and Roberts, 2007).

188 Polydrug Ecstasy users exhibit impairments in fronto-executive functioning.

Studies suggest a there is a stronger relationship between Ecstasy and frontal functioning then the effect of Ecstasy use on long-term memory and hippocampal functioning.

Several reports have found polydrug Ecstasy users presented working memory impairments using various digit, verbal, and spatial span tasks. However, the majority of these deficits was also observed in polydrug controls, and could be negated if statistical analysis used polydrug use as a covariate (Bedi and Redman, 2008; Curran and

Verheyden, 2003; Gouzoulis-Mayfrank et al., 2000; Montogomery and Fisk, 2008;

Morgan et al., 2002; Verkes et al., 2000; Wareing et al., 2004). By contrast, several reports have also failed to elucidate similar working memory deficits (Golding et al.,

2007; Hoshi et al., 2007; Roiser et al., 2007; Thomasius et al., 2003).

Quednow and colleagues (Quednow et al., 2005, 2007) conducted a series of experiments on the influence of Ecstasy on fronto-executive tasks. MDMA users were tested on several paradigms including versions of a go/no-go task, a virtual gambling task testing decision-making, the Matching Familiar Faces task (MFFT), which measures impulsivity, and a version of Rey auditory verbal learning test (RAVLT). MDMA users with extensive histories of alcohol, amphetamine and hallucinogen use were compared to cannabis users, who had minimal exposure to MDMA, and drug-naïve controls. MDMA users showed an increase in impulsive choices, and impairments in working memory and decision-making. Moreover, frontal deficits were positively correlated with cumulative dose in Ecstasy users. Lifetime use of and dosage of Ecstasy plays a role in persistent neuropsychological deficits. These deficits in impulsivity and decision-making seen in polydrug users have been replicated in several studies (Morgan et al., 2005; Hoshi et al.,

189 2007). Fox et al. (2002) failed to find decision-making deficits using CANTAB’s computerized version of the Iowa Gambling Task, however participants’ cumulative dose of Ecstasy was 50% less than the aforementioned studies.

Negative effects on executive processes have also surfaced in studies examining polydrug use alone (Stevens et al., 2007). Hanson et al. (2008) found elevated impulsivity and decision-making impairments in both Ecstasy users and polydrug users.

The groups performed identically, eliminating Ecstasy as a contributing factor. By comparison, Roiser et al. (2007) failed to find decision-making impairments when controlling for polydrug use. No differences in impulsivity were seen between Ecstasy users and polydrug users, though all drug users showed higher amounts of impulsivity compared to naïve controls. Interestingly, in a related study, errors associated with poor decision-making were present only in participants that expressed the ss genotype of the

SERT protein, whereas these impairments were absent in ll genotypic individuals (Roiser et al., 2006). The vast variability in the results across human studies could be associated with polymorphisms at the SERT protein or other locales in participants.

Fox et al. (2002) tested Ecstasy users on a series of paradigms including

CANTAB’s intradimensional extradimensional (IED) test, a computerized language independent version of the Wisconsin Card Sorting task. The IED assesses the ability to form an attentional set, the ability to shift an attentional set, and behavioral flexibility through discriminative reversal learning (RL). Ecstasy users had slower trial latencies and committed significantly more perseverative errors in reversal learning during the earlier stages of the task, but improved RL performance after the extradimensional shift.

Ecstasy users also made significantly more perseverative errors on the reversals in the

190 Wisconsin Card Sorting task, with performance remaining unaffected on attention stages

(Thomasius et al., 2003).

Surprisingly, while the human literature contains numerous studies focusing on attentional and executive functioning deficits in MDMA users, there is a scarcity of data on MDMA’s direct impact using rodent models. Most reports focused on the impact of

MDMA on working memory. MDMA intoxication resulted in an increase in omitted responses in a delayed reinforcement task using a fixed ratio lever press protocol. High doses of the drug produced a long lasting impairment on reliable acquisition of delayed reinforcement altogether (Byrne et al., 2000). Subacute doses of MDMA produced deficits in a delayed match to sample paradigm, though deficits were delay independent.

The overall impairment in accuracy of choice may have been the result of proactive interference of trials across a testing session, and in fact, introducing an intertrial interval delay in between each trial attenuated inaccurate choices (Harper et al., 2005, 2006). No significant differences in working memory errors were found in a radial arm maze test

(Kay et al., 2009).

Even fewer studies have examined MDMA’s effects on attention and impulsivity.

Dalley et al. (2007) tested rats, which self-administered MDMA, after cessation of the drug, using a 5-choice serial reaction time test (5-CSRTT). There was an initial increase in the number of premature (impulsive) choices. However, after several weeks of discontinuation of MDMA, there was an increase in omitted responses, reflecting a decrease in vigilance. This is consistent with the studies that assessed impulsivity using a visual discrimination delayed match to sample lever press task. Immediate increases in impulsive choice disappeared over several weeks (Saadat et al., 2006). Likewise, our

191 laboratory has found a lasting effect of loss of vigilance (measured by a significant increase in aborted trials) in SD rats treated with 2 x 5 mg/kg PCA (Hughes et al., 2007).

Thus, MDMA’s effects on impulsivity in rats do not mimic the reliable presence of the behavioral trait in recreational polydrug users. High impulsivity may be reliant on other drugs or factors associated with drug abuse and addiction.

To date, there is a paucity of literature on the effect of MDMA on decision making or reversal learning using rodent behavioral paradigms, though the traits have been assessed in related amphetamine analogues. Fenfluramine administration produced an increase in responding to the immediate smaller reward (impulsive choice) in a delay reward paradigm (Cherek and Lane, 2000). Masaki et al. (2006) gave rats neurotoxic doses of the amphetamine analogue PCA and tested them on a visual discrimination go/no go and reversal task. Deficits in both the acquisition and reversals of the task were exhibited.

Ecstasy’s acute and long-lasting influence on the serotonin system is well documented in human literature. Therefore, the predictions, testing, and presence of executive impairments in rodents should parallel the faceted role of 5-HT in these frontal executive traits. As reviewed earlier, 5-HT input into the prefrontal cortex (PFC) critically modulates both behavioral inhibition and decision-making. Impulsivity has been tested in rats using protocols including delayed discounting tasks and the 5-CSRTT.

Behavioral inhibition/impulsivity is associated with drug abuse. High impulsivity is a behavioral phenotype which predicts acquisition of self administration in rats, and impulsivity acts as a predictor of drug abuse in humans. Conversely, chronic application of psychoactive drugs also produce impulsive traits in rodents and humans (Belin et al.,

192 2008; Everitt et al., 2008; Dalley et al., 2008; Perry and Carroll, 2008; Winstanley, 2007;

Winstanley et al., 2010). As such, the presence of high impulsivity in Ecstasy users can represent a chicken or egg dilemma.

The medial prefrontal cortex (mPFC) modulates behavioral inhibition. Both cytotoxtic lesions and inactivation of the mPFC result in an increase in impulsive behavior. Likewise, the dorsal and ventral striatum have also been found to contribute to impulsivity in different behavioral paradigms. Disruption of the orbital prefrontal cortex

(OFC), by disparity, has little impact on impulsive choices. The neurochemical basis of impulsivity includes 5-HT, dopamine, and norepinephrine, and the direct interactions between and homeostatic levels of each of these transmitters result in normal behavioral inhibition (Boulugouris and Tsaltas, 2009; Dalley et al., 2008; Winstanley et al., 2005).

Central serotononergic denervation, produced by intracerebral injections of 5,7-

DHT into raphe nuclei induced high impulsivity in a delayed discounting task and increased premature responses in the 5-CSRTT (Harrison et al., 1997; Mobini et al.,

2000; Winstanley et al., 2004). Elevated impulsivity has been replicated in humans using central reduction of 5-HT with acute tryptophan depletion (Schweighofer et al., 2008).

Microdialysis studies recorded 5-HT efflux, and specifically, increases of 5-HT in the mPFC in rats while performing the 5-CSRTT (Dalley et al., 2008). Amphetamine administration in rats increased impulsive responding and intracerebral 5,7-DHT application attenuated amphetamine-induced impulsivity. Impulsivity was also alleviated if dopamine (DA) release is blunted in the nucleus accumbens, illustrating a complex interaction between 5-HT and dopamine in impulsivity (Boulougouris and Tsaltas, 2009;

Winstanley et al., 2005).

193 In addition, discrete manipulations of 5-HT in specific anatomical regions or by

activation of specific receptors also effect impulsivity. 5-HT2A receptor antagonism decreased impulsive responding in the 5-CSRTT. Conversely, 5-HT2C antagonists increase impulsivity and 5-HT2C agonists decreased premature responses. Specifically, these 5-HT receptor drugs exert their actions on impulsive behavior when infused into the nucleus accumbens, but have little effect when intracerebally administered into the mPFC

(Carli et al., 2006; Navarra et al., 2008; Robinson et al., 2008). Activation of 5-HT1A receptors reduced impulsivity using a fixed ratio lever response paradigm (Evenden,

1998).

Numerous studies have found serotonin to be a regulating factor in decision- making. 5,7-DHT lesions produced impairments in a delayed reward paradigm, whereby rats chose the small immediate reward over the large delayed choice. Conversely, administration of SSRIs resulted in an opposite effect, with a majority of responding towards the delayed large reward. Similar impairments have been witnessed in humans using acute tryptophan depletion (ATD) tested with the Iowa Gambling task (see Clark et al., 2004 for review).

One of the most prominently studied 5-HT modulated executive traits is behavioral flexibility. Serotonin is involved in a specific example of behavioral flexibility: reversal learning (RL). RL is defined by Clark et al. (2004), as “the adaptation of behaviour according to changes in stimulus-reward contingencies, a capacity relevant to social and emotional behaviour… exemplified by [visual] discrimination tasks.” Perseverative responding is guiding one’s selection based on a previous contingency. This reflects an inability to shift response to the currently correct

194 choice, often paired with salience or a reward. Anatomically, reversal learning is impaired following lesions of the orbitofrontal cortex (OFC), but the behavior is relatively unaffected following lesions of other mPFC regions. Perseverative responding reflects cognitive inflexibility. Furthermore, imaging studies have found increases in

OFC activity whilst participants were performing reversal stages (McAlonan and Brown,

2003; Ragozzino, 2005, 2007; Rogers et al., 2000; Schoenbaum et al., 2002).

5-HT depletion of the OFC produces these same reversal learning impairments, whereby depletions of other monoamines, such as dopamine, leave reversal learning intact (Clark et al., 2004, 2005, 2007; Walker et al., 2009). Reversal learning impairments have also been seen following tryptophan depletion (van der Plasse and Feenstra, 2008).

5,7-DHT lesions directly into the prefrontal cortex resulted in cognitive inflexibility.

Deficits in reversal learning were associated with the affectual salience of stimuli (van der Plasse et al., 2007). Studies using acute tryptophan depletion in humans found reversal learning impairments, however, interestingly, these deficits only emerge in individuals expressing the ll allelic polymorpism of the SERT protein.

Serotonin receptor profiles are central in the modulation of behavioral flexibility.

Stimulation of the 5-HT1A receptors in the prefrontal cortex attenuated perseverative responding, induced via PFC inactivation by NMDA antagonism (Carli et al., 2006). 5-

HT2 receptors play a parallel role in antagonistic role in reversal learning as witnessed in impulsivity. Boulougouris et al. (2008) found 5-HT2A antagonism increases perseverative responding, fabricating cognitive inflexibility when rats were tested on a two-lever

discrimination with serial spatial reversals. 5-HT2C antagonism improved performance on the reversal stages. This modulation of reversal learning was also witnessed when 5-

195 HT2A/5-HT2C antagonists were directly infused into the OFC, though no effect on behavioral flexibility was seen with infusion into the mPFC or nucleus accumbens

(Boulougouris and Tsaltas, 2009).

One paradigm that examines reversal learning is the intradimensional extradimensional (IED) test. The IED is modeled in rodents using an odor texture

(digging medium) discrimination. Whereby primate versions of this task use visual discriminations (colors, shapes), variants of this test have used 2 or 3 perceptual dimensions ( odor, texture, digging medium). This task extrapolates two additional distinguished attentional measures: attentional set formation and attentional set shifting.

