speedball by lindsey p. pattison wake forest … · figure 5. representative images from dat...

FUNCTIONAL ALTERATIONS IN THE DOPAMINE TRANSPORTER OF

RODENTS FOLLOWING SELF-ADMINISTRATION OF COCAINE, HEROIN AND

SPEEDBALL

BY

LINDSEY P. PATTISON

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Neuroscience

MAY 2013

Winston-Salem, North Carolina

Approved By

Scott E. Hemby, Ph.D., Advisor

Ashok N. Hegde, Ph.D., Chair

Sara R. Jones, Ph.D.

Brian A. McCool, Ph.D.

Michael A. Nader, Ph.D.

Loren H. Parsons, Ph.D.

ii

ACKNOWLEDGEMENTS

I would like to acknowledge my labmates Scot McIntosh and Amanda Grigg for

their continued assistance, but most of all their friendship. I would also like to thank my

“step-advisors,” Dr. Evgeny (Zhenya) Budygin and Dr. Steve Childers. They welcomed

me into their labs and worked extensively with me through understanding concepts and

resolving technical difficulties to ensure that I received the best training possible. And of

course, I would like to thank my advisor, Dr. Scott Hemby, for his unrelenting

professional and personal support system throughout my entire graduate career. It was

indeed a pleasure to work with an advisor who is so passionate about science and I will

greatly miss the inspiring conversations we’ve had over the years.

I would like to dedicate this dissertation to my father, Chuck Pattison, who has

been my role model as a life-long learner and has always supported my scholastic

endeavors, particularly throughout graduate school.

This research was funded by DA012498, DA021634 and DA006634 from the

National Institute on Drug Abuse.

iii

TABLE OF CONTENTS

PAGE

LIST OF ABBREVIATIONS iv

LIST OF TABLES AND FIGURES ix

ABSTRACT xii

CHAPTER

I THE DOPAMINERGIC CORRELATES OF CHRONIC COCAINE, HEROIN

AND SPEEDBALL ADMINISTRATION 1

II SPEEDBALL-INDUCED CHANGES IN ELECTRICALLY STIMULATED

DOPAMINE OVERFLOW IN RAT NUCLEUS ACCUMBENS 61

Published in Neuropharmacology September 14, 2010

III DIFFERENTIAL REGULATION OF ACCUMBAL DOPAMINE

TRANSMISSION IN RATS FOLLOWING COCAINE, HEROIN AND

SPEEDBALL SELF-ADMINISTRATION 87

Published in Journal of Neurochemistry March 19, 2012

IV EFFECTS OF COCAINE, HEROIN AND SPEEDBALL SELF-

ADMINISTRATION ON DOPAMINE TRANSPORTER BINDING SITES 118

V COMPARING IN VIVO AND IN VITRO ALTERATIONS IN DAT KINETICS

AND BINDING TO ELUCIDATE FUNCTIONAL ADAPTATIONS TO

CHRONIC SPEEDBALL SELF-ADMINISTRATION 169

APPENDIX 219

SCHOLASTIC VITAE 231

iv

LIST OF ABBREVIATIONS

[125

I]RTI-55 iodinated 3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester

[3H] tritium

[3H]CFT tritiated 3β-(4-fluorophenyl)tropan-2β-carboxylic acid methyl ester

[3H]DA tritiated dopamine

[Ca2+

]I intracellular calcium concentration

[DA] dopamine concentration

[DA]e extracellular dopamine concentration

[DA]p concentration of dopamine release per stimulation pulse

[Na+]e extracellular sodium concentration

[Na+]i intracellular sodium concentration

3-MT 3-methoxytyramine

5-HT serotonin

6-MAM 6-monoacetylmorphine

AC adenylyl cyclase

ANOVA analysis of variance

Bmax binding density

Bmax1 high-affinity binding density

Bmax2 low-affinity binding density

Bmaxtotal high- and low-binding densities combined

Ca2+

calcium ion

CaMKII calcium/calmodulin-dependent protein kinase

cAMP 3’,5’-cyclic monophosphate

Cdk 5 cyclin-dependent kinase 5

CFT 3β-(4-fluorophenyl)tropan-2β-carboxylic acid methyl ester (WIN 35,428)

Cl- chloride ion

CNS central nervous system

v

COMT catechol-O-methyl transferase

CPE carboxypeptidase

CPu caudate putamen

D1R dopamine D1 receptor subtype

D2L dopamine D2 receptor long isoform


D2S dopamine D2 receptor short isoform




DA dopamine

DA-RT dopamine reverse transport

DAT dopamine transporter

DBH dopamine-beta-hydroxylase

DOPAC 3,4-dihydroxyphenylacetic acid

DOPAL 3,4-dihydroxyphenylacetaldehyde

DOPET 3,4-dihydroxyphenylethanol

DYN dynorphin

EGFP enhanced green fluorescent protein

ENK enkephalin

EPI epinephrine

ERK1/2 extracellular signal-regulated kinases

FR fixed-ratio

FSCV fast-scan cyclic voltammetry

GABA gamma-aminobutyric acid

GABAergic gamma-aminobutyric acid-expressing cells

GABAT gamma-aminobutyric acid transporter

vi

Gαi/o inhibitory G protein alpha subunit

Gαs/olf stimulatory G protein alpha subunit

glu glutamate

GP globus pallidus

GPCR G protein-coupled receptor

GPe globus pallidus external

GPi globus pallidus internal

hDAT human dopamine transporter

HPLC high-pressure liquid chromatography

HVA homovanillic acid

i.p. intraperitoneal

i.v. intravenous

JNK c-Jun N-terminal kinase

K+ potassium ion

Kd dissociation constant

Kd1 high-affinity dissociation constant

Kd2 low-affinity dissociation constant

Km Michaelis-Menten constant, representing apparent affinity

L-DOPA L-3,4-dihydroxyphenylalanine

LeuTAa leucine transporter purified from Aquifex aeolicus

MAO monoamine oxidase

MAPK mitogen-activated protein kinase

MAT monoamine transporter

MKP3 mitogen-activated protein kinase phosphatase 3

MOPET 3-methoxy-4-hydroxyphenylethanol

MOR mu opioid receptor

mPFC medial prefrontal cortex

vii

MSN medium spiny neuron

Na+ sodium ion

NAc nucleus accumbens

NE norepinephrine

NET norepinephrine transporter

NO nitric oxide

NPY neuropeptide Y

NT neurotensin

OCT organic cation transporter

OT olfactory tubercle

PET positron emission tomography

PFC prefrontal cortex

PI3K phosphoinositide 3-kinase

PKA protein kinase A

PKB protein kinase B

PKC protein kinase C

PP1/PP2Ac protein phosphatases 1 and 2Ac

PR progressive-ratio

PTM post-translational modification

PV parvalbumin

rDAT rat dopamine transporter

RTI-55 3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester

s.c. subcutaneous

S1 primary binding site

S2 secondary binding site

SA self-administration

SERT serotonin transporter

viii

SN substantia nigra

SNc substantia nigra pars compacta

SP substance P

TH tyrosine hydroxylase

TM transmembrane

VMAT1 vesicular monoamine transporter 1

VMAT2 vesicular monoamine transporter 2

Vmax maximal reuptake rate

VP ventral pallidum

VTA ventral tegmental area

WF-23 2β-propanoyl-3β-(2-naphthyl) tropane

ix

LIST OF TABLES AND FIGURES

PAGE

CHAPTER I

Table 1. Effects of cocaine on the mesolimbic DA system 23

Table 2. Compounds used to elucidate mechanisms of cocaine/heroin

combinations 40

CHAPTER II

Figure 1. Representative traces of evoked DA signals detected by FSCV in rat NAc

before and 1 min after cocaine (1.0 mg/kg, i.v.), heroin (0.03 mg/kg, i.v.)

and speedball (1.0 mg/kg cocaine + 0.03 mg/kg heroin, i.v.) injection in

drug-naïve animals (upper panel) and topographical color plots of

voltammetric data before drug administration, with time on the x-axis,

applied scan potential on the y-axis and background-subtracted faradaic

current shown on the z-axis in pseudo-color (lower panel) 71

Figure 2. Electrically evoked DA efflux in the NAc of anesthetized drug-naïve rats

following a single i.v. injection of cocaine (1.0 mg/kg), heroin (0.03

mg/kg) and speedball (1.0 mg/kg cocaine + 0.03 mg/kg heroin) 72

Figure 3. DAT apparent affinity (Km, or Michaelis-Menten rate constant) in the NAc

of anesthetized drug-naïve rats following a single i.v. injection of cocaine

(1.0 mg/kg), heroin (0.03 mg/kg) and speedball (1.0 mg/kg cocaine + 0.03

mg/kg heroin) 74

Figure 4. DAT-mediated maximal reuptake rate (Vmax) in the NAc of anesthetized

drug-naïve rats following a single i.v. injection of cocaine (1.0 mg/kg),

heroin (0.03 mg/kg) and speedball (1.0 mg/kg cocaine + 0.03 mg/kg

heroin) 76

x

CHAPTER III

Figure 1. (A) Acquisition of self-administration during the first 14 days of training

rats to self-administer cocaine (250 µg/infusion), heroin (4.95 µg/infusion)

or speedball (250/4.95 µg/infusion cocaine/heroin) 98

(B) Mean interinfusion intervals for each group (n = 5) throughout the 25

day period of self-administration under an FR2 99

Figure 2. DA changes detected by FSCV in the NAc of anesthetized rats following

i.v. cocaine, speedball and heroin infusion 101

Figure 3. Electrically evoked DA efflux in the NAc of anesthetized rats

approximately 24 hours after the last chronic self-administration session,



Figure 4. DAT apparent affinity (Km) in the NAc of anesthetized rats (n = 5/group)

approximately 24 hours after the last chronic self-administration session,



Figure 5. (A) DAT-mediated maximal reuptake rate (Vmax) in the NAc of

anesthetized rats (n = 5/group) approximately 24 hours after the last

chronic self-administration session, following a single i.v. injection of

cocaine (1.0 mg/kg), heroin (0.03 mg/kg) and speedball (1.0 mg/kg

cocaine + 0.03 mg/kg heroin) 106

(B) Baseline, or pre-drug, Vmax calculated at three time points prior to

bolus drug infusions as compared to the baseline Vmax values of drug-

naïve rats 107

Figure 6. Comparison of electrically evoked DA efflux in NAc of drug-naïve rats

and chronically self-administering rats following a single challenge i.v.

