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Master's Theses Graduate College

12-2007

Neuropsychopharmacological Investigations of MDMA/ Combinations

John J. Panos

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Recommended Citation Panos, John J., "Neuropsychopharmacological Investigations of MDMA/Cocaine Combinations" (2007). Master's Theses. 4661. https://scholarworks.wmich.edu/masters_theses/4661

This Masters Thesis-Open Access is brought to you for free and open access by the Graduate College at ScholarWorks at WMU. It has been accepted for inclusion in Master's Theses by an authorized administrator of ScholarWorks at WMU. For more information, please contact [email protected]. NEUROPSYCHOPHARMACOLOGICAL INVESTIGATIONS OF MOMA/COCAINE COMBINATIONS

by

John J. Panos

A Thesis Submitted to the Faculty of The Graduate College in partial fulfillmentof the requirements for the Degree of Master of Arts Department of Psychology

WesternMichigan University Kalamazoo, Michigan December 2007 Copyright by John J. Panos 2007 ACKNOWLEDGEMENTS

I am forever indebted to my faculty advisor Dr. Lisa Baker forher patience, understanding, guidance, and overall support. The opportunity that Dr. Baker has granted me by accepting me into the graduate program in Psychology and her laboratory has provided the freedom to explore multiple of scientific investigation forwhich I am grateful. I would like to also thank my other committee members Dr. Alan Poling and

Dr. Brad Huitema who have aided in advancing my training as a researcher.

I would also like to thank the members of Dr. Baker's Behavioral

Neuropharmacology Laboratory for their support and assistance with this project.

John J. Panos

11 NEUROPSYCHOPHARMACOLOGICAL INVESTIGATIONS OF MDMA/COCAINE COMBINATIONS

John J. Panos, M.A.

WesternMichigan Univeristy, 2007

Despite the popularity of polydrug abuse among recreational MDMA users, relatively fewcontrolled experimental studies have documented the neurobehavioral effectsof

MDMA in combination with other abused substances. In this study, the neurochemical and behavioral effectsof MDMA/cocaine combinations were assessed using in vivo microdialysis with simultaneous measurement of locomotor activity (LMA). Rats were administered cocaine (10 mg/kg or 20 mg/kg, IP), MDMA (1.5 mg/kg or 3.0 mg/kg, IP) or combinations of cocaine and MDMA during microdialysis experiments. Microdialysis samples were collected every 30 min for three hours prior to injections. Following drug administration, six additional 30 min samples were collected over a three-hour period. Samples were analyzed by HPLC with electrochemical detection. Locomotor activity was monitored in microdialysis chambers equipped with infrared emitters and detectors. The results of this study suggest the possibility that MDMA and cocaine produce additive or synergistic neurochemical and behavioral effects. This preclinical study may have important clinical implications and may help explain the common practice of polydrug use among MDMA users. TABLE OF CONTENTS