Just as 5-HT input to the OFC selectively modulates reversal learning, attentional set and set-shifting is modulated by distinct neuroanatomical and chemical components.

Prefrontal dopamine is key in the development of attentional selection (for review, see

Robbins, 2005; Chudasama and Robbins, 2006; Robbins and Arnsten, 2009; Robbins and

Roberts, 2007). To form an attentional set, subjects must learn to respond to stimuli of a relevant perceptual dimension and learn to ignore irrelevant stimuli belonging to another perceptual dimension. In the IED tasks, this occurs at the level of a compound discrimination (CD), whereby the rewarded perceptual domain in the acquisition of a simple discrimination (odor) is presented with an irrelevant perceptual stimuli in another domain (digging medium). If subjects have no difficulty in forming an attentional set, different digging mediums should not impact their response selection, and there will be a gradual improvement across stages in which response choice is guided by exemplars of the same perceptual domain (Robbins and Roberts, 2007).

196 Attentional set shifting involves the ability to shift one’s attention (response selection) from one perceptual dimension to another perceptual dimension. In the IED, the intradimensional shift (ID) involves the refocusing of attentional selection to two new separate pairs of perceptual exemplars (2 new odor/digging medium pairs), but the rewarded selection is still an exemplar of the originally rewarded perceptual domain

(odor). Improvement from the CD to the ID indicate the formation of an attentional set.

The extradimensional shift (ED) requires subjects to respond to exemplars of the previously irrelevant perceptual domain during the stages of the task (i.e. digging medium). In the ED, the previously rewarded percept (i.e. odor) is now relevant to response selection. Both excitotoxic lesions and inactivation of the mPFC produced attentional set shifting deficits in the IED and similar strategy switching deficits in related paradigms. Howerver, reversal learning performance was left intact (Birrell and Brown,

2000; Brown and Bowman, 2002; Floresco et al., 2008; Ragozzino et al., 1999; see

Boulougouris and Tsaltas, 2009 for review).

Prefrontal noradrenergic input plays a modulating role in attentional set shifting.

Noradrenergic lesions and pharmacological manipulations of norepinephrine (NE) and

NE receptors applied directly into the PFC, or into the noradrenergic bundle arising from the locus coeruleus, selectively impaired attentional set shifting across species (for review, see Robins and Arnstein, 2009; Robbins and Roberts, 2007). Additionally, mPFC NE depletion impaired attentional shifts, however acetylcholine depletion had no impact on the executive trait (McGaughy et al., 2008). Desipramine, a norepinephrine transporter protein (NET) blocker, and Mazindol, which blocks the reuptake of both

197 dopamine and norepinephrine, facilitate set-shifting (Bondi et al., 2010; Nikiforuk et al.,

2010).

5-HT receptor modifications also play a role in attentional set shifting. 5-HT6 receptor antagonists improve attentional set shifting in rats using an odor texture IED test.

5-HT6 antagonists also enhance cognition and memory in a number of other paradigms

(Fone, 2008; Hatcher et al., 2005; Rodefer et al., 2007). Lastly, dysregulation of acetylcholine also seems to have a negative effect on reversal learning performance in the

IED task. Saporin lesions of the basal forebrain produce impairments in serial reversal learning in a modified version of an odor texture discrimination task (Tait and Brown,

2008).

This study examined the long-term effects of variable low and high dose injection regimens of MDMA on executive functioning using an odor texture (IED) discrimination test in Long Evans (LE) rats. Currently, there are very few empirical studies analyzing the effects of MDMA on attentional and executive processes. This behavioral paradigm allows experimenters to extrapolate several executive processes from one task. The IED task tests acquisition of a simple discrimination, attentional set formation, attentional set shifting, and reversal learning. In addition, perseverative responding and (a)volition was inferred via error and aborted (omitted selections) trials respectively. The odor texture

IED task has two primate analogues: the Wisconsin Card Sorting task and CANTAB’s

IED test. This paradigm is translational, so specific executive processes can be extrapolated and examined across species from rodent to monkey to human (Brown and

Bowman, 2002; Fray and Robbins, 1996).

198 In the present study, rats were pretreated with a chronic low dose of MDMA, a binge low dose of MDMA (to denote ‘stacking’ of pills in recreational use), a high neurotoxic dose of MDMA, or saline controls. After two weeks of recovery, rats were tested across 7 different stages in a 2 dimensional odor digging medium IED task.

Following the test, rats were perfused, and changes in 5-HT and dopamine were assessed using an antiserum for the serotonin transporter and tyrosine hydroxylase, similar to the first two experiments. Stages of the IED test were measured independently, so each fronto-executive measure was probed discretely.

It was hypothesized that neurotoxic MDMA dose regimens would produce behavioral inflexibility, gauged by an increase in errors during the reversal stages. 5-HT lesions in the orbital frontal cortex, and tryptophan depletion produce perseveration in reversal learning stages of the IED and related tasks (Clark et al., 2004, 2006, 2007).

Likewise, impairments have been recorded in Ecstasy users and primates during IED tests (Fox et al., 2002; Thomasius et al., 2003). A binge high dose of MDMA should result in substantial denervation of the forebrain, including the prefrontal cortex, reflecting similar serotonergic damage as intracebral 5,7-DHT lesions as observed in

Experiment 1. By comparison, there should be no deficits in the ability to acquire a simple discrimination, or in the formation of an attentional set, or set shifting. This reasoning stands, as these executive processes are reliant on other chemical transmitters systems (Boulougouris and Tsaltas, 2009; Robbins and Roberts, 2007). Therefore, the long-term effects of MDMA should have little effect on these frontal behaviors.

LE rats given high doses of MDMA may become less vigilant and display avolition. Decreased vigilance and motivation was expressed in MDMA pretreated rats

199 measured by omitted responses in the 5-CSRTT (Dalley et al. 2007). Also a significant increase in aborted (omitted) trials in the compound and reversal stages of the odor texture discrimination test has been found in Sprague Dawley rats treated with the 5-HT selective amphetamine analogue PCA (Hughes et al., 2007). An in depth analysis of the impact of MDMA on 5-HT and DA systems was discussed in Experiment 1. Rats used in experiment 1 and 2 had a survival time of one month following their last treatment. The current study’s survival time was 2 weeks. In a subsection of this chapter, the two survival points will be analyzed to see if any large regeneration and sprouting of 5-HT axonal processes was visualized across the 2 week period from the two sample groups.

Early studies found a significant, though not total recovery of 5-HT axon fibers in rats 1 year following MDMA induced lesions (Battaglia et al., 1988; De Souza and Battaglia,

1989). Haring (1991) observed the initiation of axonal sprouting of dorsal raphe (DR) fibers at 2 weeks following intra-median raphe infusion of 5,7-DHT. DR fibers began collateral sprouting, so morphologically identical DR axons were expressed in areas denervated by MR lesions, such as the hippocampus. While it is most likely that some axonal sprouting began after 14 day time point, it is predicted that such fiber innervation would not be visually outstanding one month later.

MATERIALS AND METHODS

Subjects

Long Evans rats (n=40) weighing 250-300 grams upon arrival were included in this study. Original colony breeders were obtained from Charles River (Wilmington,

MA) and were bred by Florida Atlantic University Veterinary Services. Rats were

200 originally housed in pairs until the onset of their injections schedules, and then moved to individual plastic cages and maintained on a 12hr light/dark cycle with lights on at 7

A.M. Rats had access to food and water ad libitum until the start of behavioral testing.

All protocols were approved by the Florida Atlantic University Institutional Animal Care and Use Committee and held in accordance to the National Institutes of Health guidelines for the care and use of laboratory animals.

Rats were randomly assigned to one of the five groups prior to the start of the experiment: MDMA chronic low dose (10 x 1.5 mg/kg daily for 5 consecutive days over two weeks); MDMA binge low dose (4 x 1.5 mg/kg every two hours for two consecutive days); MDMA binge high dose (2 x 7.5 mg/kg); or two groups of physiological saline administered in a chronic or binge injection schedule. Injection schedules were staggered so two weeks passed before behavioral testing began for each subject. Body weights were obtained regularly throughout the experiment.

Drugs

MDMA (gifted from the National Institute of Drug Abuse) was dissolved in physiological saline and injected at a volume of 1ml/kg.

All rats were acclimated to the testing environment prior to the start of their dose regimens. They were wheeled into the injection room 30 min prior to the start of the daily session and left in the test area a maximum of 8 hours after their last injection to ensure that the effects of the drug had dissipated before return to the vivarium. Maximum onset of the drug occurred 20-30 min after their injection. Rats were monitored every 15 minutes for signs of serotonin toxicity. Behaviors including rearings, locomotion, head shakes, body tremors, salivation, piloerection, forepaw treading, penile ejaculate,

201 increased defecations, hind limb splaying, hyperthermia, and straub tail were noted. For each rat, the presence or absence of the behavior was recorded. In addition, rats that showed signs of hyperthermia, decreased locomotion, and/or flattened body were watched carefully. If they (n=5) entered into a catatonic state, they were rescued in a cool water bath (as described in the previous experiment).

Apparatus

Rats were tested in an opaque rectangular Plexiglas apparatus (40cm wide X 70 cm length x 20cm height). Two removal opaque dividers separated the apparatus into three quadrants. One removable divider held rats in the start position, and was removed at the beginning of each trial. A stationary central opaque divider was at the opposite end of the arena, running parallel to the length of the apparatus and divided one third of the end length into two sections (choice areas). Each of these sections housed a ramekin (4 cm deep x 7 cm wide) during the test trials. Ramekins contained discriminative odor and digging medium stimuli and were affixed to the apparatus floor using Velcro to prevent them from tipping over. The second removable divider was inserted to block the two choice areas once a rat began digging in a selected ramekin to prevent access to the other ramekin. Figure 37 is a schematic illustration of the behavioral testing apparatus.

202

Figure 37: Schematic diagram of the behavioral apparatus used in experiment 3. Rats were tested in an opaque Plexiglas apparatus (40 cm wide X 70 cm length x 20 cm height). One removable divider held rats in the start position, and was removed at the beginning of each trial. A stationary central opaque divider was welded to the opposite end of the arena, running parallel to the length of the apparatus and divided one third of the end length into two sections (choice areas). Each of these sections housed a ramekin (4 cm deep x 7 cm wide) during the test trials. Ramekins contained discriminative odor and digging medium stimuli and were affixed to the apparatus floor using Velcro to keep them stationary.

203 Procedure

Habituation

Figure 38 is a flowchart outlining the timeline and procedure for the IED protocol used in experiment 3. Rats were given three days of recovery following their last treatment injection before being placed on a food restricted diet, maintaining rats at 85% of their free feeding weights (approximately 15 grams per day of rat food). One week following their last injection schedule, test room habituation began. Ramekins filled with corncob (their bedding material) were placed in their homecage with a buried fruit loop

(Kellogg, US) to familiarize them with the food reward. During the third day of room habituation, rats were habituated in the empty testing arena for a 5-min period. During habituation and testing, fruit loops were divided into 1/4th size pieces and placed at the bottom of the ramekins covered in digging medium.

Testing habituation began on the 13th day post injection schedule so that the odor discrimination test was administered 2-weeks following drug administration. During digging establishment habituation, food rewards were buried at the bottom of the two ramekins, which were filled with an unscented digging medium (recycled cellulose bedding). One ramekin was placed in the each of the choice areas in the testing apparatus. Rats were initially placed in the start area, and when the removable divider was lifted, digging establishment trials commenced. Rats were required to dig in both ramekins to retrieve each of the food rewards to perform the trial correctly. This digging behavior was established over a series of 5-min trials. On the first trial, considered exploratory, both ramekins were baited with the fruit loop placed on top of each bowl.