infusion of (A) cocaine (1.0 mg/kg), (B) speedball (1.0/0.03 mg/kg

cocaine/heroin), and (C) heroin (0.03 mg/kg), and effects on the inhibition

of DA uptake, or apparent Km, in NAc following i.v. challenge infusions

of (D) cocaine (1.0 mg/kg), (E) speedball (1.0/0.03 mg/kg cocaine/heroin)

and (F) heroin (0.03 mg/kg) in chronic self-administering rats and in drug-

naïve animals (n = 5/group) [Data from drug-naïve rats is reprinted with

permission from Pattison et al., 2011] 109-110

xi

CHAPTER IV

Figure 1. SA behaviors of rats responding under an FR5 for i.v. cocaine (250

µg/inf), heroin (10 µg/inf) or speedball (250/10 µg/inf cocaine/heroin).

(A) The number of days required obtain the maximum number of 25

infusions per day varied between drug groups 131

(B) Average interinfusion intervals for rats self-administering cocaine,

heroin and speedball were also significantly different 132

Figure 2. (A) High- and (B) low-affinity DAT binding site dissociation constants

(Kd) were determined by membrane binding assays using [125

I]RTI-55 and

unlabeled RTI-55 at varying concentrations 134

Figure 3. (A) High- and (B) low-affinity DAT binding site densities (Bmax) were

determined by membrane binding assays using [125

I]RTI-55 and unlabeled

RTI-55 at varying concentrations 135

Figure 4. Percents of (A) high- and (B) low-affinity DAT binding sites were

determined using the Bmax values by defining Bmaxtotal = Bmax1 + Bmax2,

and percent high-affinity sites = (Bmax1 / Bmaxtotal) × 100 and percent low-

affinity binding sites = (Bmax2 / Bmaxtotal) × 100 136

Figure 5. Representative images from DAT quantitative autoradiography are shown

with (A) schematics of the medial NAc (bregma+2.20mm, left) and caudal

NAc (bregma+1.20mm, right) regions of interest that determined the size

and location of densitometry, as outlined in black. (B) Sample

autoradiographs of sections at the medial NAc (left) and caudal NAc

(right) levels from saline control animals with regions of interest outlined

in black 138

Table 1. Effects of chronic cocaine, heroin and speedball SA on the binding

parameters of high- and low-affinity DAT binding sites. Mean (± SEM)

dissociation constants (Kd) and binding densities (Bmax) were determined

by nonlinear regression for two-site binding of membrane binding assays

with [125

I]RTI-55 and unlabeled RTI-55 135

Table 2. Effects of chronic cocaine, heroin and speedball SA on densities of

[3H]CFT binding to DAT in rat NAc. Mean (± SEM) autoradiography data

quantified at the medial and caudal levels of the NAc are presented as

specific binding in pmol/mg of wet-weight tissue 138

xii

ABSTRACT

Lindsey P. Pattison

FUNCTIONAL ALTERATIONS IN THE DOPAMINE TRANSPORTER OF

RODENTS FOLLOWING SELF-ADMINISTRATION OF COCAINE, HEROIN AND

SPEEDBALL

Dissertation under the direction of Scott E. Hemby, Ph.D.,

Professor of Physiology and Pharmacology

Cocaine/heroin combinations (speedball) induce a synergistic elevation in

extracellular dopamine (DA) concentrations ([DA]e) in the nucleus accumbens (NAc)

that can explain the continued patterns of abuse even after seeking treatment for heroin

addiction. To further delineate the mechanism of this neurochemical synergism, in vivo

fast-scan cyclic voltammetry (FSCV) was used to compare evoked DA release and DA

transporter (DAT)-mediated reuptake kinetic parameters following acute administration

of cocaine, heroin and speedball in drug-naïve rats, as well in rats with chronic self-

administration (SA) history of cocaine, heroin and speedball. Together, the results have

shown that speedball combinations likely induced DA autoreceptor feedback in attempt

to regulate increased [DA]e, and that there is also a significant increase in baseline

reuptake rate by DAT following chronic speedball SA. In order to deduce the potential

mechanism by which DAT is altered to increase reuptake rate, cocaine-like ligands were

used in membrane binding assays and quantitative autoradiography for DAT in the NAc

of rats to assess the long-term effects of chronic SA of cocaine, heroin and speedball on

xiii

DAT high- and low-affinity binding sites. The results show a significant increase in

affinity of the DAT low-affinity binding site and a shift in DAT binding site composition

toward a greater percent of high-affinity binding sites following chronic speedball. These

binding data suggest that long-term speedball SA induces a flexibility in the DAT

substrate binding sites toward an outward facing conformation in order to increase

maximal reuptake rate in compensation for chronically elevated [DA]e. Knowledge of

these chronic alterations in DAT function may be able to provide insight into the

development of novel pharmacotherapies for the treatment of polysubstance abuse, as

well as for cocaine and heroin addictions alone.

1

CHAPTER I

THE DOPAMINERGIC CORRELATES OF CHRONIC COCAINE, HEROIN

AND SPEEDBALL ADMINISTRATION

Lindsey P. Pattison and Scott E. Hemby

The following chapter is a portion of a review in preparation to be submitted for

publication. Lindsey P. Pattison prepared the review and Scott E. Hemby acted in an

advisory and editorial capacity.

2

Introduction

According to the latest National Survey on Drug Use and Health, cocaine is the

second-most common illicit drug used by persons 12 years or older in the United States,

with 1.5 million admitted cocaine users in 2010 alone (SAMHSA, 2011b). The number of

people in the U.S. classified as a heroin abuser or as heroin dependent rose from 214,000

in 2002 to 359,000 in 2009. In 2010 alone, 699,000 Americans received treatment for

cocaine abuse and 417,000 for heroin abuse (SAMHSA, 2011b). Among patients entering

a substance abuse clinic for intravenous drug use, heroin was reported as the most

commonly injected drug and cocaine was the second-most injected drug. Approximately

15% of these patients also admitted to injecting their secondary or tertiary drug of choice,

which is often overlooked in clinic admission surveys (SAMHSA, 2011a). This suggests

that many Americans are polysubstance abusers and that the health risks associated with

intravenous injection of illicit substances may be affecting more people than predicted.

The World Health Organization defines polydrug abuse as the use of more than

one drug or type of drug by an individual, often at the same time or sequentially, and

usually with the intention of enhancing, potentiating, or counteracting the effects of

another drug. Dependence, or the compulsive need to use a drug despite problems related

to use of the substance, on one or more of the drugs is often assumed (World Health

Organization, 2013). Such patterns of substance abuse have been on the rise over the past

decades as well as incidence of emergency room admissions related to nonfatal

polysubstance overdoses (Strang et al., 1999; Stohler, 2003; Kedia et al., 2007;

EMCDDA, 2011). The use of multiple substances has a long history in the United States,

dating back to the early 1900s when it was recorded that 11.9% of narcotic addicts in

3

New York reported combination drug abuse (Kaufman, 1976). During the 1960s,

polydrug abuse increased radically in the U.S. as noted in heroin and narcotic addicts

(Kaufman, 1976).

For the past few decades, there has been clinical and experimental attention paid

to a street phenomenon in which opioid-dependent individuals, either through regular

heroin use or maintenance on synthetic opioid agents, also co-abuse cocaine (Leri et al.,

2003a). This form of polysubstance abuse where opioids and cocaine are co-administered

either simultaneously or within a short period is often referred to as “speedballing”

(Ellinwood et al., 1976; Hunt et al., 1984; Leri et al., 2003a). In a sample of treatment-

seeking heroin abusers from Sydney, Australia, 24% also admitted to co-abusing cocaine

(Darke and Hall, 1995). In a poll of heroin-addicted patients admitted to treatment in

Texas during 2010, 18% also admitted to using cocaine (NIDA, 2011). Various rationales

have been proposed to explain the combined use of cocaine and heroin in humans. These

include a reduction in the magnitude of undesired side effects, enhancement in the

positive effects of either drug alone, induction of a euphoric feeling, beyond superior

positive effects, or independent, non-additive effects even though the drugs are

administered concurrently (Kosten et al., 1987; Foltin and Fischman, 1992; Hemby et al.,

1996). The hypothesis of an intensified euphoria with speedball parallels preclinical

results showing that cocaine and heroin potentiate the reinforcing effects of one another

in studies utilizing self-administration (SA) paradigms in non-human primate models

(Mattox et al., 1997; Rowlett and Woolverton, 1997; Rowlett et al., 2005).