ACKNOWLEDGEMENTS...... ii

LIST OF FIGURES...... V

INTRODUCTION ...... :...... 1

Background Statement...... 1

Polydrug Use...... 1

MDMA...... 3

Cocaine ...... 4

Neuropharmacology of Psychostimulants...... 5

Rationale forCurrent Study...... 7

METHOD ...... 8

Animals ...... 8

Surgical Procedures...... 8

Behavioral Procedures...... 9

Microdialysis Procedures...... 9

Histology ...... 10

HPLC-EC...... 10

Drugs ...... 11

Statistical Analysis ...... 11

RESULTS ...... 12

DISCUSSION...... 15

lll Table of Contents - Continued

CONCLUSION ...... 18

REFERENCES ...... 19

APPENDIX ...... 23

A. Institutional Animal Care and Use Committee Approval Form ...... 24

lV LIST OF FIGURES

1. Locomotor Activity ...... 12

2. Regression Lines Log of Locomotor Activity ...... 13

3. Levels ...... 14

4. Regression Lines Log of Nucleus Accumbens Dopamine Levels ...... 15

V INTRODUCTION

Background Statement

Despite increased public awareness regarding the health risks of , and abuse continues to be a major problem, especially among adolescents and young adults. The increased accessibility of "club " to young adults is a growing problem in our society. In club settings, known as "", the use of multiple drugs in combination is very common. In particular, MDMA ("Ecstasy") is commonly used in combination with other drugs, such as cocaine. Unfortunately, relatively little experimental research exists on the neurobehavioralconsequences of these drugcombinations. A review of the electronic literature databases revealed a significantgap in the literature with respect to animal models ofpolydrug abuse that correspond to human drug abuse patterns. A recent article by (Cowan et al., 2003) that examined MDMA use and polydrug use in humans stressed a need foranimal studies on the effectsof MDMA and combined polydrug administration. Therefore, the aim of the proposed study is to examine the neurobehavioral effectsofMDMA in combination with cocaine, and to determine the extent to which these effectsdiffer from those of either drug alone. This study may lead to a greater understanding ofthe neurobehavioral consequences ofpolydrug abuse.

Polydrug Use

Polydrug use has become a rampant problem in modem society (Barret,

Darredeau, & Pihl, 2006; Gouzoulis-Mayfrank & Daumann, 2006; Montgomery, Fisk,

Newcombe, & Murphy, 2005; Wish, Fitzelle, O'Grady, Hsu, & Arria, 2006). Ecstasy or

3, 4-methylenedoxyamphetamne (MDMA) has the potential of being a "gateway" drug initiating further exploration with illicit substances. Wish et al (2006) conducted a self­ report based study on college students; their results infer that MDMA users are more

1 likely to have used cocaine, , LSD, other , and . In relation to psychostimulant use, MDMA preceded cocaine use in 46% of the individuals surveyed

(Wish, Fitzelle, O'Grady, Hsu, & Arria, 2006).

The medical complications ofMDMA and cocaine use are documented and include cerebral oedema and hepatic failure (Kramer et al., 2003). Polydrug use of psychostimulants such as cocaine and MDMA may present several other possible complications forthe user which include high abuse potential, neurotoxic effectson the and serotinegic systems, and the possibility of experiencing an -type . The hypothesized neurotoxic damage may impact on cognitive and motor system function.There exists a possibility that this neurotoxic effect could result aftera single exposure to a high dose ofMDMA or even to a lower dose if

taken in combination with another psychostimulant drug. It is hypothesized that these

negative effects may result from excessive release elicited by an additive or synergistic effectof simultaneous polysubstance use (SPU).

A review of the current literature has found relatively fewstudies that examine

SPU (N = 19). Several researchers have called forfurther investigations of polysubstance use including human (Barret, Darredeau, & Pihl, 2006; Gouzoulis-Mayfrank &

Daumann, 2006; Wish, Fitzelle, O'Grady, Hsu, & Arria, 2006) and animal studies

(Gouzoulis-Mayfrank & Daumann, 2006).

Polydrug use among MDMA users is common, especially MDMA use in combination with psychostimulant drugs, such as amphetamine, , or cocaine (Khorana, Pullagurla, Young, & Glennon, 2004; Riley, James, Gregory, Dingle,

& Cadger, 2001; Williams, Dratcu, Taylor, Roberts, & Oyefeso, 1998; Winstock,

2 Griffiths,& Stewart, 2001). Presumably, users take other psychostimulants in combination with MDMA to prolong or enhance its euphoric effects. Scholey et al.,

(2004) reported significantly greater use among experienced MDMA users compared to nonusers based on an internetsurvey. Specifically, survey data revealed that cocaine use was greatest among heavy Ecstasy users (81% ) compared to novice (44%) and moderate (61%) MDMA users. Lua, Lin, Tseng, Hu, and Yeh (2003) investigated polydrug abuse with MDMA in Taiwan. These investigators analyzed urine samples frompolice detainees and foundevidence fora high rate ofMDMA use in combination with other illicit drugs. Despite the apparent prevalence ofpolydrug abuse among MDMA users, only a fewcontrolled experimental studies have documented the neurobehavioral effects ofMDMA in combination with other drugs. MDMA

3,4-Methylenedioxymethamphetamine (MDMA) is a ring substituted phenylisopropylamine, and a to amphetamine. Also known as "Ecstasy" on the street, MDMA is commonly used as a recreational drug among young adults.