Rats had the opportunity to retrieve the fruit loops. If rats retrieved both food rewards

204 before 5 min, the animals remained in the apparatus for the entire five minutes to familiarize themselves with the arena and ramekins. After the first trial, the food reward was buried in the bottom of each ramekin, and subsequent trials ended when both rewards were retrieved or when 5 min had elapsed. Testing continued until rats retrieved the food reward from both ramekins for 12 correct trials to establish reliable digging behavior. If none or only one food reward was retrieved during the five-minute trial, the trial was considered unsuccessful (aborted). If a rat aborted three trials in a row, he was placed in his homecage for a 5-minute break with access to water to re-stimulate novelty in the task. During this time, digging mediums were refreshed. Rats unable to establish digging behavior after thirty trials were excluded from the study (n=1).

Next, two simple discrimination (SD) tasks were conducted. In the simple discrimination, rats had to learn to discriminate between two exemplars in one perceptual domain to retrieve a food reward, while a second perceptual domain remained constant between the two choices. The first SD was odor discrimination: ramekins were scented with vanilla (correct choice paired with food reward) or sweet orange (no reward) and filled with recycled cellulose bedding as a digging medium. The second SD was a digging medium discrimination: unscented plastic beads (baited correct choice) versus unscented shredded foam (incorrect choice).

During habituation SD stages, the first four trials were exploratory. The first trial of each stage lasted two minutes. During this time, rats were allowed to explore the ramekins and retrieve the food reward. If digging occurred in the incorrect bowl first, the trial was marked as an error, however the rat was not removed from the testing apparatus, but allowed the entire 2 minute trial period to retrieve the food reward (make the correct

205 association). If the rat made an error during the next three trials (2-4), he remained in the apparatus until the reward was retrieved from the correct ramekin, or 2 minutes.

However, if the correct selection was made, the trial was immediately terminated, and the rat was placed in the start arena until the commencement of the next trial. After the exploratory trials (5-), trials were terminated once rats began to dig in one of the ramekins, whether rats performed a correct or incorrect choice. If a rat did not dig in either of the choices after two minutes, the trial was terminated and considered an aborted trial (omitted response). If rats committed three aborted trials in a row, they were placed in their homecage for 5 minutes with access to water, before testing recommenced. Six consecutive correct choices were required to successfully complete each SD task. Both errors and aborts were considered incorrect choices. The correct and incorrect odor and digging exemplars were not used again during testing. Rats unable to successfully complete each habituation task (acquisition of a simple discrimination) were eliminated from the study (SD1, n=2; SD2, n=3).

IED testing

Twenty four hours following habituation, rats were tested across seven stages: (1) simple discrimination (SD), (2) compound discrimination (CD), (3) reversal learning

(RL) of the compound discrimination (CD2), (4) intra-dimensional shift (ID), (5) RL of the intradimensional shift (ID2), (6) extradimensional shift (ED), and (7) RL of the extradimensional shift (ED2). Ramekins were scented using essential oils for all stages of the experiment. Table 11, adapted from Birrell and Brown (2000), lists the odor and digging medium exemplars used in each stage of this experiment.

206 In the simple discrimination (SD), choice selection differed along one dimension: odor (O1 rewarded). Recycled cellulose bedding, the same digging medium in digging establishment, was used for both choices in the SD. In the compound discrimination

(CD), the same odor (O1) was rewarded, however during each trial, odors were paired with a possibility of one out of two levels of the irrelevant dimension (digging medium)

In the compound discrimination, rats must learn to form an attentional set, and respond to the correct odor, regardless of the digging medium it is paired with was reflected if rats displayed a high rate of errors or omitted responses in selecting the correct odor choice in the light of inconsistent digging exemplars.

The third stage was a CD reversal. The same odor digging medium pairs remained constant from the previous stage, however the food reward was now paired with the previous incorrect (O2) odor exemplar. In the reversal learning stages, an assessment of behavioral flexibility, rats had to extinguish previously correct choices and shift their behavioral selection to the newly rewarded (but previously incorrect) choice.

A significant number of errors during this stage was indicative of perseveration.

207

Figure 38: Outline and timeline of the intradimensional extradimensional (IED) odor texture discrimination paradigm.

208 Table 11 Odor and Digging Medium exemplar pairs presented in each intradimensional extradimensional (IED) stage are listed in the table adapted from Birrell and Brown (2000). The exemplar and percept paired with a reward for each stage is italicized. See list for abbreviations.

209 In the intradimensional shift (ID) stage, two new odor and digging mediums were introduced as novel medium pairs, however selection was still based on the previous perceptual domain (attentional set). Rats needed to attend to the correct odor choice (O3) and ignore irrelevant digging mediums. The 5th stage of the task was the ID reversal test, where the incorrect odor from ID (O4) was now baited and the proper choice. The extradimensional shift (ED), stage 6, was a test of attentional set shifting. The ED stage introduced another pair of novel odor/digging medium pairs. However, this time rats had to shift their behavioral selection to the somatosensory perceptual domain, whose exemplars were irrelevant throughout the previous stages in the IED test. For the ED and

ED reversal stages, the odors now represented the irrelevant perceptual domain, and the correct digging medium was the correct (baited) selection (M5 for ED1; M6 for the ED reversal). A high rate of errors and/or an increased number of trials to complete criterion reflected deficits in attentional set shifting.

During each stage trial, rats were given access to two bowls, which varied with respect to odor and/or digging medium depending on the stage. Ramekins were the same color for each rat and remained uniform throughout the experiment. Only one bowl (the correct choice) was baited with the food reward. As in habituation, the first four trials were exploratory trials. If a choice was not made after two minutes, the trial was aborted

(omitted response). A series of six consecutive correct trials was required to complete each stage. If the rat could not complete 6 consecutive correct responses after thirty trials, the rat failed the stage (n=2; two high binge MDMA rats failed at the ID reversal stage). If rats were responding correctly at trial 30, rats were allowed to continue until they completed the stage or made an incorrect choice to terminate the test. The

210 odor/digging medium pairs in stages 1-7 were presented in a consistent order across all rats. The location and the correct exemplar pair (i.e. O1 paired with M1 or O1 paired with M2) was selected pseudorandomized for every trial in all stages to prevent conditioned place learning.

Immunohistochemical Analysis

Immediately following testing, rats were anesthetized, perfused, their brains removed and prepared for immunohistochemical procedures. A detailed description of the immunohistochemical protocols used in these series of experiments is located in the methods section of experiment 1.

Data Analysis

Univariate analysis of variance (ANOVA) was conducted on the total amount of trials to establish digging behavior, and the mean total number of trials, mean errors, and aborts during the habituation simple discrimination. If significant results were present in the habituation stage, it indicated deficits in acquisition of the SD. ANOVAs were also conducted on mean total number of trials to complete each stage, mean errors for each stage, and mean aborted trials for each IED stage, with a between subject factor of experimental group. Mean number of errors, aborted trials, and total trials per stage were used to determine performance on each stage and assessed fronto-executive functioning, specifically attentional set formation, attentional set shifting, and behavioral flexibility to assess if significance differences existed between different dose regimens of MDMA treated rats and saline controls.

Impaired performance in any IED stage could be a result of several executive deficits. For example, if treatment groups showed significant impairments on an IED

211 reversal stage by illustrating a greater number of total trials to meet criterion, preservative behavior was analysed by looking for a concurrent increase in errors. Aborts were used as a measure of volition, and are similar to omitted responses recorded in other attentional paradigms such as the 5-CSRTT. A significant increase in aborts (omitted responses) in a treatment group may reflect deficits in motivation to complete the task.

Completion of each stage required six consecutive responses. If a treatment condition exhibited a significantly larger number of trials to complete a specific stage, but there were no differences in the amount of errors or aborts to correlate with this stage, deficits in forming an attentional set, attentional shift, or in reversal learning can not be directly associated with impairments in motivation and perseveration, per se.

Univariate analysis of variance was also conducted for the mean latency of trials in each stage, and average median of latency for trials in each stage. Latencies were collected by stopwatch. Latency data was measured in seconds. While this was a self- paced task, a consistent decrease in latencies of trials for a treatment group, regardless of correct or incorrect responses, may reflect impulsive responding. Because of the nature of the IED task and its non-automation, rats may have become distracted on one trial during a stage. If this occurred, mean latency data would be skewed, and average latencies could be largely affected by outliers. To correct for this, the average median latencies for both correct and error trials was also analysed using an ANOVA.

Multivariate repeated measures analysis was also conducted across stages to assess if improvement in learning and or decrement or fatigue effects were present.

Multivariate tests were conducted on total trials, errors, and aborts. The within subjects factor in each analysis was each stage with a between subject factor of treatment

212 condition. Next, a repeated measures multivariate analysis was completed for total number of trials and errors with the within subjects factor of (CD2, ID2, and ED2) and a between subjects factor of treatment condition to see if any reversal learning would stabilize or improve across the three RL stage presentations. This repeated measures multivariate analysis was also completed on the CD to ID and ID to ED stages, to see if there were differences in attentional set, or shifts when the shift required intrasensory percepts versus when the shift required the rat shift attention to a new sensory percept.

For all univariate and multivariate measures, homogeneity of variance was calculated using Levene’s test to see if there were differences in variances between groups. In repeated measures multivariate analysis, Mauchly’s test of sphericity tested whether or not covariance could be assumed. Because of the relatively small sample sizes in each group, if homogeneity of variance could not be met in univariate analysis, it indicated significant differences in variance across subjects. Data were then analyzed using the nonparametric Kruskall Wallis ANOVA test. In the Kruskal Wallis test, data points are converted into ranks, which then uses the median to determine if differences between the groups are present. If the results from the Kruskall Wallis test were significant between subject’s differences, Mann Whitney U pair comparisons tests were run on each combination of treatment groups.

2 Estimates of effect size were calculated using partial eta squared (np ). All measures calculated used an alpha level of 0.05 to reach statistical significance. Statistics were run in SPSS 170.0 and 18.0 for Mac. In multivariate repeated measures analysis, pairwise comparisons using the Bonferroni test were used to look at differences across the within subjects factors. If sphericity could not be assumed, the Greenhouse Geisser

213 correction was used. To examine the between subjects factor of treatment condition, post hoc analysis was conducted using the Tukey HSD test. If homogeneity of variance could not be established, data were examined using the Tamahane’s T2 test.

The pattern and density of SERT innervation of the forebrain was assessed in subjects across experimental groups similar to methods described in Experiment 1. For immunohistochemical verification, lightfield photomicrographs at 100x magnification were taken from a best representative case for each MDMA treatment group and a control rat through key forebrain areas. Photomicrographs were taken with Q Imaging ICAM camera mounted on a Nikon Eclipse E600 microscope. Photomicrographs were captured in Nikon NS Elements at 100x, as greyscale tiff file images. Images were then opened in

Adobe Photoshop for Creative Suite 4 for Mac (Mountain View, CA) where further adjustments to brightness and contrast were corrected for illustrative purposes.

Survival times following MDMA treatment differed between the two behavioral tasks. LE rats used in experiment 1 had a survival time of one month following their last dose injection before they were perfused. The survival period in this experiment was two weeks. The immunohistochemical results section of the current experiment compared the degree of serotonin axonal denervation in the binge MDMA high dose at two weeks versus binge MDMA high dose LE rats with a survival period of one month.

RESULTS

Physiological Effects of MDMA

The behavioral profiles and activity of MDMA chronic low dose, MDMA low dose, and MDMA binge high dose rats did not differ from those in the previous 214 experiment. See experiment 2 for a detailed description of the physiological effects of

MDMA on rats.

Behavioral Testing

Digging Establishment/Habituation

The habituation phase was held on a single day and included three stages. The first stage was digging establishment. In order to ensure rats would reliably dig to retrieve a food reward from the ramekins, rats completed 12 correct 5 minute digging trials. The means and standard deviations for total number of trials needed to complete this stage are displayed in Table 12. Figure 39 is a bar graph displaying the mean number of total trials to establish digging for each treatment condition. No significant differences in mean number of trials to establish digging behavior were found, F(4, 35)= 2.642, p >

2 .05, np =.232, however the value approached significance.