Compared to cocaine-dependence only, speedball users show different

psychopathologies, including increased profiles of depression, hysteria, psychopathic

4

deviation, paranoia, schizophrenia and social introversion (Malow et al., 1992; Trujillo et

al., 2011). Compared to opioid-dependence only, treatment-seeking patients with co-

dependence on cocaine and opioids tend to practice more risky drug injection and sexual

behaviors (Grella et al., 1995; Hudgins et al., 1995), exhibit antisocial personality

disorders (King et al., 2001), engage in more income-generating crimes (Strug et al.,

1985; Cross et al., 2001), and most importantly, these speedball users have worse

treatment outcomes than persons using heroin alone (Perez de los Cobos et al., 1997;

Preston et al., 1998; DeMaria et al., 2000; Downey et al., 2000; Sofuoglu et al., 2003;

Tzilos et al., 2009). Specifically, of methadone-maintained individuals that co-abused

cocaine, a 1995 study of 652 patients reported that 52% of patients continued to use

cocaine regardless of the dose of methadone (Hartel et al., 1995), and another in 1998

found that methadone was effective in significantly reducing heroin intake in 80% of

patients while only reducing cocaine intake in about 50% of patients (Magura et al.,

1998). Besides attempts of approved pharmacotherapies for heroin dependence, clinicians

have combined opioid-addiction treatment with contingency management and shown

modest success in speedball abusers who were less-severe cocaine users (Weinstock et

al., 2010). There are no approved pharmacotherapies for cocaine addiction, but some

research supports the use of psychostimulant treatment for cocaine dependence [see

(Mariani and Levin, 2012) for review]. Even so, sustained-release amphetamine

treatment reduced cocaine- but not speedball-seeking in buprenorphine-maintained

speedball polydrug abusers (Greenwald et al., 2010). These adverse ramifications of

cocaine and heroin co-abuse provide compelling evidence for preclinical research to

unmask the neurobiological substrates associated with prolonged speedball abuse,

5

compared to heroin or cocaine alone. Certainly more targeted treatment methods are

desired for this class of polysubstance abusers, both of the behavioral therapy and

pharmacotherapy nature. Much of the neurochemical research on speedball abuse

described to date has involved an important neurotransmitter system relating to addiction,

the mesolimbic dopamine system.

Dopamine neurobiology and pharmacology

Mesolimbic dopamine pathway

Dopamine (DA) as a neurotransmitter is known to be implicated in motivated

behavior and reward-driven learning (Mirenowicz and Schultz, 1994; Schultz et al., 1997;

Ikemoto and Panksepp, 1999), and it has been known for decades that DA plays a

significant role in the brain processes underlying the reinforcing actions of drugs of abuse

(Schiff, 1982; Dackis and Gold, 1985). In the central nervous system (CNS), the

dopaminergic system includes four neuronal pathways: the mesocortical, mesolimbic,

nigrostriatal and tuberoinfundibular pathways. The tuberoinfundibular pathway of the

hypothalamus projects from the arcuate nucleus to the median eminence where DA

regulates secretion of prolactin from the pituitary gland (Gore, 2008). The nigrostriatal

pathway is involved in the production of movement, and especially fine-tuning motor

movements, as part of the basal ganglia loop. DA cells project from the substantia nigra

pars compacta (SNc) to the neostriatum, and dysregulation of this pathway, specifically

loss of DA cells, results in Parkinsonism (Mink, 2008). The dopaminergic cell bodies of

the mesocortical pathway originate in the ventral tegmental area (VTA) and project to the

6

prefrontal cortex (PFC), particularly the dorsolateral prefrontal cortex in primates. This

DA pathway is thought to be involved in motivation and emotional responses, and

dysregulation of the mesocortical pathway has been implicated in schizophrenia and

other psychoses (Miller and Wallis, 2008). The fourth, and most pertinent DA pathway to

addiction, is the mesolimbic pathway, projecting from the VTA to the striatum,

particularly the nucleus accumbens (NAc) of the ventral striatum. The striatum is the

most densely innervated DA target in the brain (Yao et al., 2008).

The endogenous role of the mesolimbic system is a rewarding response for

behaviors such as eating, drinking and sex, but this pathway is also hijacked by drugs of

abuse (Wise, 1982; Koob and Bloom, 1988; Robbins et al., 1989; Koob, 1992; Koob et

al., 2008), particularly those with psychomotor stimulant properties (Wise and Bozarth,

1987). The NAc has been the primary region of focus for drug addiction research for

decades. Many peer-reviewed publications on the neurocircuitry of addiction have begun

referring to the brain reward circuitry as the mesocorticolimbic pathway, in a sense

combining the two latter DA pathways described above (Feltenstein and See, 2008; Chen

et al., 2009; Van den Oever et al., 2012). In this combined reward circuitry, dopaminergic

cells bodies of the VTA project to both the NAc and PFC, while the PFC sends glutamate

projections back to both the NAc and VTA (Carr and Sesack, 2000). Specifically the

medial PFC (mPFC), as well as the subcortical amygdala, sends dense glutamatergic

projections to the NAc (Voorn et al., 2004). The multiple cell types from these projection

areas converge to form a striatal microcircuit of synapses in the NAc that connect the

brain regions most affected by drugs of abuse. The union of dopaminergic and

glutamatergic inputs onto NAc neurons suggests that the NAc functions as a limbic-

7

motor interface where it can process the relevance of salient stimuli to initiate a

behavioral response (Sesack et al., 2003), and thus lies at the center of attention in drug

addiction research.

The gamma-aminobutyric acid (GABA)-expressing (GABAergic) medium spiny

neuron (MSN) is the predominant cell type within the NAc, making up 90-95% of total

striatal neurons. GABAergic MSNs also generate neuropeptides, about half of which

produce dynorphin (DYN) and substance P (SP), and the other half enkephalin (ENK)

and neurotensin (NT) (Smith et al., 2013). These cells are aptly named for the densely

populated spines located on their dendrites (Yao et al., 2008). Virtually every MSN

borders DA terminals due to the dense dopaminergic varicosities, which average only 4

µm distance between release sites (Gonon, 1997). Glutamatergic inputs from the PFC and

amygdala also innervate striatal MSNs and a significant subpopulation of their dendritic

spines are simultaneously contacted by dopaminergic and glutamatergic axons (Freund et

al., 1984; Smith et al., 1994). MSNs of the NAc project to the ventral pallidum (VP),

globus pallidus (GP, internal and external: GPi and GPe), substantia nigra (SN) and VTA

(Lobo, 2009; Smith et al., 2013). The reciprocal GABAergic projections from NAc back

to VTA are thought to mediate a long-loop feedback to regulate DA neuron activity, but

do so indirectly by innervating non-dopaminergic VTA neurons (Xia et al., 2011).

Besides MSNs, the remaining approximately 5-10% of striatal neurons are interneurons,

which modulate MSN activity. The four types of striatal interneurons are cholinergic

interneurons, parvalbumin (PV)-expressing GABAergic interneurons, calretinin-

expressing GABAergic interneurons and neuropeptide Y (NPY)/nitric oxide (NO)-

expressing GABAergic interneurons (Tepper et al., 2008; De Mei et al., 2009). Though

8

glutamate (Glu), GABA and acetylcholine all modulate MSN activity as well, the main

correlate thought to underlie addictive behaviors related to drugs of abuse is DA release

in the NAc.

It was first discovered that DA neurons fired in response to salient stimuli,

including intracranial self-stimulation and tones paired with food reinforcers, about half a

century ago by Olds and colleagues (Olds, 1958; Olds and Olds, 1969; Phillips and Olds,

1969). The proof that enhanced striatal DA neurotransmission was directly related to

administration of addictive drugs (alcohol, opiates, barbiturates, nicotine, amphetamine

and cocaine) came with the approach of in vivo microdialysis with electrochemical

detection (Di Chiara and Imperato, 1986; Imperato et al., 1986; 1988b; Pettit and Justice,

1989, 1991). At the same time, in vivo voltammetry for neurochemical detection of

catecholamine release and reuptake was also being used to study the enhancement of DA

neurotransmission by cocaine and amphetamine (Caviness and Wightman, 1982; Ewing

et al., 1983). More recent advances in electrophysiology and electrochemistry have also

allowed for the observation of real-time increases in striatal DA signaling of behaving

animals while responding for drugs of abuse (Carelli and Wightman, 2004).

Investigations of the specific dynamics of DA signaling regulation, from synthesis and

release to receptor activation and reuptake, have been a major focus of polydrug abuse

and addiction research in general.