Originally documented in Merck's laboratory records, MDMA was referredto as

"Methylsafrylamin" (Bemschneider-Reif, Oxler, & Freudenmann, 2006; Freudenmann,

Oxler, & Bemschneider-Reif, 2006), patented by Merck in 1912 under its chemical structure as an intermediate product in the development ofa vasoconstrictive drug

(Bemschneider-Reif, Oxler, & Freudenmann, 2006; Freudenmann, Oxler, &

Bemschneider-Reif, 2006; Pentney, 2001). During the 1970's, MDMA was used as an adjunct to psychotherapy (Greer & Tolbert, 1986; Meehan, Gordon, & Schechter, 1995), reportedly to enhance communication and "self-examination". A psychotherapy study

3 reported that subjective effectsof MDMA include altered states ofconsciousness with emotional components such as empathy, acceptance, and insight (Kemmerling, Haller, &

Hinterhuber, 1996). In the early 1980's, MDMA became a popular recreational drug and was sold legally, typically through mail order (Eisner, 1989; Ray & Ksir, 1999). Citing nationwide abuse and the potential health problems ofMDMA, the Drug Enforcement

Agency (DEA) established MDMA a Schedule I controlled substance in 1988 (Lawn,

1988). Despite increased public awareness ofthe health risks associated with MDMA, its use has continued to rise in recent years, particularly among young people.

MDMA users have consistently reported the subjective effectsof this substance to include elevated , feelingsof closeness and intimacy, increased empathy, insightfulness, mild alterations in perception, accelerated thinking, jaw clenching, and

suppression (Greer & Tolbert, 1986; Grinspoon & Bakalar, 1986; Peroutka,

Newman, & Harris, 1988). MDMA has been of great interest to behavioral

pharmacologists due to its unique profileof subjective effects.

Cocaine

Cocaine is an obtained from the ofthe (Vetulani, 2001).

Coca leaves have a long history ofuse by civilizations in the southern hemisphere (Das,

1993; Johanson & Fischman, 1989; Vetulani, 2001). There is evidence that coca leaves

were used in religious ceremonies, as medicine, and to relieve the pain ofhunger

(Johanson & Fischman, 1989). It was not until afterthe process ofchemically isolating

cocaine from the coca in the was developed that the implications of

cocaine as a powerfulpsychostimulant became apparent in the western hemisphere

(Johanson & Fischman, 1989). The purificationof cocaine fromits naturally occurring

4 plant formis a multi step process. In this process, the intermediate precursor cocaine­ sulfateis extracted by washing crushed coca leaves in or an aromatic . Cocaine-sulfateis then transformedinto cocaine-hydrochloride, a water soluble white powder. Cocaine-HCl can be administered by snorting or by dissolving in water and then injecting or drinking the solution. The snorting or injection of cocaine produces rapid psychostimulant and euphoric effects.

Neuropharmacology of Psychostimulants

The pharmacological classificationof psychostimulants includes a large variety of compounds, including but not limited to cocaine and t�e . In general, the actions of these compounds fallunder the psychomotor theory of

(Wise & Bozarth, 1987). The psychomotor stimulant theory of addiction is heavily based on the principles of Skinnerian operant reinforcement. Wise and Bozarth (1987) state;

"The crux of the theory is that the reinforcing effectsof drugs, and thus their addiction liability can be predicted fromtheir ability to induce psychomotor activation" (p. 474). In advancing beyond the standard Skinnerian approach of considering psychostimulants as environmental variables, the psychostimulant theory of addiction examines the activation of dopaminergic pathways and its relevance to forwardlocomotion (Wise & Bozarth,

1987).

The psychostimulants, amphetamine and cocaine, have been indicated in the activation of reward circuitry and in particular activation of dopamine pathways in the nucleus accumbens (Wise & Bozarth, 1984). Furthermore, the psychostimulants cocaine and the amphetamines increase catecholamine levels in the extracellular fluid space (Bozarth, 1986).