The first habituation (HB) simple discrimination (SD1) stage used odor as the rewarded percept and second HB simple discrimination (SD2) used digging medium as the reward contingency. Table 12 displays the means and standard deviations for total trials, errors, and aborted trials for each treatment condition for SD1 and SD2. Figure 40 displays bar graphs depicting the mean number of trials to completion, errors, and aborted trials for each treatment condition for the two habituation simple discriminations.

No significant differences in the total number of trials to completion, F(4, 35)= .352, p >

2 2 .05, np = .039; total errors, F(4, 35)= .605, p > .05, np =.065, or total aborts, F(4, 34)=

2 .522, p > .05, np =.058, were present between treatment condition for the odor SD1 stage.

Results did reveal a significant between-subjects effect for the total number of

2 trials to completion in digging medium SD2, F(4, 35)= 3.188, p < .05, np = .267. 215 MDMA chronic low dose rats needed significantly more trials to complete the second simple discrimination (M difference = 2.50, p < .05) than rats in the control chronic dose.

Despite this significance, all rats completed this stage with a mean fewer than 10 trials, demonstrating rats learned the SD2 proficiently. There was no significant difference between treatment conditions in the mean number of errors, F(4, 35)= .705, p > .05,

2 2 np =.075 or aborted trials, χ (4, N = 40) = 6.686, p > .05 committed during SD2.

Odor texture IED test

The IED testing session, comprised of 7 independent stages, which commenced

24 hours subsequent to the habituation session. Table 13 lists the means and standard deviations of total trials to completion of stage, errors, and aborts for each treatment condition across all seven stages in the IED test. Figure 41 is a bar graph which illustrates the mean number of total trials for the completion of each stage for each treatment condition. Figure 42 shows the mean number or errors committed during each of the seven stages for each of the drug treatment conditions. Figure 43 displays the mean number of aborted trials across treatment conditions for each of the test stages.

Stage 1 required completion a simple discrimination (SD), using an odor exemplar (O1) as the reward contingency. No significant differences in total trials to

2 complete the stage, F(4, 35)= .893, p > .05, np =.093 were observed. There was also no

2 significant difference in errors committed, F(4, 35)= .656, p > .05, np =.07, between the experimental groups during the SD stage. No difference in mean aborts during the SD stage were noted, χ2(4, N = 40) = 4.992, p > .05, when using the nonparametric Kruskal

Wallis test. Mean rank values for trials, errors, and aborts for stages which did not meet

216 the assumption of homogeneity of variance used in the Kruskal Wallis test are listed in

Table 14.

The second stage of the IED test introduced a compound discrimination (CD), where two levels of odor and digging medium exemplars were presented in pairs, with

O1 still the reward contingency. There was a significant difference in the total trials to complete the CD stage between drug treatment conditions, F(4, 35)= 2.76, p < .05,

2 np =.240. MDMA binge high dose rats performed significantly more trials than the control chronic condition to complete the CD stage (M difference= 3.75, p < .05). The difference between the MDMA high dose and control binge group approached significance (M difference = 3.375, p = .074). No significant differences existed in the mean number of total trials to reach criterion between the three MDMA treatment conditions.

ANOVA statistics also observed a significant difference in the mean errors

2 performed during the CD stage, F(4, 35)= 4.46, p < .01, np =.338. Post hoc analysis confirmed that the MDMA binge high dose group committed significantly more errors than both the control binge (M difference= 2.375, p < .01) and control chronic (M difference = 2.125, p < .05) conditions. MDMA binge high dose rats also had more errors than both the MDMA chronic low dose (M difference =1.625, p= .09) and MDMA binge low dose groups (M difference= 1.75, p= .058), however, this difference approached, but did not reach significance. No significant between-subjects effects were found for mean CD aborted trials, χ2(4, N = 40) = 8.562 , p > .05.

217 Table 12 Mean (Standard Deviations in parentheses) values for total number of trials completed to complete digging establishment and total number of trials to completion, errors, and aborted trials in the first habituation odor simple discrimination (SD1) and the second habituation digging medium simple discrimination (SD2) for each of the treatment conditions.

218

Figure 39: Mean number of total trials (y axis) to complete digging establishment for each of the treatment conditions (x axis) during the habituation phase. No between- 2 subjects effect of drug treatment were found, F(4, 35)= 2.642, p > .05, np =.232.

219 Table 13 Mean (Standard Deviations in parentheses) values trials to reach stage completion, errors, and aborted trials for the seven stages of the Intradimensional Extradimensional (IED) test: Simple Discrimination (SD), Compound Discrimination (CD), Compound Reversal (CD2), Intradimensional shift (ID), Intradimensional Reversal (ID2), Extradimensional shift (ED), Extradimensional reversal (ED2).

220

Figure 40: Mean total number of trials to reach stage completion (A), errors (B) and aborts (C) for each treatment conditions for each of the two habituation simple discrimination stages: an odor simple discrimination (SD1) and digging medium simple discrimination (SD2). No significant differences in the total number of trials to 2 completion, F(4, 35)= .352, p > .05, np = .039; total errors, F(4, 35)= .605, p > .05, 2 2 np =.065, or total aborts, F(4, 34)= .522, p > .05, np =.058, between-treatment condition in the SD1 stage were found. There was a significant difference between-treatment conditions for the total number of trials to completion in SD2, F(4, 35)= 3.188, p < .05, 2 2 2 np =.267, but no differences in errors F(4, 35)= .705, p > .05, np =.075, or aborts, χ (4, N = 40) = 6.686, p > .05. Chronic low dose needed significantly more trials to complete the second simple discrimination the control chronic dose. Despite this significance, all rats completed this stage < 10 trials.

221 Table 14 Mean rank values for median number of trials to complete stage, median errors, and median aborted trials in stages in the intradimensional extradimensional test for which homogeneity of variance could not be assumed. The nonparametric Kruskal-Wallis test transformed data points to ranks, which were then used to examine whether median ranks across treatment conditions statistically differed from one another. Abbreviations: Simple Discrimination (SD), Compound Discrimination (CD), Compound Reversal (CD2), Intradimensional shift (ID), Intradimensional Reversal (ID2), Extradimensional shift (ED), Extradimensional reversal (ED2).

222 The third stage of the IED test was a RL test of the compound discrimination

(CD2), in which the previously unrewarded odor (O2) exemplar was now the reward contingency. Significant between-subject effects were found for total trials to complete

CD2 criterion, χ2(4, N = 40) = 21.124 , p < .001. Mann Whitney U tests found MDMA binge high dose rats performed a significantly greater number of trials than the control binge, control chronic, MDMA chronic low dose, and MDMA binge low dose treatment groups.

Significant between-subject differences were also found in the mean number of

2 errors committed during CD2, F(4, 35)= 29.408, p < .001, np =.771. MDMA binge high rats performed significantly more errors during the CD2 than the control binge, control chronic, MDMA chronic low dose, and MDMA low binge treatment conditions. There were no other significant differences in errors between the other treatment conditions.

No between-subjects differences in aborted trials were noted, χ2(4, N = 36) = 6.209, p >

.05.

The 4th stage in the IED test was the intradimensional (ID) shift. Two novel odor digging medium pairs were introduced. The reward contingency was now paired with a new odor (O3) exemplar, an intra (sensory) dimensional shift in attention. The nonparametric Kruskal-Wallis test found no differences in the mean total number of trials for completion of the ID stage between treatment conditions, χ2(4, N = 40) = 5.54, p >

.05, but a significant between-subjects effect was found for errors, χ2(4, N = 40) = 11.273, p < .05. A Mann Whitney U pairwise comparison test found MDMA high binge rats committed significantly more errors during the ID stage than the control binge, U=13.50, z= -2.019, p= .05, control chronic, U=7.00, z= -2.768, p < .01, and the MDMA chronic

223 low dose groups, U=10.50, z= 12.346, p < .05. However, there was no difference in the number of ID errors between the MDMA binge low and high dosed rats. No significant

2 differences in mean number of ID aborted trials, F(4, 35)= .561, p > .05, np =.06.

Stage 5 of the IED test was the ID reversal (ID2). The reward contingency was now the previously incorrect exemplar O4. Significant between-subjects effects were present for the total number of trials to reach completion, χ2(4, N = 40) = 18.904, p < .01.

Pairwise comparisons using the Mann Whitney U test found the MDMA binge high group needed a significantly greater number of trials to reach criterion than the control binge, control chronic, MDMA chronic low, and MDMA binge low dose treatment conditions. However, no significant differences in total trials to criterion existed between the control binge, control chronic, and chronic and binge low dose MDMA groups.

A significant difference in mean errors, χ2(4, N = 40) = 22.549, p < .001, was also found, however, there were no significant difference in ID2 aborts, χ2(4, N = 40) =

13.395, p >.05. Pairwise comparisons using the Mann Whitney U test found MDMA binge high dose rats performed more errors during the ID2 than control binge, U=.00, z=

-3.414, p < .001, control, chronic, U=.00, z= -3.414, p< .001, MDMA chronic low dose,

U= 2.00, z= -3.167, p < .01, and MDMA binge low dose rats, U=1.50, z= -3.232, p <

.001. MDMA binge low dose rats performed significantly more errors than control binge rats, U=10.50, z= -2.38, p < .05. There were no other significant differences between the other treatment conditions. Two MDMA binge high dose rats failed the IED test at this stage, and were excluded from the last two ED stages.

224

Figure 41: Mean number of total trials to reach criterion for each of the stages of the intradimensional extradimensional (IED) test: simple discrimination (SD); compound discrimination (CD); compound discrimination reversal (CD2); intradimensional shift (ID); intradimensional reversal (ID2); extradimensional shift (ED); extradimensional reversal (ED2). MDMA high binge dosed rats needed significantly greater number of trials to reach completion in the CD stage as compared to the control chronic group, F(4, 2 35)= 2.76, p < .05, np =.240.. MDMA high binged rats also performed significantly more trials than the control binge, control chronic, MDMA low chronic, and MDMA low binge groups in the CD2, χ2(4, N = 40) = 21.124 , p < .001, and ID2 reversal stages, χ2(4, N = 40) = 18.904, p < .01. MDMA high binge rats also performed significantly more trials in the ED2 stage than the control groups and the MDMA low chronic condition, χ2 (4, N = 38) = 9.981, p < .05. Brackets (*) indicate groups significantly different than the MDMA binge high dose group (**).

225

Figure 42: Mean errors committed during each of the seven stages in the intradimensional extradimensional (IED) test: simple discrimination (SD); compound discrimination (CD); compound discrimination reversal (CD2); intradimensional shift (ID); intradimensional reversal (ID2); extradimensional shift (ED); extradimensional reversal (ED2). MDMA high binge dosed rats committed significantly more errors than 2 the control groups during the CD stage, F(4, 35)= 4.46, p < .01, np =.338, and during the ID stage, , χ2(4, N = 40) = 11.273, p < .05. MDMA high binged rats also committed 2 2 significantly more errors in the CD2, F(4, 35)= 29.408, p < .001, np =.771, and ID2, χ (4, N = 40) = 22.549, p < .001, reversal stages than all treatment conditions. MDMA high binge dosed rats also committed significantly more errors during the ED2 reversal stage as compared to the control binge, control chronic, and MDMA chronic low dose treatment groups, χ2 (4, N = 38) =12.891, p < .05. Brackets indicate groups (*) significantly different than the MDMA binge high dose group (**).

226

Figure 43: Mean aborted trials during each of the seven stages in the intradimensional extradimensional (IED) test: simple discrimination (SD); compound discrimination (CD); compound discrimination reversal (CD2); intradimensional shift (ID); intradimensional reversal (ID2); extradimensional shift (ED); extradimensional reversal (ED2). If a rat did not dig for the reward in the two minute trial period, the omitted response was noted as an abort. There were no significant differences in aborted trials between treatment conditions in any of the seven stages of the IED test. The large variation in aborts in the ID2 stage in MDMA binge high dose rats was attributed to rats (n=2) who failed the IED test at this stage.

227 The 6th stage was the extradimensional shift (ED), whereby two novel odor/digging medium pairs were introduced and now, the digging medium exemplar

(M5) was rewarded. No significant between subjects differences were found in trials to

2 meet stage criterion, F(4, 33)= 1.029, p > .05, np =.111, mean errors, F(4, 33)= 1.076, p >

2 2 .05, np =.115, or in the mean aborted trials, χ (4, N = 38) = 6.318, p > .05, in the ED stage.