9

Dopamine synthesis and metabolism

DA is classified in the catecholamine and phenethylamine families of chemicals

and is part of the monoamine class of neurotransmitters in the CNS. DA can be

synthesized in vivo in neurons and adrenal gland cells from three possible precursors: L-

tyrosine, L-phenylalanine and L-3,4-dihydroxyphenylalanine (L-DOPA). Its precursors

are able to cross the blood-brain barrier, but DA itself cannot. The pathway of DA

synthesis is L-phenylalanine to L-tyrosine by phenylalanine hydroxylase, L-tyrosine to L-

DOPA by tyrosine hydroxylase (TH), and L-DOPA to DA by aromatic L-amino acid

decarboxylase (Deutch and Roth, 2008). DA is also the precursor for norepinephrine

(NE, noradrenaline) and subsequently epinephrine (EPI, adrenaline), via dopamine-beta-

hydroxylase (DBH) and phenylethanolamine N-methyltransferase, respectively (Deutch

and Roth, 2008). Outside from neuronal synthesis, DA is produced in the adrenal glands

in order to generate the major catecholamine neurotransmitter of the adrenal system, EPI.

TH is the rate-limiting enzyme in catecholamine synthesis under basal conditions, but

when NE- or EPI-containing cells are activated, DBH becomes rate-limiting (Deutch and

Roth, 2008). Not all three of these catecholamines (DA, NE and EPI) are typically

present in the same brain region or type of neurons, and expression of the aforementioned

enzymes determines which catecholamine is the predominant neurotransmitter for a given

population of neurons.

DA is produced in the cytosol of neurons and transported into synaptic vesicles by

vesicular monoamine transporter 2 (VMAT2). In adrenal cells, VMAT1 is responsible for

vesicular packaging of DA, where it can be converted into NE within synaptic vesicles by

DBH (Deutch and Roth, 2008). In dopaminergic cells, DA remains stored in synaptic

10

vesicles until an action potential occurs in the neuron, forcing the vesicles to merge with

the cell membrane in the process of calcium (Ca2+

)-dependent exocytosis where DA is

released into the synapse (Schwarz, 2008). In certain situations, DA can be reverse-

transported into the synapse (Sulzer et al., 1995) and DA not stored in vesicles can be

released from the cytosol somatodendritically (Cheramy et al., 1981; Greenfield, 1985).

Once in the extracellular space, DA can act upon pre- and postsynaptic receptors to,

respectively, regulate further DA release and propagate the signal from pre- to

postsynaptic neurons. Upon dissociation from DA receptor complexes, DA is transported

back into the presynaptic neuron via the dopamine transporter (DAT), and sometimes by

the other monoamine transporters (MATs), norepinephrine transporter (NET) and

serotonin transporter (SERT), depending on the brain region and types of neurons

expressed (Deutch and Roth, 2008). Once returned to the cytosol, DA can either be

repackaged into synaptic vesicles by VMAT2 or enzymatically degraded into inactive

metabolites by monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT).

DA is degraded into 3,4-dihydroxyphenylacetaldehyde (DOPAL) by MAO, and DOPAL

is further metabolized into either 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde

dehydrogenase or into 3,4-dihydroxyphenylethanol (DOPET, or hydroxytyrosol) by

aldose reductase, the latter to a lesser extent. Finally, COMT reduces DOPAC and

DOPET to homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylethanol (MOPET),

respectively. COMT can also directly metabolize DA into 3-methoxytyramine (3-MT),

which is then degraded into HVA by MAO. HVA and MOPET metabolites of DA are

excreted in the urine (Deutch and Roth, 2008). These metabolites can also be measured in

laboratory animals modeling human drug abuse and have shown alterations following

11

treatment with drugs that act upon the mesolimbic DA system (Trulson and Ulissey,

1987; Wilson et al., 1996). Throughout its lifetime, from synthesis to release and

eventually to degradation, DA can have both excitatory and inhibitory effects on

neurotransmission, depending on which DA receptors are expressed in a given neuron.

Dopamine receptors

DA receptors have long been proposed to exist as two discrete populations,

broadly based on either excitation or inhibition of neurotransmission via opposing effects

on adenylyl cyclase (AC) and thus 3’,5’-cyclic monophosphate (cAMP) production

(Kebabian and Calne, 1979; Missale et al., 1998). The D1-like family of DA receptors

includes the D1 and D5 receptors (D1R and D5R) and the D2-like family includes D2,

D3 and D4 DA receptors (D2R, D3R and D4R). All DA receptors are G protein-coupled

receptors (GPCRs) and contain seven hydrophobic α-helix amino acid stretches, called

transmembrane (TM) domains, with TM sequence homology within each family (Probst

et al., 1992). D1-like DA receptors couple to stimulatory G proteins (Gαs/olf) to activate

AC and increase cAMP production to activate protein kinase A (PKA) and subsequent

downstream signaling systems (Monsma et al., 1990; Yao et al., 2008; Sako et al., 2010).

When cAMP levels are increased by activation of AC, PKA is in turn activated and

begins phosphorylation of cytoplasmic and nuclear proteins that regulate cellular

metabolism and ion channel functioning, and desensitize GPCRs, leading to action

potential propagation and the cellular response of neurotransmitter release (Choi et al.,

1993; Hofmann et al., 1994). The D2-like receptors couple to inhibitory G proteins

12

(Gαi/o) to inhibit AC and decrease cAMP levels (Picetti et al., 1997; Beaulieu and

Gainetdinov, 2011). D2-like receptors also influence intracellular calcium ([Ca2+

]i) levels

and a number of Ca2+

-dependent intracellular signaling processes through G protein-

independent pathways (Yao et al., 2008; De Mei et al., 2009). D2R activation leads to

reduction in Ca2+

currents (Yan et al., 1997; Missale et al., 1998) and modulation of

[Ca2+

]i likely regulates DA biosynthesis, given that Ca2+

activates the kinase

Ca2+

/calmodulin-dependent protein kinase (CaMKII) that in turn phosphorylates and

activates the rate-limiting enzyme in DA synthesis, TH (Braun and Schulman, 1995).

Besides differences in stimulatory and inhibitory signaling, D1- and D2-like

families also differ in subcellular location. D1Rs and D5Rs are found exclusively on

postsynaptic cells receiving DA signaling, such as GABAergic MSNs of the striatum

(Civelli et al., 1991). In contrast, D2Rs, D3Rs and D4Rs are located both pre- and

postsynaptically on dopaminergic neurons and DA target cells, respectively (Jackson and

Westlind-Danielsson, 1994; Tarazi et al., 1998). In addition to having dual localization,

D2Rs are a heterogeneous population formed by two splice variants. The long and short

isoforms (D2L and D2S) differ by 29 amino acids encoded by alternative splicing of the

sixth exon, which inserts into the G protein-interacting third intracellular loop of the

receptor (Gingrich and Caron, 1993; Jackson and Westlind-Danielsson, 1994). Though

D2L and D2S may be expressed both pre- and postsynaptically, D2S is responsible for

presynaptic regulation of DA synthesis and release and modulation of DAT reuptake

activity, while D2L mediates postsynaptic G protein-dependent signaling and Ca2+

-

dependent cascades (De Mei et al., 2009).

13

Of all DA receptor subtypes, D1Rs and D2Rs have the most widespread and

highest levels of expression (Vallone et al., 2000). Within the NAc, D1Rs and D2Rs are

located postsynaptically on MSNs, and D2Rs are also presynaptically expressed

throughout dendrites and axons on dopaminergic neurons originating from the VTA and

SN (Civelli et al., 1991; Jackson and Westlind-Danielsson, 1994; Tarazi et al., 1998).

There has been a dichotomous debate over the years concerning NAc MSN classification

as either distinctly D1R- or D2R-expressing (Gerfen et al., 1990; Gertler et al., 2008)

versus co-localization of both D1Rs and D2Rs within a MSN (White and Wang, 1986;

Surmeier et al., 1996; Mizuno et al., 2007); however, the debate is still evolving (Smith et

al., 2013). It is also possible that within the NAc there exists a population of MSNs with

distinct D1R or D2R expression as well as a population of MSNs with D1R and D2R co-

localization. To further complicate the issue of DA receptor localization in the NAc,

D2Rs are also expressed on striatal cholinergic interneurons (Hersch et al., 1995;

Alcantara et al., 2003) and neurons originating in the GP that innervate striatal PV-

expressing interneurons (Bevan et al., 1998). The current overall opinion is that

convergence of multiple inputs utilizing various neurotransmitters onto NAc MSNs,

which also differentially express DA (and many other) receptors, leaves researchers of

addiction and other neurological disorders involving the mesolimbic DA pathway with an

immense task to further unravel this complex system.

14

Reuptake by dopamine transporter

Once DA has activated DA receptors within the proximity of the release site,

extracellular DA is transported back into presynaptic terminals by its principal

monoamine transporter. DAT is an extrasynaptic membrane protein (Nirenberg et al.,

1996; Pickel et al., 2002; Mengual and Pickel, 2004) that is responsible for reuptake of

DA from the extracellular space back into the cytosol (Deutch and Roth, 2008). DAT is

expressed almost exclusively in dopaminergic neurons (Torres et al., 2003). In situ

hybridization has localized DAT mRNA to cell bodies of the SN and VTA in the rat brain

(Hoffman et al., 1998). Consistent with the terminal projections from the SN and VTA,

immunoreactivity assays in mouse brain have localized DAT protein expression to

presynaptic DA terminals in the striatum (particularly the NAc), OT, nigrostriatal bundle,

lateral habenula, mPFC and layers I, II and III of the cingulate cortex, as well as less-

dense reactivity in axons and dendrites in the SN and VTA (Ciliax et al., 1995; Freed et

al., 1995; Hersch et al., 1997).