5 Nucleus accumbens dopamine has been implicated in the study of motivation and reward (Salamone, 1996; Salamone, Correa, Mingote, & Weber, 2003; Sokolowski,

Conlan, & Salamone, 1998). It is well documented that most drugs of abuse, including cocaine, (Doyon et al., 2003; Mcdowell & Kleber, 1994; Yan, 1999) and

MDMA (Bankson & Yamamoto, 2004; Hser, Huang, Chou, Teruya, & Anglin, 2003;

Koch & Galloway, 1997), increase extracellular dopamine (DA) in the nucleus accumbens (NAc ). Acute administration of MDMA increases the release of both DA and

5-HT in awake-behaving rats (Gough, Ali, Slikker, & Holson, 1991; Hiramatsu & Cho,

1990; Kankaanpaa, Meririnne, Lillsunde, & Seppala, 1998; Yamamoto & Spanos, 1988), and NAc DA release may be modulated by 5-HT (Filip & Cunningham, 2002; Koch &

Galloway, 1997). Recent studies have shown that MDMA acts at nerve terminals to modulate release and re-uptake mechanisms of DA and 5-HT (Bankson & Yamamoto,

2004).

Cocaine exerts its actions primarily through blocking DA re-uptake in the . It is well documented that cocaine increases extracellular DA levels in the nucleus accumbens shell (David, Zahniser, Hoffer, & Gerhardt, 1998). Previous microdialysis studies have shown that both systemic and local injections of cocaine increase synaptic 5-HT in the NAc (Teneud, Baptista, Murzi, Hoebel, & Hernandez,

1996). and DA interactions on open-fieldand stereotypical behaviors in rats following cocaine administration, and the functionalimportance of increased monoamine release on behavioral activation have also been established (Broderick & Phelix, 1997).

At the present time, there are no published microdialysis studies on the effects of acute

MOMA/cocaine combinations.

6 Rationale for Current Study

It is of great importance to closely approximate human drug use when developing an animal testing model of drug abuse. The current study simultaneously examined locomotor activity and neurochemical changes using in vivo microdialysis sampling techniques to investigate the combined effectsof MDMA and cocaine. The locomotor activity (LMA) testing paradigm is based upon the activation of brain mechanisms associated with movement. Example behaviors areincreased exploratory behavior and rearing. Due to the association of motor system activation with drugs that produce reward, the locomotor activity test is often used as a preliminary assessment prior to more extensive investigations of the behavioral effectsof drugs. In vivo microdialysis and High

Performance Liquid Chromatography (HPLC) are commonly used to analyze and quantify extracellular levels of in the behaving organism. The analysis of drug-induced changes in neurotransmitter levels combined with measures of LMA offers a valuable strategy to assess simultaneously the neurochemical and behavioral actions of drugs. With respect to drugs of abuse, it is of particular interest to examine the release of monoamines in the mesocorticolimbic system. Two factorsthat are highly related when examining the abuse potential of a drug are behavioral activation and the release of dopamine in the nucleus accumbens. By using both LMA and microdialysis, the present study investigated behavioral and neurochemical effectsof acute MDMA­ cocaine combinations.

7 METHOD

Animals

Eighty-four male Sprague-Dawley rats served as subjects (Sasco, Portage, Ml).

Each rat weighed approximately 500 g at the start ofthe experiment. All rats were individually housed with standard rat chow and water available ad libitum. Rats were

maintained on a 12L: 12D cycle (lights on at 0700h/lights offat 1900 h),at a constant

temperature (20 ± 2° C) and humidity (50 ± 5%). Animals were allowed to recover for fourdays prior to testing. Six animals were eliminated fromthe study due to surgical complications or problems related to microdialysate sample volume. All procedures were approved by WesternMichigan University's Institutional Animal Care and Use

Committee. In accordance with university policy, all effortswere made to minimize pain and distress and the number ofanimals used.