The last stage of the IED test was the reversal of the extradimensional shift

(ED2). A significant between subjects effect was found for mean total trials to completion of stage, χ2 (4, N = 38) = 9.981, p < .05 and for mean errors committed, χ2 (4,

N = 38) =12.891, p < .05. MDMA binge high dose rats had a greater mean number of trials to reach completion of the ED2 stage as compared to the control binge, U=7.00, z=

-2.207, p < .05, control chronic, U=1.00, z= -3.016, p=.001, and the MDMA low chronic dose, U= 2.00, z= -2.869, p < .01. MDMA binge high dose rats needed a greater number of trials to reach criterion than MDMA binge low dose rats, however this difference only approached significance, U=9.00, z= -1.954, p= .059. No other treatment conditions had significant differences in total trials. MDMA binge high dose rats also performed a significantly greater number of errors than the control binge, U=1.00, z= -3.026, p= .001, control chronic, U= 3.50, z= -2.722, p < .01, and MDMA low chronic dose, U= 1.00, z= -

3.023, p= .001. There was no significant difference in errors between the MDMA binge low dose group, U= 12.00, z= -1.592, p > .05. Errors committed by the MDMA binge low dose group during ED2 did not significantly differ from the other treatment groups and there were no significant differences between any of the other treatment conditions.

228 There were no significant differences in ED2 mean aborted trials, F(4, 33)= .94, p > .05,

2 np =.023.

Repeated multivariate analyses were conducted using IED stages as a within subjects factor to look for improvement or decrement of performance across stages. All treatment conditions needed significantly less trials to complete the SD stage as

2 compared to the CD stage, F(1, 35)= 7.25, p < .05, np =.172, but no significant stage by

2 drug treatment condition interaction, F(4, 35)= .1.128, p > .05, np =.114 was present (data not shown). All rats also took significantly greater number of trials to complete the CD2

(first reversal stage of CD) as compared to the number of trials for completion of the

2 compound discrimination, F (1, 35)= 18.248, p < .001, np =.343. A significant stage by treatment interaction was also present across CD to CD2, F(4, 35)= 5.299, p < .01,

2 np =.377.

No significant differences existed between the mean number of trials to completion of the compound discrimination stage as compared to the intradimensional

2 stage, F(1, 35)= .08, p > .05, np =.002, with no significant stage X treatment interaction,

2 F(4, 35)= 3.169, p > .05, np =.061. Finally, there was no significant difference in the mean number of trials to stage completion across the extradimensional stage in comparison to the compound or intradimensional stage, F(2, 66)= 1.797, p > .05,

2 np =.052, and no significant stage X treatment interaction, F(8, 66)= 1.023, p > .05,

2 np =.110 (data not shown).

No significant within-factor differences in total trials across the three reversal

2 stages was observed, F(2, 66)= .877, p > .05, np =.026, however a significant stage X

2 treatment condition interaction was found, F(8, 66)= 2.467, p < .05, np =.23. There were 229 no significant within-subjects effect of stage for mean total trials, F(2, 66)= .877, p > .05,

2 2 np =.026, or mean errors, F(2, 66)= 1.171, p > .05, np =.034, however there was a significant interaction between stage and treatment condition, F(8, 66)= 6.681, p < .01,

2 np =.308.

The significant stage X treatment interaction present across reversal stages was the result a progressive reduction in the mean number of trials and errors performed across the three reversal stages in the MDMA high binge condition. Figure 44A is a bar graph illustrating the mean number of total trials across the reversal stages for the

MDMA high binge rats. Rats in the high binge MDMA dose condition needed fewer trials to reach completion in the extradimensional reversal as compared to the intradimensional reversal, however this only approached significance (p= .061). A significant within-stage effect was discovered across the three reversal stages for mean

2 number of errors in the MDMA high binge condition, F(2, 10)= 4.675, p < .05, np =.483.

Figure 44B displays the mean number of errors performed during each of the reversal stages. Rats given the MDMA high binge dose performed significantly fewer errors in the ED reversal than the CD reversal (p < .05), showing a possible improvement of performance across reversal presentations, however the 2 cases which failed the ID2 stage were not calculated in the ED2 data.

Latencies

The latency to complete each trial was recorded for every trial across each of the stages for subjects. Latency to complete each trial was recorded (seconds; using a stop watch) for digging selection of a trial, regardless of whether the rat selected the correct or incorrect ramekin. If the trial was aborted, the latency recorded for the trial was 120

230 seconds. Mean latencies per stage were then calculated for each subject and compared via univariate measures across treatment conditions. Mean latencies for each treatment condition are displayed in Table 15. No significant differences across treatment conditions for latencies to complete each trial were found across the following stages:

2 habituation (HAB) SD1, F(4, 36)= .316, p > .05, np =.034, HAB SD2, F(4, 36)= .875, p

2 2 2 >.05, np =.089, SD, χ (4, N = 40) = 4.065, p > .05, CD2, χ (4, N = 40) = 8.781, p < .05 ,

2 2 2 ID, F(4, 36)= .146, p > .05, np =.168, ID2, F(4, 36)= .378, p > .05, np =.108, ED, χ (4, N

2 = 38) = 3.898, p < .05, and ED2, F(4, 34)= .508, p > .05, np =.056. Significant differences in mean latencies of trial were found only in the compound discrimination

2 stage, F(4, 36)= 2.717, p< 05, np =.232. The MDMA chronic low dose condition displayed significant increased latencies as compared to the control chronic condition, however no other significant between subject effects were noted.

Average median latencies for completion of each trial during the 7 stages were also calculated to rule out the possibility of single (or multiple) trial distractibility skewing latency data points during the experimenter controlled IED task, which was not automated. Average median latencies for each treatment condition across the 7 stages is displayed in Table 16. Median latencies for trial completion were shorter than mean latencies across each stage, but not significantly so. Table 20 displays the average median latencies for each stage across every treatment condition. There were no significant differences in treatment condition in average median latency of trial

2 completion during the following stages: HAB SD1, F(4, 36)= .657, p > .05, np =.068,

2 2 2 HAB SD2, F(4, 36)= .811, p >.05, np =.083, SD, χ (4, N = 40) = 7.214, p > .05, CD, χ

(4, N = 40) = 5.63, p >.05, ID2, χ2 (4, N = 40) = 7.077, p > .05, ED, χ2 (4, N = 38) =

231 2 7.157, p < .05 , and ED2, F(4, 34)= .809, p > .05, np =.087. Significant differences in median latencies across treatment condition were found in the CD2, χ2 (4, N = 40) =

16.91, p < .01 and ID stages, χ2 (4, N = 40) = 13.256, p = .01. Mann Whitney U pairwise comparisons distinguished rats in the control chronic condition had significantly faster average median latencies compared to the MDMA chronic low dose in the CD2 and ID stage, and compared to the MDMA binge low dose conditions during the ID stage. MDMA binge high dose rats also showed significantly faster latencies to complete trials compared to the MDMA chronic low dose, and MDMA binge low dose groups during the CD2 and ID stages.

Immunohistochemistry

A detailed account of the pattern of labeling of serotonergic innervation of the forebrain, and serotonergic denervation of the forebrain produced only by the high binge dose of MDMA (2 x 7.5 mg/kg) was discussed at length in Experiment 1. MDMA chronic low dose and MDMA binge low dose injection schedules produced no long-term degeneration of serotonergic axons as observed by the pattern of innervation of SERT immunolabelled fibers. As with Experiment 2, a significant loss of SERT forebrain innervation was observed in the MDMA binge high dose condition. Figure 45 is a high magnification brightfield photomontage showing the pattern of SERT forebrain innervation in the prelimbic cortex in a representative control and MDMA binge high dose rat. Notice the substantial loss of SERT labeled fiber expression across PL in the

MDMA treated rat as compared to the control. Figure 46 is a brightfield photomontage of the pattern of SERT innervation across the dorsal and ventral striatum in a control and

MDMA binge high dose rat. Note the dense innervation of both the dorsal striatum (CP)

232 and nucleus accumbens in the control rat, and strong reduction of labeled fibers in the

MDMA binge high dose rat.

Survival time between Experiment 2 (one month) and Experiment 3 (two weeks) varied by 14 days, due to differences in training periods of the behavioral paradigms.

SERT immunohistochemistry of subjects in the MDMA binge high dose group from the two experiments was examined to determine the possibility of additional axonal damage or axonal sprouting. Figure 47 is a brightfield photomontage through the caudal aspect of the ventral orbital cortex in a representative MDMA binge high dose subject from

Experiment 2 (A) and Experiment 3 (B). Figure 48 is a brightfield photomontage through the ventral hippocampus in a representative MDMA binge high dose subject from Experiment 2 (A) and Experiment 3 (B). MDMA binge high dose rats in

Experiment 2 and Experiment 3 illustrated no large differences in the reduction of SERT fiber expression across the forebrain. There were also no notable differences in the morphology of the remaining SERT labelled fibers.

233

Figure 44: Performance of rats in the MDMA high binge experimental group across the three reversal stages in the intradimensional extradimensional (IED) test: compound discrimination (CD2), intradimensional shift (ID2), and extradimensional shift (ED2). No significant differences in mean number of trials to stage completion (A) across the reversal stages were observed, however rats committed significantly fewer perseverative errors (B) during the last reversal (ED2) as compared to the initial compound discrimination reversal, illustrating a possible improvement in reversal learning across 2 stage exposure, F(2, 10)= 4.675, p < .05, np =.483.

234 Table 15 Mean (Standard Deviations in parentheses) latencies to complete each trial across treatment conditions across for each habituation and test stage of the intradimensional extradimensional (IED) test: Habituation simple discrimination 1 (HABSD1); Habituation simple discrimination 2 (HABSD2); simple discrimination (SD); compound discrimination (CD); compound discrimination reversal (CD2); intradimensional shift (ID); reversal of intradimensional shift (ID2); extradimensional shift (ED); and reversal of extradimensional shift (ED2).

235 Table 16 Average (Standard Deviations in parentheses) median latencies (seconds) of trials for across each habituation and test stages of the intradimensional extradimensional (IED) test for all treatment conditions: Habituation simple discrimination 1 (HABSD1); Habituation simple discrimination 2 (HABSD2); simple discrimination (SD); compound discrimination (CD); compound discrimination reversal (CD2); intradimensional shift (ID); reversal of intradimensional shift (ID2); extradimensional shift (ED); and reversal of extradimensional shift (ED2).

236

Figure 45: High magnification brightfield photomontage showing the distribution of SERT immunopositive fibers through the prelimbic cortex (PL) for a representative control (A) and MDMA binge high dose (B) subject from experiment 3. Note the reduction of SERT immunopositive fibers in PL in the MDMA binge high dose rat as compared to very dense fiber labeling in Layer 1 and dense innervation of PL throughout in the control subject. Scale bar, 100 µm.

237

Figure 46: Lightfield photomontage showing the distribution of SERT fiber innervation in the dorsal and ventral striatum for a representative subject from the control and MDMA binge high dose condition. A large reduction in SERT immunolabelled fibers was witnessed in MDMA binge high dose rats in the dorsal striatum (CP, caudate putamen) and nucleus accumbens (ACC), structures which receive a dense plexus of SERT fibers in control rats. See list for abbreviations. Scale bar, 500µm.

238 DISCUSSION

Long Evans rats given a high binge dose of MDMA (2 x 7.5 mg/kg i.p.) demonstrated fronto-executive deficits on an odor texture discrimination intradimensional extradimensional (IED) test, which correlated with a large loss of 5-HT innervation of the forebrain as witnessed by immunohistochemical analysis using an antisera for the SERT protein. A low dose of MDMA administered chronically (10 x 1.5 mg/kg) or modeled binge consumption (2 days of 4 x 1.5 mg/kg spaced 2 hours apart) did not affect performance on the IED test and had no effect on SERT expression in the forebrain. The neurotoxic high binge dose of MDMA produced selective impairments in reversal learning, exhibited by a significant increase in perseverative errors during the reversal stages. Additionally, these MDMA binge high dosed rats exhibited initial impairments in attentional set, as witnessed by a significant increase in the number of trials to completion and errors committed during the compound and intradimensional shift discriminations in comparison to control subjects but not drug treatment groups. This neurotoxic dose of

MDMA had no effect, however, on the acquisition of a simple discrimination, or attentional set shifting.