The cDNA and amino acid sequences of DAT were initially described in 1991

when DAT mRNA was isolated and cloned using primers for NET and GABA

transporter (GABAT) from rat ventral midbrain tissue (Kilty et al., 1991; Shimada et al.,

1991). Of the mRNA transcripts resulting from the hybridized cDNA clones, the protein

product which conferred [3H]DA uptake in transfected cells was 619 amino acids in

length with a nonglycosylated molecular weight of 69 kDa. The rat DAT (rDAT)

sequence shows 67% amino acid identity and 81% similarity with human NET and 45%

identity and 67% similarity with rat GABAT (Shimada et al., 1991). Rat DAT and human

DAT (hDAT) exhibit 93% amino acid identity, but hDAT contains 620 amino acids

15

(National Center for Biotechnology Information, 2013) and displays an approximate 75

kDa molecular weight in wild-type form (Li et al., 2004). Similar to both NET and

GABAT, the DAT protein contains 12 putative TM-spanning domains across species.

Both amino- and carboxyl-terminals are located intracellularly, with several predicted

protein kinase sites. DAT also contains potential sites for N-linked glycosylation on its

second extracellular loop. Four N-glycosylation sites were identified from rat cDNA on

the second extracellular loop (Kilty et al., 1991; Shimada et al., 1991), and three N-

glycosylation sites were identified on the same loop in DAT coding regions from Old

World macaque monkeys, which exhibit 98.9% sequence homology to the cloned hDAT

(Miller et al., 2001). Human DAT is proposed to exhibit three N-linked glycosylation

sites on the second extracellular loop, as well (Beuming et al., 2008). N-glycosylation of

DAT promotes stable expression in the plasma membrane, allowing for proper binding

and transport function. In fact, blockade of glycosylation or removal of canonical

glycosylation sites results in both reduced surface expression and intracellular DAT

levels. Although non-glycosylated DAT is still expressed in the plasma membrane, it is

less stable at the cell surface due to more rapid endocytosis than glycosylated DAT, and

the non-glycosylated form shows a sharp reduction in maximal DA reuptake rate by DAT

(Li et al., 2004).

DAT is a member of the sodium/chloride (Na+/Cl

-)-dependent transporter of the

monoamine family. It uses the electrochemical gradient of the cell created by the

sodium/potassium (Na+/K

+) ATPase to drive transport by coupling the reuptake of one

DA molecule with inward transport two sodium ions (Na+) and the outward transport of

one chloride ion (Cl-) (Krueger, 1990; Gu et al., 1994). The binding of two Na

+, one Cl

-

16

and one DA is sequential (Torres et al., 2003). Chen at al. showed in gramicidin-

perforated DAT-expressing cells that the intracellular concentration of Na+ ([Na

+]i) plays

an important role in affecting the access of DA to external binding sites of DAT on

depolarized plasma membranes (Chen and Reith, 2004). In order for the transport to be

energetically favorable, extracellular Na+ concentration ([Na

+]e) must be greater than

[Na+]i to perpetuate transport with the concentration gradient. To date, the crystal

structure of DAT has yet to be described, but in 2005 the crystal structure of a bacterial

leucine transporter from Aquifex aeolicus (LeuTAa), which is a homologue to Na+/Cl

--

dependent neurotransmitter transporters, revealed the LeuTAa core determinants of

substrate binding and architecture of the internal and external gates required for transport,

which may be suggestive of DAT binding sites and gating control (Yamashita et al.,

2005). TM domains one, three, six and eight are thought to be implicated in transporter

substrate binding for both LeuTAa and DAT (Yamashita et al., 2005; Beuming et al.,

2008; Merchant and Madura, 2012). Specific breaks in the helical structures of TM1 and

TM6, along with nearby residues of TM3 and TM8, are predicted to comprise the protein

core where substrate and Na+ would bind (Yamashita et al., 2005).

Molecular modeling of free energy profiles of LeuTAa support a primary and

secondary binding pocket, and comparison of the free energy profiles for DA and cocaine

in DAT suggests that the binding site of cocaine is located in the secondary binding

pocket of DAT (Merchant and Madura, 2012). Other studies utilizing computational

modeling of amino-acid mutations combined with site-directed in vitro mutations at

various locations on DAT also support distinct or minimally-overlapping DA and cocaine

binding sites, suggesting that cocaine’s role in blocking DA reuptake may be caused by

17

an allosteric modulation instead of direct binding site blockade (Lin and Uhl, 2002; Uhl

and Lin, 2003; Loland et al., 2004; Volz and Schenk, 2005; Chen et al., 2006; Huang et

al., 2009) [but see (Beuming et al., 2008)]. Using different experimental techniques of

radioligand binding, it has been shown for some time with certain ligands that DAT

contains both a high- and low-affinity binding site (Madras et al., 1989a; Madras et al.,

1989b; Boja et al., 1992; Pristupa et al., 1994; Staley et al., 1994; Gracz and Madras,

1995; Rothman et al., 1995; Letchworth et al., 1999; Katz et al., 2000; Mash et al., 2002).

Whether such high- and low-affinity binding sites may represent distinct binding sites of

DA and cocaine on the DAT has not yet been experimentally investigated. Yet, the

affinities of DAT ligands for high- and low-affinity DAT binding sites may be

dynamically regulated by drugs of abuse (Kish et al., 2001; Samuvel et al., 2008).

DAT function is dynamically regulated and modulated by several processes,

including phosphorylation-dependent and –independent post-translational modifications

(PTMs). These PTMs can change intrinsic activity, alter turnover, control exocytic fusion

with the plasma membrane and regulate sequestration of DAT from the plasma

membrane (Ramamoorthy et al., 2011). Activity of several enzymes, particularly protein

kinases, has been linked to DAT functional regulation. The enzymes include, but are not

limited to: PKA, protein kinase C (PKC), phosphoinositide 3-kinase (PI3K), mitogen-

activated protein kinase (MAPK), extracellular signal-regulated kinases (ERK1/2),

protein kinase B (PKB, or Akt), CaMKII, cyclin-dependent kinase 5 (Cdk 5),

carboxypeptidase E (CPE), c-Jun N-terminal kinase (JNK), tyrosine kinases, mitogen-

activated protein kinase phosphatase 3 (MKP3), and protein phosphatases (PP1/PP2Ac)

(Foster et al., 2003; Moron et al., 2003; Bolan et al., 2007; Hoover et al., 2007; Foster et

18

al., 2008; Mortensen et al., 2008; Gorentla et al., 2009; Price et al., 2009; Zhang et al.,

2009). These regulatory enzymes are often activated or inhibited by other DA receptors

or associated proteins as a mechanism of dopaminergic system modulation. The predicted

intracellular domain of hDAT contains 15 serine, 9 threonine and 5 tyrosine residues as

potential phosphorylation sites for protein kinases (Foster et al., 2006). The most well-

studied PTM is phosphorylation by PKC (Huff et al., 1997; Vaughan et al., 1997; Zhang

et al., 1997; Chang et al., 2001; Page et al., 2001; Lin et al., 2003; Gorentla and Vaughan,

2005; Foster et al., 2008). Increased phosphorylation by PKC activation leads to

downregulation of DA transport activity as shown by decreased reuptake rate with no

change in apparent affinity of DAT (Zhu et al., 1997), attributable to increased

sequestration of DAT leading to decreased cell surface expression (Vaughan et al., 1997;

Holton et al., 2005). Other PTMs yet to be investigated may also contribute to regulation

of DAT binding sites.

Other more direct mechanisms can lead to DAT involvement in DA release,

known as DA reverse transport (DA-RT) or lateral release, as compared to synaptic

vesicle exocytosis of DA as described previously, also referred to as quantal release

(Leviel, 2011). Unlike quantal DA release which is dependent on neuronal firing, the

proposed mechanism of DA-RT is an exchange-diffusion process. Opazo et al. have even

linked PKC activation to glutamate-induced DA-RT (Opazo et al., 2010). Other means of

DA-RT are described as the mechanism of action of a class of abused drugs aptly referred

to as DA releasers, such as amphetamine (Pifl et al., 1995). Many other drugs of abuse,

including cocaine and heroin, also have far-reaching effects on the dopaminergic system

of the mesolimbic pathway, particularly on DA release and DAT reuptake mechanisms.