Surgical Procedures

Rats were injected with 1.0 mg/kg - (Calbiochem, , CA)

15 min. prior to being anesthetized with 51.0 mg/kg (Sigma­

Aldrich, St. Louis, MO). Animals were then placed in a Kopf small animal stereotaxic device (David Kopflnstruments, Tujunga, CA) and maintained at 37.5° C using a

Gaymar T/PUMP heat therapy pump (Gaymar Industries Inc, Orchard Park, NY).

Removable guide cannulae (Bioanalytical Systems Inc, West Lafayette, IN) were stereotaxically implanted in the nucleus accumbens (AP+ 1.70, ML -1.50, DV -6.20) with the incisor adjusted to achieve the flat skull position (Paxinos and Watson,

8 1998). Guide cannula were secured in place with three small jewelers screws

(Bioanalytical Systems Inc, West Lafayette, IN) and dental cement (PERM, Hygenic

Corp, Akron, OH).

Behavioral Procedures

Locomotor activity assessment and in vivo microdialysis procedures were conducted in six identical Plexiglas chambers (16"1 x 16"w x 16"h) equipped with a

Versamax® Activity Monitoring System (Accuscan Instruments Inc., Columbus, OH).

All locomotor data were collected in 30 minute intervals that coincided with microdialysis sample collection times.

Microdialysis Procedures

BAS BR-2 microdialysis probes (Bioanalytical Systems Inc, West Lafayette,IN) were flushed with artificial cerebral spinal fluid (aCSF) ( 147.2 mM NaCl, 1.2 mM

CaCh, 2.7 mM KCl, 1.0 mM MgCh ) at a flowrate of 0.5 µI/min 12 hours prior to calibration using BAS BEE syringe pump (Bioanalytical Systems Inc, West Lafayette,

IN). At 0600h probes were placed in a calibration solution ( 1.5 µm DOP AC , 15 nM DA,

760 nM HVA, 300 nM 5-HIAA, 15 nM 5-HT) at a flow rate of 1.5 µI/min, a 30 minute calibration sample was collected. At 0700 - 0800h microdialysis probes were inserted into the guide cannulae and the rats were tethered to an Instech fluidswivel (Plymouth

Meeting PA) attached to a counter-balanced arm and placed in the locomotor activity chambers. Microdialysis flowrates were maintained at 1.5 µ1/min for the entire experiment. Following insertion, the probes were allowed to equilibrate forthree hours prior to testing. At approximatelyl 100 h, microdialysis sample collection began. Samples

9 were collected at 30 minute intervals and immediately flashfrozen and stored in a -80 ° C freezeruntil HPLC-EC analysis. The sampling regimen consisted of six 30 minute baseline samples, one 30 minute vehicle sample, one 30 minute sample, and fivepost injection 30 minute samples.

Histology

At the conclusion of the microdialysis procedure, rats were euthanized by injection of a solution containing sodium pentobarbital (50 mg/kg) dissolved in a 60% ethanol and then perfused at a constant pressure of 300 mm/hg with a 10% solution followed by a 10% formalin solution using a PerfusionOne pressurized perfusionsystem

(myNeuroLab, St Louis, MO). The microdialysis guides were removed afterthe perfusionand the were removed fromthe skulls and stored in 10% formalin.A

Vibratome 1500 sectioning system (Ted Pella Inc, Redding, CA) was used to slice coronal sections at a thickness of 70 µm. Coronal sections were mounted on microscope slides and histological placement was confirmed.All probe placements were within the nucleus accumbens target.

HPLC-EC

Dopamine was detected by reverse phase high performanceliquid chromatography coupled to electrochemical detection. The HPLC-EC system consisted of an ESA Coulochem II Model 5200 detector, ESA 582 solvent delivery module, ESA

540 autosampler and PC running ESA 501 chromatography software,MD-150/RP-C18 column (particle size 3µm, 3.0 x 150 mm i.d.) and a model 5014 analytical cell (ESA,

Chelmsford, MA). The detector settings were: guard cell 350 mv , CHI: -175 mv and

10 CH2: 250 mv, all filtersettings were set to 5 sec. A commercially prepared mobile phase,

MD-TM (ESA, Chemlsford, MA) was used consisting of75 mM sodium dihydrogen phosphate monohydrate, 1.7nm 1-octanesulfonic acid, 100 µ1/L triethyllamine, 25 µm

EDTA, 10% acetonitrile, and adjusted to a pH = 3.0 with phosphoric acid.