As predicted, there were no differences across treatment conditions in the digging establishment or habituation stages. The MDMA chronic low dose, MDMA binge low dose, and MDMA binge high dose regimen did not impair rats’ ability to retrieve a reward by digging in a ramekin, nor did it impair the ability to acquire the appetitive simple discrimination of odor or digging medium percept. Five out of 45 rats failed the

IED task during the digging establishment habituation phase, demonstrating an attrition

239 of 11% and success rate of 88.89% for completion of the behavioral task. Only one rat failed the digging establishment. One rat from each of the treatment conditions (1 control, 1 MDMA chronic low dose; 1 MDMA binge low dose; 1 MDMA binge high dose) comprised the four rats failed at either the first (odor) or second (texture) simple discrimination during habituation. 75% of these rats failed due to persistent trial omissions, aborting large number of trials, demonstrating a lack of vigilance in performing the task. The failure rate in this study reflects a single session for training and habituation. For example, in Izquierdo et al. (2010) recorded only a 90% success rate at training after giving rats 1-3 daily attempts at training on their odor digging medium paradigm.

There were marginal, but significant impairments in the formation of an attentional set observed in the compound discrimination stage on the IED test (CD1 –

Stage 2). The MDMA binge high dose group performed significantly worse during this stage, committing more errors than both the control groups, however, there were no significant differences between MDMA treated groups during this stage. The increased number of errors, rather than aborted responses indicates a failure to attend to the odor percepts and disregard the irrelevant digging mediums. This is in contrast to previous studies, which used intracerebral localized 5-HT lesions to study similar behavioral flexibility paradigms. These studies found specific deficits in reversal learning, but no deficits in the CD stage (Clark et al., 2004, 2005, 2007; Robbins et al., 2006; Walker et al., 2009a). Impairments of attentional set have been associated with dopamine (DA) lesions in the prefrontal cortex. DA lesions improve ED set shifting (Boulougouris and

Tsaltas, 2009; Crofts et al., Roberts et al., 1994; Walker et al., 2009a, 2009b). Here,

240 MDMA treated rats exhibited no long-term depletions of dopamine as measured via imunohistochemical analysis with an antisera for tyrosine hydroxylase (Experiment 1).

The reason for the set impairment is unknown, however performance at the intradimensional (ID) shift stage improved slightly. No significant differences in trials to completion existed at the ID, however, the MDMA high binge dose group did perform significantly more errors than both control conditions and the MDMA low chronic dose.

It may be possible that widespread serotonergic depletion across the scope of the forebrain may be disrupting dopaminergic functioning, resulting in impairments in attentional set formation. 5-HT efflux and receptor activation is involved in dopamine release. Pharmacology of current psychotropic compounds include a combination of 5-

HT and DA receptor profiles. 5-HT2 receptor activation is involved in the release of dopamine in response to administration of amphetamine analogues (Bortolozzi et al.,

2010; Dupre et al., 2008; Kapur and Reminton, 1996; Meltzer and Huang, 2008; Schmidt et al., 1990, 1994). Dysregulation of dopamine activity via 5-HT axonal degeneration throughout the forebrain may underlie the impairment in attentional set observed in this study.

There was a significant and selective deficit in the reversal learning across each of the three reversal learning stages for the MDMA high binge group in comparison to all other groups. Specifically, the MDMA high binge group needed significantly more trials to completion, and made significantly more errors during the CD, ID, and ED reversals.

Two MDMA binge high dose rats failed to complete the IED task at the level of the ID reversal stage. Each of these two rats could not make six consecutive responses of the

241 correct choice after > 30 trials. The data points, therefore, of the two rats were not included in the data analysis for the last two ED stages.

The specific deficit in reversal learning is consistent with data on rats and primates, which find that 5-HT input into the orbital frontal cortex is involved in behavioral flexibility (Clark et al., 2004, 2005, 2007; Dalley et al., 2004; McAlonan and

Brown, 2003; Robbins, 2005; Robbins et al., 2006; Robbins and Roberts, 2007; Roberts and Parkinson, 2006; Rolls, 2006). The MDMA high binge dose group exhibited decreased SERT fiber expression not only in the OFC, but across the prefrontal cortex, cortical mantle, basal forebrain, hippocampus, and thalamus. This loss of SERT immunoreactive fibers was not witnessed in rats treated with the chronic or binge low doses of MDMA, indicating these low doses produced no detectable neurotoxic or behavioral effects at the timepoint examined.

This is one of the first studies to examine forebrain 5-HT depletion on reversal learning using an odor texture discrimination task in rodents. Previous studies have used localized 5-HT lesions in the prefrontal cortex and pharmacological manipulation of 5-

HT receptor profiles to examine behavioral flexibility and other frontal executive functions (Barnes et al., 1990; Carli et al., 2006; Clark et al., 2004, 2005, 2007; Fone,

2008; McAlonan and Brown, 2003; Robbins et al., 2006; Rodefer et al., 2007). Very few studies have examined the effects of MDMA and other 5-HT specific amphetamine analogues on these tasks of attention and behavioral flexibility in rats. The MDMA high binge group had a significant increase in perseverative errors during each of the reversal stages in the IED task. This demonstrated an inability of extinction in responding to the previously rewarded stimulus, and increased responding to the non-rewarded stimulus.

242 Lapiz-Bluhm et al. (2008) found chronic stress and central serotonin depletion using para-chlorophenylalanine, produced impairments of reversal learning on a similar attentional set-shifting paradigm. This corresponded to a reduction of 5-HT in the orbital frontal cortex. Reversal learning impairments were rescued after SSRI administration in rats increased 5-HT activity (Danet et al., 2010; Lapiz-Bluhm et al., 2008).

Our results are consistent with the recent findings of Kay et al. (2011), who found behavioral inflexibility in SD rats treated with 4 x 10 mg/kg MDMA in a modified reversal learning radial arm maze task. Rats were trained on a 4 arm baited 8 arm radial arm maze. MDMA treated rats significantly increased reference memory errors during the acquisition of this task. During the reversal phase, the unbaited arms were now baited, and rats were trained to disregard previously learned reward locations. Visiting baited arms were now noted as reference memory errors. MDMA treated rats showed an increase in reference memory errors during the reversal phase (Kay et al., 2011).

As Boulougouris and Tsaltas (2009) stated, “behavioural inflexibility may take the form of impulsivity (hastily responding with no regard for consequences) or compulsivity (needless response repetition).” For that reason, significant differences in the total number of trials in reversal learning stages are not automatically assumed to be associated with perseverative responding. Response selection during trials, the number of errors and aborts (omitted responses) were also measured. If an impairment in behavioral flexibility reflected deficits in impulsivity, rats should show no overt pattern in responding for the correct or incorrect choice, but rather select the ramekin based on

“first come, first serve” basis. Therefore, in this example of behavioral dysfunction, no significant differences in either errors or omitted responses should be present even though

243 significantly more trials were needed to complete the stage in impaired treatment conditions. If the behavioral inflexibility is associated with compulsive, perseverative responding, a significant increase in errors (selection of the previously rewarded irrelevant choice) would be detected in subjects. Lastly, if animals showed a significant amount of aborted (omitted) responses in a reversal learning stage, the deficits may be associated with loss of volition due to incorrect responding and/or attentional fatigue.

This would result in an increase in aborted trials rather than errors. Most rodent studies using the odor texture IED task have not isolated each of these distinct trial completions

(Birrell and Brown, 200; Egerton et al., 2005; Featherstone et al., 2008; Liston et al.,

2006; McAlonan and Brown, 2003).

The MDMA high binge group also displayed a significant reduction in the number of errors committed during the reversal stages from the compound discrimination reversal to the ED reversal, though these errors were still significantly greater in comparison to the other treatment conditions. Similar improvements in reversal learning across trials has also been seen after methamphetamine pretreatment using a similar IED protocol (Izquierdo et al., 2010). Here, the improvement in reversal learning may be contributed to an across stage attenuation of the MDMA associated reversal learning deficits. These findings are in contrast to localized lesions of the OFC on reversal learning, which find an increase in the number of errors in the ED reversal as compared to the CD and ID reversal stages (McAlonan and Brown, 2003). This difference in reversal learning stages was not witnessed in the control or low dose MDMA conditions.

Two rats failed the ID reversal stage, and therefore were not included in the ED reversal stage data. It may be possible, if they were able to complete the ID reversal, a larger

244 number of errors and trials to completion would be reflected in the data of the MDMA binge high dose rats, and a trend in improvement would not be witnessed.

There were no deficits in the ED attentional set shifting between any of the

MDMA treatment conditions and control conditions. This was predicted as attentional set shifting has been associated with homeostatic functioning of the medial prefrontal cortex and relies on proper catecholaminergic functioning. No long lasting impact was present on DA and NE levels following MDMA treatment. Previous studies have found depletions of 5-HT in the mPFC and OFC have little impact on attentional set shifting

(Boulougouris and Tsaltas, 2009; Clark et al., 2004, 2005, 2007; Robbins et al., 2006).

Attentional set shifting has been found to be sensitive to the effects of both stress and d- amphetamine (Fletcher et al., 2005, 2008; Liston et al., 2006).

Reversal learning deficits have also been witnessed in Ecstasy human users. The

IED and Wisconsin Card Sorting task, human analogues of the behavioral paradigm used in this study, have both yielded significant impairments for Ecstasy users on the reversal stages, with no impairments in attentional set formation or set shifting (Fox et al., 2002,

Thomasius et al., 2003). The specific impairment in behavioral flexibility is seen in the current study and in Ecstasy users, ingesting variable amounts of MDMA and other psychoactive substances. This relationship may in fact indicate serotonergic hypofunctioning in recreational Ecstasy users, however no causal associations may be made, and the comorbidity of polydrug use and psychiatric disease cannot be ruled out.

In addition, most Ecstasy users included in these studies have also shown impairments in both impulsivity and decision-making (Hanson et al., 2008; Hoshi et al., 2007; Morgan et al., 2005; Quednow et al., 2006, 2007; Roiser et al. 2007).

245

Figure 47: Brightfield photomontage showing the distribution of SERT fiber innervation through the caudal ventral orbital (VLO) cortex for a representative MDMA binge high dose rat from experiment 2 (A) and experiment 3 (B). Note the similar pattern and intensity of SERT fiber labeling across the two subjects. See list for abbreviations. Scale bar, 250 µm.

246

Figure 48: Brightfield photomontage showing the distribution of SERT fiber innervation through the ventral hippocampus for a representative subject from the MDMA binge high dose group in Experiment 2 (A) and Experiment 3 (B). Note the similarity in the pattern and intensity of fiber labeling throughout the CA1 field of Ammon’s horn (CA1) in the two subjects. Scale bar, 250 µm.

247 While this study does not have measures that can directly infer impulsivity, there was no consistent decreased latency per choice in any of the MDMA treatment conditions, indicating no trend for impulsive choice in the IED task. This is congruent with other animal studies which found no long-term impact on impulsivity following

MDMA administration in rats (Dalley et al., 2007; Saadat et al., 2006). The incongruence between human and animal data is unknown, but impulsivity present in Ecstasy users may result from an accumulation of premorbid factors. A large variance in latencies may also be associated with the demands of the IED task, which allows rats to self-pace trials.

While there are presently no other studies, which have examined the impact of

MDMA pretreatment on performance using an odor texture discrimination task, reports have explored the effects of other amphetamines. Most recently, Izquierdo et al. (2010) found reversal learning impairments using a variation of the odor texture discrimination task and a delayed lever press visual discrimination task after administration of a binge regimen of methamphetamine (METH). This behavioral impairment correlated with a loss of dopamine expression in the striatum. This study did not assess the effect of

METH on 5-HT content in the forebrain. METH produces axonal degeneration of monoamines indiscriminately (Axt and Molliver, 1991; Brown and Molliver, 2000;

Brunswick et al., 1992; Guilarte et al., 2003). Rats exposed to amphetamine displayed impairments in both attentional set shifting, and also performed more errors during the reversal trials. The deficit in attentional set shifting was attenuated when D1 agonists were infused into the PFC (Fletcher et al., 2005).