19

Pharmacology of cocaine and heroin in the brain

Effects of cocaine administration on the mesolimbic dopamine system

To understand the neurobiochemical effects of chronic speedball abuse on the DA

reward system, one must first comprehend the independent pharmacological mechanisms

of action of cocaine and heroin alone. Cocaine is a crystalline tropane alkaloid that is

obtained from leaves of Erythroxylon coca, a shrub-like tree native to western South

America. Although indigenous Americans have been chewing coca leaves for centuries,

the pure alkaloid was first isolated in 1860 and it was the first local anesthetic to be

discovered (Gold et al., 1992). As an anesthetic, cocaine blocks the initiation or

conduction of a nerve impulse at the site of local application by binding to voltage-

sensitive Na+ channels (Ritchie and Green, 1990), but it’s most prominent effect is

stimulation of the CNS. In the brain, cocaine inhibits reuptake of the monoamine

neurotransmitters serotonin (5-HT), NE and DA by binding to and blocking their

respective transporters, SERT, NET and DAT (Calligaro and Eldefrawi, 1987; Ritz and

Kuhar, 1989; Ritz et al., 1990; Bennett et al., 1995). The direct effect of MAT blockade

by cocaine is acute elevation of the extracellular concentration of the monoamine in that

brain region (Di Chiara and Imperato, 1988b; Pettit and Justice, 1989; Bradberry et al.,

1993; Florin et al., 1994). Cocaine’s well-known mood and cognition enhancing effects

create a euphoric feeling (Fischman et al., 1976; Dackis and Gold, 1985; Wise and

Bozarth, 1985), as well as physiological effects of increased locomotion and stereotypy

(Sahakian et al., 1975; Kuczenski et al., 1991; Ushijima et al., 1995), have been linked to

DAT inhibition by cocaine and subsequent increases in extracellular DA concentrations

20

([DA]e) (Wilson and Schuster, 1973; Johanson et al., 1976; de la Garza and Johanson,

1982; Fowler et al., 1989; Freed et al., 1995).

The technique of in vivo microdialysis in combination with high-pressure liquid

chromatography (HPLC) chemical detection of dialysates from rodent NAc has

previously demonstrated significant increases in [DA]e (averaging about 400% of

baseline) following acute, experimenter-delivered subcutaneous (s.c.) or intraperitoneal

(i.p.) injection of cocaine (Di Chiara and Imperato, 1988b; Zernig et al., 1997; Gerasimov

and Dewey, 1999), and during intravenous (i.v.) SA of cocaine after chronic exposure

(Pettit and Justice, 1989; Wise et al., 1995b; Hemby et al., 1997; Hemby et al., 1999;

Smith et al., 2006). In vivo fast-scan cyclic voltammetry (FSCV) studies have also

provided valuable information about cocaine-DAT interactions, as this technique affords

the ability to characterize the primary mechanisms regulating [DA]e: release, reuptake

and diffusion of DA (Greco and Garris, 2003). Numerous voltammetric reports have

confirmed potentiated increases in [DA]e with cocaine (and electrical stimulation), and

more importantly validated the mechanism by which the competitive competition of

cocaine for DAT increases [DA]e (Mateo et al., 2004; Budygin, 2007; Oleson et al., 2009;

Ferris et al., 2011; Pattison et al., 2011; Pattison et al., 2012). This is demonstrated by

increases in the Michaelis-Menten kinetic constant of apparent affinity (Km), which is

inversely related to the affinity of DAT for endogenous DA. Together, in vivo

microdialysis and voltammetry techniques have elucidated the direct mechanism of DAT

blockade by cocaine to result in elevated [DA]e in the NAc.

A number of other neurobiological consequences of chronic cocaine have been

examined, as well. DA receptor expression appears to be dynamically regulated,

21

especially by length of exposure, dose of cocaine and duration since last exposure.

Cocaine administration has shown increased (Nader et al., 2002; Beveridge et al., 2009),

decreased (Kleven et al., 1990; De Montis et al., 1998; Moore et al., 1998a) and

unchanged (Peris et al., 1990; Meador-Woodruff et al., 1993) striatal D1R density.

Similar patterns of differential expression of D2Rs following cocaine were also observed

as increased (Trulson and Ulissey, 1987; Kleven et al., 1990; Peris et al., 1990; Beveridge

et al., 2009), decreased (Kleven et al., 1990; Volkow et al., 1990; Moore et al., 1998b;

Nader et al., 2002; Bailey et al., 2008) and unchanged (Meador-Woodruff et al., 1993;

King et al., 1994) D2R striatal density. Some more reliable trends of repeated cocaine

exposure are increased DAT density (Little et al., 1993; Staley et al., 1994; Wilson et al.,

1994; Hitri et al., 1996; Letchworth et al., 1997; Little et al., 1998; Little et al., 1999;

Letchworth et al., 2001; Mash et al., 2002; Ben-Shahar et al., 2006; Bailey et al., 2008;

Miguens et al., 2008; Beveridge et al., 2009) and increased DAT uptake activity (Daws et

al., 2002; Mash et al., 2002; Samuvel et al., 2008) in striatal regions of the

mesocorticolimbic system. These latter alterations in DAT may reduce the basal

concentration of DA and subsequent cocaine-stimulated DA release, and alter the

responsiveness of DA receptors and second-messenger systems, possibly leading to

cocaine tolerance and continued abuse.

Some of these changes in DA receptor expression following cocaine are

reversible and depend on time since last cocaine exposure (Kleven et al., 1990; Peris et

al., 1990; Volkow et al., 1990; Wilson et al., 1994; Beveridge et al., 2009). Many other

experimental factors contribute to observed variances in results using animal models,

including: route of drug administration, dose of drug, contingency of drug delivery,

22

duration of exposure, total drug intake, length of withdrawal, methods used to study

receptor systems (i.e. agonists vs. antagonists, and direct vs. indirect measures of

expression or activity), and even environmental situations of the laboratory animals. Due

to the intrinsic nature of these experimental features of modeling human substance abuse,

researchers must continue to exhaust different patterns of drug use and experimental

variables in order to fully map the time course and extent of alterations in the

mesocorticolimbic DA system following cocaine exposure. Though there are a few

agreed-upon trends in DA system alterations, we cannot yet predict specifically defined

sequelae of chronic cocaine abuse. Though not exhaustive, many of these results are

described in detail in Table 1.

23

Table 1: Effects of cocaine on the mesolimbic DA system

Effect Measured

(Reference) Species

Cocaine Exposure

Paradigm

Length

of W/D

Methods of

Analyses Main Findings

General DA System

DA synthesis

(Trulson and

Ulissey, 1987)

R ED coc; 10 mg/kg i.p.;

2×/d; 15 d

60 d DOPA & HVA

accumulation

DEC DA synthesis in STR, LFB & MDB

(coc vs. sal)

DA-stimulated AC

activity (De Montis

et al., 1998)

R SA or YK coc; 1.1

mg/kg/inf i.v., FR2,

6 hr/d, 30 d

18 hr Activity assay

([3H]cAMP)

DEC AC activity in NAc (SA & YK coc vs.

sal) & in OT (YK coc vs. sal); NC in AC

activity in CPu, PFC or Hippo

DA tissue levels

(Wilson et al.,

1996)

Hu coc use for 1+ yr Tox HPLC

quantification of

DA & metabolites

DEC DA in rCaud & cCaud; trend dec DA

in NAc (coc vs. ctl)

D1 Receptor

D1R density

(De Montis et al.,

1998)

R SA or YK coc; 1.1

mg/kg/inf i.v., FR2, 6

hr/d, 30 d

18 hr memb bind

([3H]SCH 23390)

DEC D1R density in NAc (both coc vs. sal)

D1R density &

affinity

(Kleven et al.,

1990)

R ED coc; 10 mg/kg or 20

mg/kg i.p.; once/d; 15 d

20 min

or 2 wk

memb bind

([125

I]SCH 23982)

DEC D1R density in STR (coc vs. sal all Tx;

lo vs. hi, 2 wk w/d), NAc (coc vs. sal, 20

min w/d) & FC (lo vs. sal, 2 wk w/d);

INC D1R in FC (hi vs. sal, 20 min w/d);

NC in D1R affinity

23

24

Effect Measured

(Reference) Species

Cocaine Exposure

Paradigm

Length

of W/D

Methods of


D1R density

(Peris et al., 1990)


once/d; 1 d or 8 d

1 d, 3 d

or 1 wk

QAR

([3H]SCH 23390)

NC in D1R density in STR, NAc or VTA

D1R density

(Moore et al.,

1998a)

NHP SA coc; 0.03 mg/kg/inf

i.v., FR5; 4 hr/d, 18-22

mo

a few

hrs

QAR

([3H]SCH 23390)

DEC D1R density in cDLCPu, cVMCPu,

cNAc shell (coc vs. naïve);

NC in rNAc & rDSTR

D1R density

(Nader et al., 2002)


(lo) or 0.3 mg/kg/inf

(hi) i.v.; 30 inf/d; FI3

min; 5 d (initial) or

100 d (chronic)

a few

hrs

QAR

([3H]SCH 23390)

INC D1R density in cDLCaud, cVMCaud,

cOT, cNAc shell (hi & lo coc vs. naïve) & in

cDPut & cCPut (hi coc vs. naïve)

D1R density

(Beveridge et al.,

2009)


i.v.; 30 inf/d; FI3 min;

100 d

30 or

90 d

QAR

([3H]SCH 23390)

INC D1R density in all areas of Caud & Put,

rNAc, cNAc shell & core & OT (INC in coc

30 d w/d vs. sal; NC in 90 d w/d)

D1R density

(Meador-Woodruff

et al., 1993)

Hu Chronic coc use; no

records or duration

Tox QAR

([3H]SCH 23390)

NC in D1R density in CPu, NAc or SNr (vs.

ctl)

D1R mRNA

expression

(Meador-Woodruff

et al., 1993)