Drugs

Cocaine-hydrochloride and 3,4-Methylenedioxymethamphetamine (MDMA) were obtained fromthe National Institute on Drug Abuse (Rockville, MD). Sodium pentobarbital was purchased fromSigma-Aldrich (St. Louis, MO). Atropine sulfatewas purchased fromCalbiochem (San Diego, CA). All drugs were dissolved in 0.9% sterile saline and administered by intraperitoneal injection.

Statistical Analysis

The dependent measures of interest in this study included distance traveled (cm) and dopamine concentrations during each 30 minute sampling period. These data were graphed over the 13 sampling periods, which included six baseline samples, one sample followingthe vehicle injection, and six samples followingthe drug injection. For dopamine concentrations, averages were calculated from the baseline samples and data points were expressed as a percentage of the baseline average. Analysis of both locomotor activity and dopamine concentrations was performedby calculating individual regression line equations fortime periods 8 to 13 foreach individual subject. The slopes of the lines were grouped according to drug treatment condition. The slope of the regression lines fortreatment groups based on the LOG of dopamine concentrations were

compared using a One-Way ANOV A and Fisher-Hayter pairwise comparisons were

calculated for treatment effects.

11 RESULTS

Figure 1 represents the total distance traveled during each 30 minute sampling period. Note that drug injections were administered at the beginning ofthe eighth

sampling period. Both doses ofcocaine produced significantincreases in locomotor

activity. In comparison, MOMA produced only moderate increases in locomotor activity.

The combined effectsof either MOMA dose with 10 mg/kg cocaine were similar to the

effectsofthis dose ofcocaine when administered alone. However, the combined

administration of20 mg/kg cocaine and 3.0 mg/kg MOMA produced greater locomotor

activity compared to either dose alone.

Loomotor Atvt

-8·JOMS.O!Tv'kg+COCO"V11g -· . 1.l,+C0l +JOMS.01Tv'kg+COC1017R +�1.ln+C10n ♦JOMS.Onv'kg+COC20nv'kg E .....Un+C2n .!!. a:

l! I-

1 2 :a 4 I I 7 I I 10 11 12 1J a 4 • , 1 • , 10 11 12 12 1 2 S 4 f I 1 I I 10 11 12 'IS t .. ..,_ t-­ t .. ..,_ Sampling Time (3 min bins) Samplin Time (30 min bins) Sampling Time (30 min bins)

Figure 1. Locomotor Activity

The slope ofthe regression lines (see Figure 2) for treatment groups based on the

LOG ofdistance traveled (cm) were compared by using a One-Way ANOV A. A

significant drug effectwas obtained (F(7, 70) = 4.54, p<.01). Fisher-Hayter pairwise

comparisons were calculated for treatment effectsand qFH exceeded the critical value

12 (q.os,J-t, N-J) for [MDMA 1.5 vs. MDMA 1.5 + COC 10.0], and [MDMA 1.5 vs. MDMA

3.0 + coc 20.0].

-- -.•.oc•• 1-0••-•UCOC•:--•«�• 1 -- i' �------­ !' ------=-::.::

I 9 10.. _, II ll 13

Figure 2. Regression Lines Log ofLocomotor Activity

Figure 3 shows extracellular dopamine (DA) levels in the nucleus accumbens

(NAc) represented as a percentage ofthe average baseline concentration foreach 30

minute sampling period. When administered alone, cocaine increased NAc DA levels to

265% or 450% ofbaseline following a dose of 10 or 20 mg/kg, respectively. MDMA increased NAc DA levels to 155% or 194 % following1.5 or 3.0 mg/kg, respectively.