The present study failed to find any physiological, behavioral, and neurotoxic effects of pretreatment of low chronic or binge doses of MDMA. The MDMA low binge

248 dose ( 2 days of 4 x 1.5 mg/kg), was used to model “binge” recreational use of Ecstasy over an occasion (Baumann et al., 2007). These rats demonstrated no lasting effects on frontal executive functioning and no neurotoxic 5-HT depletion of the forebrain.

Additionally, a chronic low dose of MDMA (10 x 1.5 mg/kg) served to see if a low dose over a brief period of time would impact behavior. Currently, MDMA is being explored for therapeutic uses in PTSD, and little is known about lasting effects of low doses on clinical populations. MDMA chronic low dose administration also resulted in null behavioral and immunohistochemical findings. These results exhibit the effect of pretreatment of MDMA on behavior; studies in human Ecstasy users do not reflect these findings. It is likely that several confounds, including premorbid exposure to psychiatric disease, comorbid polydrug use, genetic polymorphisms, and the length and amount of drug ingested impact individual results. Chronic exposure to drug use and compulsive drug seeking behavior also result in genetic, morphological, and cognitive impairments over time, which may be attributed to Ecstasy related neuropsychological detrimental effects (Rogers and Robbins, 2003).

No other attention or motivational differences were found for any of the MDMA treatment conditions in the present study. The odor texture discrimination task tests the acquisition of a simple exemplar discrimination, formation of an attentional set, attentional set shifting, and behavioral flexibility. In addition, the present study added an additional measure: aborted trials tested attentional vigilance. Aborted trials were trials in which a response was omitted, similar to omitted responses in the 5-CSRTT. Two studies have demonstrated a long-term impairment in attentional vigilance after administration of amphetamine analogues. Rats with access to self administration of

249 MDMA showed an initial impairment in impulsive choice in a 5-CSRTT, however, after a prolonged period of cessation, these rats committed significantly more omitted responses, demonstrating an impairment in selective attention (Dalley et al., 2007). Our laboratory previously reported impairments in the CD and reversal stages in rats treated with a high dose of PCA using a similar odor digging medium paradigm (Hughes et al.,

2007). These deficits were a result of an increase in both errors and also an increase in aborted (omitted) trials in rats.

A few caveats to this study may have impacted the results. First, the perceptual domains of stimulus were presented in a consistent order for all rats, rather than randomly. The first five stages of the IED task asked rats to attend to the odor percepts, while the last two stages required the rats to respond to the digging mediums. A bias towards of selective attention of the perceptual domains of odors or mediums could have affected the performance on the initial stages of the task or during the extradimensional task. Second, previous studies using pseudorandomized presentation of percepts, and those IED which have used randomized orders with three perceptual domains (odor, texture of ramekins, and digging mediums) found rats needed a greater number of trials to complete the extradimensional attentional shift than the intradimensional attentional shift for controls (Birrell and Brown, 2000; McAlonan and Brown, 2003). In the current study, there was no large within subjects differences between the ID and the ED. The reason for these discrepancies is unknown, but could be related the lack of randomizing percept presentation, indicating rats’ natural bias to attend to digging mediums.

In summary, administration of a MDMA binge high dose in LE rats produced selective impairments in behavioral flexibility. A substantial loss of 5-HT input into the

250 forebrain was observed in this group. This loss of 5-HT immunoreactive fibers was not seen in MDMA chronic low dose or MDMA binge low dose rats. The lack of neurotoxicity in the low dose treated rats was also mirrored by an absence of behavioral deficits in attention or reversal learning using an odor texture discrimination task. These results suggest that moderate doses of MDMA may not be neurotoxic to serotonergic fibers, and therefore behavioral deficits associated with MDMA use may not persist after cessation of use. However, large neurotoxic doses of MDMA may impair fronto- executive functioning associated with homeostatic 5-HT levels.

251

GENERAL DISCUSSION

The preceding experiments were conducted to test in rats the behavioral and neurotoxic effects of doses of MDMA in rats that are more reflective of human use. The results demonstrate that low doses of MDMA, administered in a chronic or binge regimen, have no long-term effect on serotonergic projections. Likewise these doses did not produce any behavioral deficits in hippocampal dependent place learning using a water maze protocol, or the intradimensional extradimesional (IED) task that examined executive processes.

The neurotoxic and behavioral effects of a high binge dose of MDMA were also examined. MDMA produced a significant denervation of serotonergic input to the forebrain as measured by a reduction in SERT immunoreactivity. The greatest loss of

SERT fiber innervation was observed in the prefrontal cortex, piriform cortex, remaining neocortex, hippocampus, dorsal striatum, ventral striatum, ventral pallidum, lateral septum, anterior, midline, and lateral thalamic nuclei, and lateral geniculate nucleus.

Despite previous studies, which noted a specific denervation of fine 5-HT axons originating from the dorsal raphe nucleus, MDMA binge high dose rats displayed a pattern of 5-HT fiber loss which included structures primarily innervated by the median

252 raphe, including the hippocampus, entorhinal cortex, caudal hypothalamus. Likewise,

SERT fiber innervation remained relatively unchanged in the globus pallidus, substantial innominata, and some preoptic nuclei, known to receive strong inputs from DR.

There are conflicting reports as to whether large doses of MDMA, which do produce substantial serotonergic forebrain axonal deneveration, impair place learning in the rat. Previous studies using similar and larger cumulative doses in Sprague Dawley rats have found impairments in MDMA pretreated rats in retention of platform location during the probe trial, with no impairments in acquisition (Sprague et al., 2003; Able et al., 2006). More recent studies, however, have found this effect to be ameliorated, however this could be due to pretraining because the MWM was administered within a battery of tests (Skelton et al., 2008). Encoding and retrieval impairments were noted in

LE rats following multiple doses of 15 mg/kg (Camarasa et al., 2008). In periadolescent rats, low doses of MDMA administered immediately before training sessions increased escape latencies and a high dose of the drug produced impairment in retention during the probe trial (Arias-Cavieres et al., 2010; Cunningham et al., 2009).

Here, 2 x 7.5 g/kg MDMA administered two weeks prior to training had no impact on the acquisition or retention of location of a hidden submerged platform, despite significant loss of serotonergic axons throughout the forebrain including the hippocampal formation. Likewise, there were no behavioral deficits found in the MDMA chronic low dose, MDMA binge low dose, and MDMA binge high dose groups in a spatial reversal and serial reversal drug challenge. These results confirm the findings of previous research that found neurotoxic doses of MDMA did not produce impairments in spatial reversal learning in the MWM, despite the large loss of serotonergic innervation to the

253 orbital medial prefrontal cortex (Able et al., 2006; Skleton et al., 2008). A high binge dose of MDMA does impact spatial reversal learning in a radial arm maze task (Kay et al., 2011).

Low doses of MDMA administered during daily MWM training sessions negatively impeded both encoding, with increased latencies to platform location over trials, and in retention, measured by decreased percentage of time spent in target quadrant during a probe trial (Arias-Cavieres et al., 2010). Experiment 2 was the first study to test the effects of a low dose of MDMA (2.0 mg/kg) on retention of place learning in MDMA pretreated rats during the serial reversal phase, without administering the drug during the training sessions. There was no immediate impact of a low dose of MDMA in rats previously exposed to low or high doses of MDMA in the ability to recall the platform location during the probe trial.

Differences in rat strain could account for incongruence between current and previous studies. Some studies that reported selective deficits in platform retention used

SD rats. This strain has been noted to be especially sensitive to the neurotoxic effects of the drug as compared to other strains (Chu et al., 1996; Colado et al., 1995; Malpass et al., 1999). As discussed in Experiment 1, there is an absence of data contrasting the pharmacokinetics and neurotoxicity of MDMA directly across LE and SD rats. However, the pharmacological profile of LE rats may be similar to other strains that show less sensitivity to MDMA. This can be argued by the large serotonergic forebrain denervation produced by only two injections of 7.5 mg/kg. However, the fiber loss was not as extensive as that reported in studies during the 1980’s and 1990’s, which could account for the differences in the amount of remaining serotonergic fibers present following a

254 high binge dose of the drug (Battaglia et al., 1988; Mamounas and Molliver, 1988;

Molliver et al., 1991; O’Hearn et al., 1989).

The differences in the dosing regimens might also account for differences in behavior during the MWM test. For example, retention deficits were found after administration of 4 daily injections of 2 x 15 mg/kg s.c. in LE rats (Camarasa et al.,

2008). The high binge regimen in this experiment was selected to reduce the mortality attrition rate while producing comparable results in 5-HT fiber degeneration. The current findings are comparable to other studies that found 5-HT depletion with other known neurotoxins had little impact on spatial navigation and place learning. Both central 5-HT depletion and 5-HT receptor antagonism does not produce deficits in the MWM and other place learning paradigms, and SSRIs have even been shown to impair MWM encoding

(Adams et al., 2008; Dringenberg and Zalan, 1999; Hritchu et al., 2007; Majilessi et al.,

2002, 2003). While behavioral effects of place learning may not directly be associated with serotonergic functioning, extremely high doses of MDMA may have affected other cellular or neurochemical processes, thereby impacting mnemonic functioning.

By contrast, large doses of MDMA impact executive processes measured in the

IED test. Previous studies have not examined the effects of MDMA on attention and behavioral flexibility using an odor texture discrimination task in rodents. This test was developed by Verity Brown as an analogue to the Wisconsin Card Sorting task and

CANTAB’s language independent IED task for both nonhuman and human primates

(Birrell and Brown, 2000; Brown and Bowman, 2002). The IED task was selected for two reasons: 1.) so that direct executive measures could be compared translationally with human data on Ecstasy users, and 2.) to test MDMA pretreated rats on a frontal measure

255 known to be mediated by proper serotonergic functioning. Similar to the MWM task, the

MDMA chronic low dose and MDMA binge low dose group demonstrated no behavioral deficits across the IED test. This corresponded with no reductions in SERT immunoreactivity in subjects.

MDMA binge high dose rats expressed specific impairments in all three of the reversal learning stages. Moreover, this impairment was attributed to a significant increase in perseverative errors. This deficit corresponded to a large reduction in serotonergic fiber innervation in several fronto-striatal networks, including the orbital and medial prefrontal cortices, and dorsal and ventral striatum. These findings correspond with a large body of literature which found both depletions of 5-HT after 5,7-DHT was intrercerebroventricularly or directly infused into the orbital frontal cortices in both rats and primates produced behavioral inflexibility in versions of the IED task (Clarke et al.,

2005, 2006, 2007; Dalley et al., 2004; Robbins, 2005; Robbins et al., 2006; Robbins and

Roberts, 2007; Roberts and Parkinson, 2006; Rolls, 2006). Likewise, systemic 5-HT depletion using the related compound PCA in SD rats also produced behavioral inflexibility (Hughes et al., 2007).

Izquierdo et al. (2010) recently found that the amphetamine analogue, methamphetamine (METH) also selectively impaired LE rats on a similar version of an odor texture IED task. This was correlated with a reduction in the expression of the dopamine transporter protein in the dorsal striatum. While they did not examine the direct effects on serotonin, METH is a known neurotoxin to dopamine, norepinephrine, and serotonergic terminals (Gibb et al., 1994; Seiden and Ricaurte, 1987). The behavioral effects could be associated with 5-HT impairments or an additive

256 monoaminergic hypofunctioning. Polydrug Ecstasy users have also demonstrated impairments in executive functioning, including selective reversal learning deficits using

IED analogue paradigms (Fox et al., 2002; Montogomery and Fisk, 2008; Quednow et al., 2005, 2007; Thomasius et al., 2003). However, whether these deficits can be contributed premorbidly to psychiatric and neurological conditions, or whether these may be the results of other psychoactive substrates or an additive effect of MDMA and other drugs is unknown.