Hu Chronic coc use; no

records or duration

Tox in situ

hybridization

NC in D1R mRNA expression in CPu or

NAc (vs. ctl)

24

25

Effect Measured

(Reference) Species

Cocaine Exposure

Paradigm

Length

of W/D

Methods of


D2 Receptor

D2R density

(Bailey et al., 2008)

M ED coc; 15 mg/kg i.p.,

3×/day, 14 d (steady) or

escalating doses (15-30

mg/kg) over 14 d (esc)

30 min QAR

([3H]raclopride)

DEC D2R density in DLCPu, DMCPu,

VMCPu, VLCPu, NAc shell & core (steady

& esc coc vs. sal)

D2R density &

affinity

(Kleven et al.,

1990)

R ED coc; 10 mg/kg or 20


20 min

or 2 wk

memb bind

([125

I]spiperone)

DEC D2R density in STR & FC (both coc

vs. sal, 20 min w/d);

INC D2R in NAc (coc vs. sal, 20 min w/d);

NC in D2R affinity

D2R density

(Peris et al., 1990)


once/d; 1 d or 8 d

1 d, 3 d

or 1 wk

QAR

([3H]spiperone)

INC D2R density in rNAc for coc vs. sal (8 d

coc, 1 d w/d)

D2R density &

affinity (Trulson

and Ulissey, 1987)


twice/d; 15 d

60 d memb bind

([3H]spiroperidol)

INC D2R density in STR, LFB & MDB

(coc vs. sal);

NC in D2R affinity

D2R density &

binding (King et

al., 1994)

R ED coc; 40 mg/kg/day

int s.c. or cont inf; 14 d

7 d memb bind

([3H]spiperone)

NC in D2R density or affinity (all Tx vs. sal)

D2R density

(Moore et al.,

1998a)


i.v., FR5; 4 hr/d,

18-22 mo

a few

hrs

QAR

([3H]raclopride)

DEC D2R density throughout all regions of

Caud, Put, OT & NAc shell & core (coc vs.

naïve); NC in SN & VTA

25

26

Effect Measured

(Reference) Species

Cocaine Exposure

Paradigm

Length

of W/D

Methods of


D2R density

(Nader et al., 2002)




min; 5 d (initial) or 100

d (chronic)

a few

hrs

QAR

([3H] raclopride)

DEC D2R density in rDLCaud, rCCaud,

rDPut, cDLCaud, NAc shell & core (hi & lo

coc vs. naïve) & in rVMCaud, rCPut,

cDMCaud, cCCaud, cVMCaud, cDMPut,

cCPut, cVMPut (hi coc vs. naïve)

D2R density

(Beveridge et al.,

2009)



100 d

30 or

90 d

QAR

([3H]raclopride)

INC D2R density in rVPut & rNAc (coc 30 d

w/d vs. sal); NC in D2R density in remaining

Put, cNAc or OT

D2R density

(Meador-Woodruff

et al., 1993)

Hu chronic coc use;

duration NR

Tox QAR

([3H]raclopride)

NC in D2R density in CPu, NAc or SNc

(coc vs. ctl)

D2R availability

(Volkow et al.,

1990)

Hu chronic coc use

(1-10 yrs)

< 1 wk

or > 1

mo

PET imaging

([18

F]N-methyl-

spiroperidol)

DEC D2R availability in STR (coc, < 1 wk

w/d vs. ctl) & NC in D2R availability in

STR (coc, > 1 mo w/d vs. ctl)

D2R mRNA

(King et al., 1994)


int s.c. or cont inf; 14 d

7 d RT-PCR trend dec in D2R mRNA expression in CPu

(int s.c. coc vs. sal)

D2R mRNA

(Meador-Woodruff

et al., 1993)

Hu Chronic coc use;

duration NR

Tox in situ

hybridization

NC in D2R mRNA expression in CPu, NAc

or SNc (vs. ctl)

26

27

Effect Measured

(Reference) Species

Cocaine Exposure

Paradigm

Length

of W/D

Methods of


D2R-stimulated G

protein activity




(15-30 mg/kg) over 14 d

(esc)

30 min QAR ([35

S]GTPγS,

stimulated by

quinerolane)

INC D2R-G protein activity in VLCPu &

NAc shell in esc coc vs. sal;

NC in D2R-G protein activity in steady coc

vs. sal

DAT

DAT density




(15-30 mg/kg) over 14 d

(esc)

30 min QAR

([3H]mazindol)

INC DAT density in DLCPu, DMCPu,

VMCPu, VLCPu, OT, NAc shell & core

(esc coc vs. sal)

DAT density

(Wilson et al.,

1994)

R SA coc; 0.25 mg/kg/inf

i.v.; FR1; 24 hr/d; 7 wks

4 hr or

3 wk

QAR

([3H]WIN 35,428

&

[3H]GBR 12,935)

INC DAT density in STR, NAc shell & core,

VTA & SN (coc 4 hr w/d vs. sal); DEC DAT

density in STR, NAc shell & core, VTA &

SN (coc 3 wk w/d vs. sal)

DAT density &

affinity (Hitri et al.,

1996)


int i.p. or cont inf; 14 d

7 d QAR

([3H]WIN 35,428)

& memb bind

([3H]GBR 12,935)

INC DAT density in CPu (cont coc vs. sal;

WIN binding); DEC DAT density in PFC

(int coc vs. sal; GBR binding); NC in DAT

density in STR (GBR); NC in DAT affinity

DAT density

(Letchworth et al.,

1997)

R ED coc; 10, 15 or 20


1 hr QAR

([3H]mazindol)

INC DAT density in rNAc (lo coc vs. sal);

NC in DAT density in STR (mid & hi coc

vs. sal)

27

28

Effect Measured

(Reference) Species

Cocaine Exposure

Paradigm

Length

of W/D

Methods of


DAT density

(Ben-Shahar et al.,

2006)

R SA coc; 0.75 mg/kg/inf

i.v.; FR1 TO 20 s; 1 hr/d

(brief) or 6 hr/d (ext);

8+ d

14 d QAR

([3H]WIN 35,428)

INC DAT density in dSTR & NAc core;

NC in DAT density in NAc shell, mPFC or

VTA (brief coc vs. sal; NC in ext coc)

DAT density

(Miguens et al.,

2008)

R SA & YK coc; 1

mg/kg/inf i.v.; FR5 TO

30 s; 2 hr/d; 5+ wk

0, 1, 5,

or 10 d

QAR

([3H]WIN 35,428)

INC DAT density in CPu, mPFC, NAc shell

& core, VTA & SN (SA coc vs. YK coc &

vs. sal at all w/d points)

DAT density &

affinity

(Letchworth et al.,

1999)


once/d; 8 d

1 hr QAR & memb bind

([3H]PTT &

[125

I]RTI-55)

NC in DAT density in CPu, OT or NAc;

NC in DAT affinity in STR memb

(coc vs. sal)

DAT density

(Letchworth et al.,

2001)




min; 5 d (initial), 100 d

(chronic) or 1.5 yrs

(long-term)

a few

hrs

QAR

([3H]WIN 35,428)

INC DAT density in rNAc, cVCaud, cVPut,

cNAc shell & core, cOT (lo coc vs. sal);

INC DAT density in all cCaud & cPut areas,

cNAc shell & core, cOT (hi coc vs. sal);

INC DAT density in cVCaud, cNAc shell &

core, cOT (long-term coc vs. sal);

DEC DAT density in DLCaud & cCPut (lo

coc, initial vs. sal)

DAT density

(Beveridge et al.,

2009)



100 d

30 or

90 d

QAR

([3H]WIN 35,428)

INC DAT density in rCCaud, rDMCaud,

rVMCaud, rDPut, rCPut, rNAc, cCCaud,

cCPut, cVPut, cNAc shell & OT (coc 30 d

w/d vs. sal; NC in 90 d w/d)

28

29

Effect Measured

(Reference) Species

Cocaine Exposure

Paradigm

Length

of W/D

Methods of


DAT density &

affinity (Little et

al., 1993; Little et

al., 1998; Little et

al., 1999)

Hu chronic coc abuse or

dependence;

duration NR

Tox QAR & memb bind

([3H]WIN 35,428)

INC DAT density in Caud, Put & NAc;

NC in DAT affinity (coc vs. ctl)

DAT density &

affinity (Staley et

al., 1994)

Hu COD victims; chronic

daily coc use;

duration NR

Tox memb bind

([3H]WIN 35,428)

INC DAT density in Put memb (coc vs. ctl);

NC in DAT affinity

DAT density &

affinity (Mash et

al., 2002)


dependence;

duration NR

Tox QAR

([3H]WIN 35,428)

INC DAT density in vSTR memb

(coc vs. ctl); NC in DAT affinity

DAT density &

affinity (Wilson et

al., 1996)

Hu coc use for 1+ yr Tox memb bind

([3H]WIN 35,428)

NC in DAT density or affinity (coc vs. ctl)

DAT protein

expression (Wilson

et al., 1996)

Hu coc use for 1+ yr Tox Western blotting DEC DAT protein expression in Caud &

NAc, NC in DAT protein in Put (coc vs. ctl)

DAT mRNA &

protein expression

(Letchworth et al.,

1997; Letchworth

et al., 1999)