When administered in combination with 1.5 mg/kgMDMA, 10 mg/kgcocaine increased

NAc DA levels by 370% and cocaine 20 mg/kgincreased these levels by 532%. When

administered in combination with 3.0 mg/kgMDMA, 10 mg/kg cocaine increased NAc

DA levels by 316% and cocaine 20 mg/kgincreased these levels by 700%. Thus, the

combined actions ofMDMA and cocaine appeared to increase NAc DA levels to a

greater extent than either drug alone.

13 Nucleu .cunbn Dmin Lvls

i IS.Or+CO! • ,-JUf+COf . KS.On♦C1n 1.lf +C 10 n +M:JM ♦KS.OnCXr .'lj ♦-1.ln+C»"

Sin Tm (3 rn bn) Sin Tm (3 rn bn) Smpin rime(3 min bn)

Figure 3. Nucleus Accumbens Dopamine Levels

The slope of the regression lines (see figure4) fortreatment groups based on the

LOG of dopamine concentrations were compared by using a One-Way ANOVA. A significant drug effect was obtained F(7, 64) = 4.90, p<.01 Fisher-Hayter pairwise comparisons were calculated fortreatment effects. qFH exceeded the critical value ( q_os,J-

1, N-J) for [MDMA 3.0 vs. MDMA 3.0 + COC 20.0], [MDMA 3.0 vs MDMA 3.0 + COC

10.0], and [MDMA 1.5 vs. MDMA 3.0 + COC 20.0]. These results are indicative of cocaine's actions to produce a dose-dependent temporal and quantal augmentation of extracellular dopamine elicited by injection of MDMA 3.0 mg/kg.

14 MOMA 1,1 .../kl COMSJNATIONI MOMA J.O .../kl COMIINATIONI l.2S ----.u••• - ,,.�---�I-- --..eoc:-. .... ,-1 I.DO ------I:::: I ::0.7S ------.-=-=-1;-=,-,��1:::: I ------==B�� ------···· :. i ... -�---- 1 o.so ··:·:· � g.,, ------g .... --- ... - ---. --

I t 10 II U 13 ��,_,__ ...,.., ...... �� ...... -.-.,- .._,..-., ---'., I ____ t 10"""' 11 12 U

Figure 4. Regression Lines Log ofNucleus Accumbens Dopamine Levels

DISCUSSION

Cocaine and MDMA produced the anticipated increase in extracellular NAc DA levels and locomotor activity resulting fromactivation of the mesocorticolimbic system

(Bozarth, 1986; Bozarth & Wise, 1986). In concordance with the literature, MDMA produced locomotor system activation and dopamine effluxto a lesser extent than cocaine (Koch & Galloway, 1997). With respect to a combinatorial effectofMDMA and cocaine, MDMA 3.0 mg/kg co-injected with cocaine 20 mg/kg produced dopamine levels and locomotor activity greater than that of either drug alone. These results are indicative of a possible MD MA/cocainesynergistic effect which is dose dependent.

The individual effectsof cocaine and MDMA on extracellular dopamine efflux are well documented. Cocaine increases dopamine effluxin the nucleus accumbens shell

(Morgan, Horan, Dewey, & Ashby, 1997; Morgan et al., 1997; Shimada, Yamaguchi, &

Yanagita, 1996) (Bradberry, 1994, 2002). MDMA has also been demonstrated to enhance dopamine transmission (Cadoni et al., 2005; Green, Meehan, Elliott, O'Shea, & Colado,

2003; Koch & Galloway, 1997), although the mechanisms related to the effluxof extracellular dopamine levels differ. It is important to distinguish the action ofcocaine fromthe action ofamphetamine-analogs such as MDMA. Cocaine's action ofblocking

15 monoamine transporters generates an increase in extracellular dopamine, whereas amphetamine analogs reverse activity and in turnincrease dopamine efflux (Metzger, Hanson, Gibb, & Fleckenstein, 1998). It is hypothesized that

MDMA may affectdopamine release by several differentmechanisms; (1) by producing action analogous to amphetamine on dopamine terminals (Cadoni et al., 2005) (2) and releasing serotonin by acting on 5-HT2 receptors (Bradberry, 1994; Cadoni et al., 2005;

Koch & Galloway, 1997).