The MDMA binge high dose group also displayed an initial small deficit in attentional set during the compound (CD) and intradimensional (ID) stages in the IED task. This group had significant increases in errors in comparison to control rats, but did not differ significantly in comparison to the experimental treatment groups. Attentional set refers to the ability to direct one’s attention towards a reward or goal associated stimulus and disregard irrelevant stimuli. In the IED paradigm, attentional set is measured across the CD to the ID stage. Formation of a correct attentional set will be demonstrated if rats perform better on the subsequent ID stage following CD presentation

(Roberts et al., 1994). Though the MDMA binge high dose group performed worse in comparison to controls across both stages, this group showed improvement on the ID stage as compared to the CD stage, indicating an attentional set establishment. This laboratory has previously found an initial impairment in attentional set formation in the

CD stage following 2 x 10 mg/kg PCA in SD rats, however the deficit was associated with a decrease in vigilance as measured by an increase in the number of aborted trials, rather than an increase in errors (Hughes et al., 2007). Previous studies have found dysregulation of dopamine results in impairments in attentional set using IED paradigms

257 (Boulougouris and Tsaltas, 2009; Crofts et al., 2001; Robbins, 2007; Roberts et al.,

1994). The difficulty in completion of the CD and ID stage could be associated with a serotonergic hypofunction produced by axonal denervation of the frontal cortex. Rather, the poor performance of this attentional measure may be associated with the 5-HT modulation of dopaminergic activity.

Despite a substantial loss of SERT labeled fibers across the piriform cortex, olfactory tubercule, somatosensory cortex, dorsal lateral geniculate nucleus, and visual cortex, there was no notable effect of a binge high dose of MDMA on sensory function.

This is evident by no impairments in performance in the MDMA binge high dose group in any measure of the water maze, indicating similar visual acuity as compared to the other treatment conditions. In addition, unaltered performance on the simple discriminations in both the habituation and IED test, indicating these subjects were able to differentiate both odor stimuli and tactile digging mediums, incating preserved sensory perception.

The main findings of these experiments involve the absence of changes in the behavioral and histological profile of the MDMA chronic low dose and MDMA binge low dose groups. A plethora of studies to date have examined the behavioral and neurotoxic effects of MDMA using preclinical rodent models and in recreational polydrug Ecstasy users. The results of these experiments have produced conflicting results. In humans, both cognitive and structural (via neuroimaging) impairments have been elucidated in polydrug Ecstasy users, however these two factors are not always positively correlated in studies (Gouzoulis-Mayfrank and Daumann, 2006; Reneman et al., 2001, 2002; Thomasius et al., 2003, 2006).

258 Current studies using large doses on rats use allometric scaling to determine a dose given to a rat akin to human intake. This method was developed to consider metabolic rate, size, and circulation of blood, and therefore drugs to sites of action. The

α algorithm for allometry is based on the formula: Metabolic rate = B0M , where by B0 is a normalization constant, M is body mass, and α is the Kleiber’s law coefficient of .75

(Savage et a., 2008). Smaller animals, with less surface area, higher metabolic rates, and increase in circulation should increase dosage relative to larger animals (Boxenbaum,

1982; Sharma and McNeill, 2009). Support for this dosing scale was confirmed by a series of studies at the same time of the earlier neurotoxicity reports, on monkeys, which found doses of 5-10 mg/kg MDMA injections in primates produced similar patterns of denervation (Kleven et al., 1989; Ricaurte et al., 1988, 1989). These dose regimens do not affect these primate’s neurocognitive abilities, as they performed similar to controls on a battery of tests, which examined both long-term memory and prefrontal functioning, despite a reduction in 5-HTIAA (Taffe et al., 2002, 2003).

Allometric scaling has been challenged by other methods of dosing which factor pharmacokinetics of the drug into the equation, as the administration, absorption, binding, metabolism, and excretion of the drug play an instrumental role in the effective dose of a drug. A number of formulas have been derived to correct for each factor as a means of providing more clear representation for translating doses across species

(Sharma and McNeill, 2009). Effects scaling uses discriminative biobehavioral and physiological measures to outline appropriate doses across species. Studies have found that a dose of 1.5 mg/kg in rats and humans produces parallel changes in respiration, increases in heart rate, plasma monoamine concentrations, and in neurohormonal levels

259 (Baumann et al., 2007). This indirect route of considering the changes in the processing and clearance of the drug may provide a more accurate translational dose across organisms.

Despite the difficulties in determining a dose, which translates recreational consumption of MDMA from humans to rodents, the current study also examined the effects of a low chronic dose of MDMA. This (sub)chronic dose (10x) was administered over a period of two weeks, with injections separated by 24 hours. Due to its rampant and escalating use, MDMA was classified as a Schedule 1 narcotic in 1985. Only recently, has its proposed therapeutic use been studied in a number of FDA administered clinical reports. MDMA’s most promising therapeutic avenue is in the treatment of posttraumatic stress disorder, a severe anxiety disorder, whose onset is triggered by a traumatic event. Currently, the most common pharmacological treatment for PTSD includes SSRIs and benzodiazepines. In addition, several studies are now exploring the effectiveness of short-term medications administered immediately after the trauma to offset development of PTSD entirely (Shalev, 2009).

Medications are relatively ineffective unless coupled with behavioral therapy. In the treatment of PTSD, the emotional anxiety and fear response needs to become disconnected with the memory of the trauma. In a controlled setting, psychologists work with patients to overcome this association (McAllister and Stein, 2010). The process is known as extinction of the memory, and has been modeled in preclinical studies using fear-conditioning paradigms. While the encoding, and retrieval of fear memories involves the amygdala in rodents, the medial prefrontal cortex is involved in fear extinction, and lesions of the mPFC will produce impairments in extinction (Chang et al.,

260 2009; Shin and Liberzon, 2010). That is, the fear response will not diminish days after the cessation of paired shock in the conditioned environment in rats with ventral mPFC lesions. Likewise, studies of veteran populations have elucidated that individuals with traumatic brain injuries which involved damage to the prefrontal and amygdalar areas are less likely to develop PTSD than those without, illustrating a human correlate (Koenigs and Grafman, 2009).

There are several clinical studies which now target medications which can impact extinction of fear memories in PTSD. MDMA was first postulated as a possible treatment nearly twenty years ago. Grob et al. (1992) cited the use of the drug concomitantly with psychotherapy in the 1980’s to outline a possible role in treatment of psychiatric disorders. From them, several researchers have reviewed the pros and cons of its possible use. MDMA’s subjective and behavioral effects include openness, relaxation, euphoria, and empathy, with a “reduction in fear and defenselessness” (Doblin, 2002).

Studies use MDMA in a therapy session, under the premise that a reduction in fear and anxiety will lessen the fear response and reaction during recall and reactivation of the trauma. Reactivation of this memory under a more relaxed state could lead to degradation of the memory trace and thereby facilitate extinction, or extinction of the fear response (Cukor et al., 2009; Parrott, 2007; Sessa, 2005, 2007).

Controlled studies of low doses of MDMA on human participants displayed an absence of negative physiological or behavioral effects (Johansen and Krebs, 2009;

Parrott, 2007). Likewise, there are now positive reported findings on clinical studies of

MDMA administered during behavioral cognitive therapy in PTSD patients. Bouso et al.

(2008) recently tested a single dose of 1.5-1.75 mg/kg (based on weight) MDMA in

261 women in behavioral cognitive therapy suffering from PTSD. MDMA was congruently administered with an experimental therapy session. A significant reduction in PTSD symptomology was found, along with no detrimental somatic or psychological effects.

However, a limitation to this study was the absence of a placebo group or a blind or double blind study, as placebo treatment alone has also decreased negative PTSD symptoms (Parrott, 2007).

Literature on preclinical effects of the drug on fear memories is nearly absent in rats. A study using d-amphetamine found no effects on the facilitation or inhibition of extinction of fear conditioning in mice. Leon et al. (2009) examined the effects of

MDMA on avoidance learning in rats treated with mild chronic stress, and found the enhancement of encoding and retrieval, which they deduced could be loosely interpreted as enhanced learning and more effective strategies in handling stress. However, this study injected MDMA intracerebrally, which eliminated the possibly of its toxicity at any dose. It is imperative that further studies be conducted on the impact of the extinction of fear conditioning in rats, and the possibility of the pharmacological rescue of MDMA in response to neuronal damage which may impair the process. While the subjective effects of MDMA point to a possible therapy, there is no empirical evidence that indicates

MDMA leads to extinction of the behavioral fear response.

Spatial reversal learning, as tested in the reversal phase of the MWM is an example of extinction, rather than behavioral flexibility. It is also reliant on proper functioning of the medial prefrontal cortex (de Bruin et al., 2004; LaCroix et al., 2002;

Salazar et al., 2004; Wolf et al., 1987). In the MWM, rats must disregard the previously learned platform location, and navigate to the new location. Perseveration for the old

262 platform location during training or probe sessions indicates a failure to dismiss the previously learned location. Previous studies concur with our finding that low and high doses of MDMA do not negatively impede spatial reversal learning in the MWM.

However, there was no augmentation of this behavior, leaving no empirical data supporting a role of MDMA in facilitation of extinction.

In addition to PTSD, clinical trials are being conducted to assess MDMA’s effectiveness in the reduction of anxiety and pain in terminal cancer patients and has been proposed as an adjunct therapy in other anxiety disorders and depression (Johansen and

Krebs, 2009; Parrott, 2007). Whether these or future studies will determine that it is efficacious in the treatment of psychiatric disorders, the purported neurotoxicity and proper interspecies dosing scales need to be addressed in preclinical studies to parse out the multiple confounds associated with data gathered from recreational drug users.

There are several limitations to the experiments, some of which have already been addressed above. One of the most important factors to consider is that these experiments only sought to correlate serotonergic denervation as a marker of neurotoxicity. The subjects in these studies may show a number of other negative structural, molecular, cellular, or electrophysiological changes following low or high doses of MDMA. For example, MDMA is known to impair long-term potentiation (LTP), and has a negative impact on a number of neurogenic factors including reductions in BDNF and in hippocampal neurogenesis (Adori et al., 2010; Arias-Cavieres et al., 2010; Catlow et al.,

2010; Hernandez-Rabaza et al., 2006; Martinez-Turillas et al., 2006; Moyano et al., 2004,

2005). Disruptions of each of these has been associated with hippocampal and mnemonic

263 dysfunctioning and behavioral differences seen in the experiments by comparison to previous reports could be related with further augmentation of other neural substrates.

While effects scaling may provide a better example of interspecies dosing than using allometry, there are pharmacokinetic differences in the metabolism of the drug across rodents and humans. Metabolism of MDMA undergoes auto inhibition, whereby an hour after consumption, self-inhibition of metabolizing the drug occurs, and plasma levels rise. No such auto-inhibition has been documented in rats (Green et al., 2009).

This is important as while an initial dose of 1.5-2.0 mg/kg may be equivalent in rats and humans, recreational users who ‘stack’ Ecstasy pills, or partake in a binge consumption, will show increases in the drug’s plasma concentration much larger than rats. Most studies using effect scaling have not taken this into consideration. Shorter time intervals between injections in rodents may be a method of resolving the differences in metabolism of the drug.

The lack of effect for low doses of MDMA should not be interpreted that recreational use of the drug is not neurotoxic. Both preexisting neurological conditions and genetic polymorphisms influence the impact of Ecstasy on drug users. In addition,

Ecstasy pills contain a number of different substances, including cocaine, methamphetamine, caffeine, and ephedrine (Sang Leung and Cottler, 2008; Vogels et al.,

2009). Any given dose of Ecstasy then could contain a number of agents producing compounding effects. Finally, and most discussed, Ecstasy users are polydrug users.

Not only has an additive effect of detriment been recorded by congruent use of Ecstasy with other psychoactive substances, but also a number of these illicit substances have been known to produce behavioral deficits on their own. These factors contribute to the

264 escalating epidemic of social, psychological, and cognitive decline associated with abusers of designer amphetamines.

265

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