The neurobehavioral actions of MDMA and cocaine, when co-administered, have not been thoroughly investigated. The results of the current study bear some similarity to findingsreported by Morgan et al. ( 1997) who administered cocaine to rats fourhours followingthe administration of the cocaine analog RTI-55. RTI-55 has been demonstrated to have a high binding affinityfor the and the . Our results are consistent with those of Morgan and colleagues

(1997), who demonstrated an increase in NAc dopamine levels of 458% over basal levels following 20 mg/kg cocaine and a 758% increase in dopamine levels following the administration of both RTI-55 and cocaine. In addition, the RTI/cocaine data parallel our findings with respect to an augmented temporal duration of the drug combinations on extracellula NAc dopamine levels. Given the results of Morgan et al (1997) it is hypothesized that MDMA may augment the effects of cocaine via dopamine transporters.

These results have important implications for futurelines of research. Future research should include the investigation of the appetitive and aversive properties of

MDMA/cocaine combinations. Although these combinations produce increases in extracellular dopamine, which is highly relevant in relation to reward mechanisms,

16 excessive dopamine release may induce a psychological state similar to a drug-induced amphetamine psychosis, which in tum may be experienced as an aversive condition. To examine the aversive and appetitive effects of these combinations, the conditioned place preferenceparadigm may be employed. To assess the abuse potential of these drug combinations in animal models, a self-administration ·study could be conducted to examine whether these combinations will be readily administered and if the rate of administration is greater forthe combination compared to either drug alone. Further neurochemical investigations ought to be conducted simultaneously with behavioral experiments to assess the relationship between dopamine release and behavioral effects of MD MA/cocaine combinations. In addition to the aforementioned studies, immunohistochemical investigations of the dopamine transporter and hydroxylase activity could be examined. These neurochemical and behavioral studies may also be extended to examine the chronic effectsof combined MDMA and cocaine administration with respect to their possible neurotoxic consequences.

These preclinical data may generalize to the population of polydrug abusers who

frequently use MDMA in conjunction with other drugs of abuse. The preclinical data

demonstrated an increased effect of cocaine's action on dopamine efflux. This increase in

extracellular dopamine may augment the subjective feeling of that humans

experience in association with psychostimulant use. MDMA may also extend the

temporal effects of psychostimulant actions in humans, causing a prolonged subjective

euphoric effect. The increase in intensity and temporal elongation of drug action may

contribute to the abuse potential of polydrug use.

17 CONCLUSION

Recent reports ofhuman polydrug abuse have demonstrated the popularity of

MDMA use in conjunction with cocaine (Chinet, Stephan, Zobel, & Halfon, 2007;

Marsden et al., 2006) MDMA and cocaine are psychostimulants that individually produce forward locomotion and elevations in extracellular dopamine. In combination, these drugs produce a significant increase in extracellular dopamine levels and locomotor

activity greater than either drug alone. The relevant importance of investigating the

neurobehavioral effectsofthis particular drug combination is becoming readily apparent.

Further study of the neurochemical, neurobehavioral, and neurotoxic actions of

MDMA/cocaine combinations is warranted.

18 REFERENCES

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22 APPENDIX

Institutional Animal Care and Use Committee Approval Form

23 .-WESTERN MICHIGAN UNIVERSIT Institutional Animal Care and Use Committee

Date: · October 15, 2004

To: Lisa Baker, Principal Investiga )/ / ; From: Robert Eversole Chair , � Re: IACUC Protocol No. 04-09-01

Your protocol entitled '�crodialvsis Studies Of Polydrug Administration,, has received approval from the Institutional Ammal Care and Use Committee. The conditions and duration of this approval are specified in the Policies of Western Michigan University. You may now begin to implement the research as described in the application.

The Board wishes you succ�ss in the pursuit of your research goals.

Approval Termination: October 15, 2005

Watwood Hall, KalamazooMl 49008-5456 ,o, o-,,e 24 PHONF• (f;lf;l �R7.R?O1 c.v. 1c1c1