R ED coc; 10, 15, 20


(1997); ED coc; 25

mg/kg i.p.; 2×/d; 8 d

(1999)

1 hr in situ

hybridization &

Western blotting

DEC DAT mRNA expression in VTA &

SNc (dose-dep coc vs. sal);

NC in DAT protein expression in

STR memb

29

30

Effect Measured

(Reference) Species

Cocaine Exposure

Paradigm

Length

of W/D

Methods of


DAT mRNA

expression

(Little et al., 1998)

Hu Chronic coc abuse or

dependence; duration

NR

Tox in situ

hybridization

DEC DAT mRNA expression in mMDB;

NC in lMDB (coc vs. ctl)

DAT uptake rate &

affinity

(Daws et al., 2002)


one inj only

90 min [3H] DA uptake INC DAT uptake rate in NAc synap

(coc vs. sal);

NC in DAT affintiy

DAT uptake rate &

affinity (Samuvel et

al., 2008)

R SA coc; 0.6 mg/kg/inf;

FR1; 2 hr/d; 10+ d

3 wk [3H] DA uptake INC DAT uptake rate in CPu & NAc synap;

INC DAT affinity in NAc synap (coc vs. sal)

DAT uptake rate &

affinity

(Mash et al., 2002)


dependence;

duration NR

Tox [3H] DA uptake INC DAT uptake rate in STR synap

(coc vs.ctl);

NC in DAT affinity

VMAT2 density &

affinity

(Little et al., 1999)

Hu Chronic coc abuse or

dependence;

duration NR

Tox QAR

([3H]DTBZ)

DEC VMAT2 density in STR (coc vs. ctl);

NC in DAT affinity

VMAT2 density &

affinity (Wilson et

al., 1996)

Hu coc use for 1+ yr Tox memb bind

([3H]DTBZ)

DEC VMAT2 density in Caud & Put,

trend dec in VMAT2 density in NAc;

DEC VMAT2 affinity in Put (coc vs. ctl)

Abbreviations and definitions: W/D, withdrawal

M, mice

R, rats

NHP, nonhuman primates

Hu, humans

COD, cocaine overdose

coc, cocaine group

sal, saline control group

naïve, controls with no drug or saline

ctl, age-matched human controls

inf, infusion

i.v., intravenous

i.p., intraperitoneal

s.c., subcutaneous

30

31

SA, self-administration

YK, yoked, non-contingent i.v. drug

ED, experimenter delivered drug

FR, fixed-ratio

FI, fixed-interval

TO, time out

int, intermittent dose

esc, escalating dose

cont, continuous inf via osmotic pump

ext, extended access

s, seconds

min, minutes

hr, hours

d, days

wk, weeks

mo, months

NR, not reported

Tox, unknown withdrawal length but

positive toxicology exam

memb bind, radioligand binding in

membrane preparations

QAR, quantitative autoradiography on

slide-mounted sections

HPLC, high-performance liquid

chromatographic electrochemical

analyses

RT-PCR, reverse transcription

polymerase chain reaction of

isolated mRNA

DTBZ, dihydrotetrabenazine

INC, significantly increased

DEC, significantly decreased

NC, no significant change

trend dec, strong trending decrease but

not statistically different

hi, high dose

lo, low dose

mid, middle dose

all Tx, all treatments

synap, synaptosomes

LFB, limbic forebrain

FC, frontal cortex

PFC, prefrontal cortex

mPFC, medial PFC

OT, olfactory tubercle

cOT, caudal OT

Hippo, hippocampus

MDB, midbrain

mMDB, medial MDB

lMDB, lateral MDB

VTA, ventral tegmental area

SN, substantia nigra

SNr, SN pars reticulata

SNc, SN pars compacta

STR, striatum

vSTR, ventral STR

dSTR, dorsal STR

rDSTR, rostral dorsal STR

NAc, nucleus accumbens

rNAc, rostral NAc

cNAc, caudal NAc

CPu; caudate putamen (R or M)

VMCPu, ventromedial CPu

VLCPu, ventrolateral CPu

DLCPu, dorsolateral CPu

DMCPu, dorsomedial CPu

cVMCPu, caudal VMCPu

cDLCPu, caudal DLCPu

Caud, caudate (NHP or Hu)

rCaud, rostral Caud

cCaud, caudal Caud

cVCaud, caudal ventral Caud

VMCaud, ventromedial Caud

DMCaud, dorsomedial Caud

DLCaud, dorsolateral Caud

rVMCaud, rostral VMCaud

rDMCaud, rostral DMCaud

rDLCaud, rostral DLCaud

rCCaud, rostral central Caud

cCCaud, caudal central Caud

cVMCaud, caudal VMCaud

cDMCaud, caudal DMCaud

cDLCaud, caudal DLCaud

Put, putamen (NHP or Hu)

rVPut, rostral ventral Put

rDPut, rostral dorsal Put

rDMPut, rostral dorsomedial Put

rCPut, rostral central Put

cVPut, caudal ventral Put

cDPut, caudal dorsal Put

cDMPut, caudal dorsomedial Put

cVMPut, caudal ventromedial Put

cCPut, caudal central Put

31

32

Effects of heroin administration on the mesolimbic dopamine system

Like cocaine and other drugs of abuse, heroin can also affect [DA]e, but via

indirect mechanisms. Heroin was first synthesized in 1874 from morphine, a potent

analgesic used medicinally to treat severe pain, often in chronic or terminal cases.

Morphine is one of the most abundant opiates derived from the opium poppy plant,

Papaver somniferum (Sawynok, 1986; Schultz et al., 1997). Upon entering the body,

heroin is hydrolyzed rapidly into 6-monoacetylmorphine (6-MAM) and then further

hydrolyzed into morphine in the liver. Although heroin and 6-MAM can enter the brain

more readily than morphine (Sawynok, 1986), morphine is considered the active

compound in heroin due to its high affinity for opiate receptors in the brain (Jaffe and

Martin, 1990). Acutely, heroin produces subjective effects including the ability to

overcome inhibitions, relieve tensions and produce an intense euphoric effect, while

long-term heroin use provides relief of negative psychological and physiological

symptoms of opioid dependence (Mirin et al., 1976; Tschacher et al., 2003). The positive

subjective effects of acute heroin use are attributed to its mechanism of action within the

VTA and to less well-described NAc mechanisms.

Morphine binds to mu opioid receptors (MORs) located presynaptically on

GABA-releasing interneurons (Goldstein and Naidu, 1989; Klitenick et al., 1992; Devine

et al., 1993). These GABAergic interneurons are responsible for placing a tonic inhibition

on postsynaptic dopaminergic projection neurons of the mesocorticolimbic pathway.

When morphine binds to MORs, neuronal activity is decreased and the tonic inhibition of

GABA onto DA neurons is reduced. Stimulated release of GABA is also reduced upon

MOR activation (Di Chiara and North, 1992). This in turn increases the firing rate of

33

VTA dopaminergic cells (Johnson and North, 1992; Kiyatkin and Rebec, 1997;

Steffensen et al., 2006). Acute activation of MORs on VTA GABAergic interneurons

results in increased somatodendritic DA release in the VTA (Klitenick et al., 1992) as

well as increased synaptic release of DA, resulting in increased NAc [DA]e (Di Chiara

and Imperato, 1988a; Spanagel et al., 1990; Hemby et al., 1995; Wise et al., 1995a). After

a long period of speculation and studies that localized MORs to the striatum but not to a

specific cell type (Svingos et al., 1997; Ambrose et al., 2004), it has recently been shown

with whole-cell patch clamp electrophysiology of D1- and D2-enhanced green

fluorescent protein (EGFP+) MSNs that there are indeed MORs located on GABAergic

MSNs within the NAc (Ma et al., 2012). Because the NAc also provides long-loop

feedback to the VTA, heroin-induced MOR activation on GABAergic MSNs is also

disinhibitory and likely contributes to the increased firing rate of dopaminergic

projections.

In vivo microdialysis measures of NAc [DA]e following heroin SA have reported

mixed results. Using high unit doses of heroin during i.v. SA in rodents, Wise et al. were

able to obtain significantly elevated accumbal [DA]e levels comparable to those reached

during cocaine SA (Wise et al., 1995a). Moderate increases of baseline [DA]e (about

150%) have also been observed during heroin SA at unit doses that maintain stable

responding in rodents (Wise et al., 1995a; Leri et al., 2003b). However, under our

laboratory conditions, there was no significant difference in NAc [DA]e levels from

baseline following extended SA of heroin in rodents (Hemby et al., 1995; Hemby et al.,

1999; Smith et al., 2006). Conversely, acute experimenter-delivered i.v. administration of

moderate to high doses of heroin did significantly increase NAc [DA]e to about 250%

34

(Hemby et al., 1995). Acute experimenter-delivered s.c. and i.p. heroin has also shown

only modest elevations in NAc [DA]e (50-70%) over a moderate range of doses (Di

Chiara and Imperato, 1988b; Gerasimov and Dewey, 1999). This variance is likely due to

both length of exposure (acute vs. chronic) and contingency of heroin administration

during dialysate perfusion. Conversely, Lecca et al. reported that rodents performing i.v.

heroin SA showed moderately increasing levels of NAc [DA]e elevations over the time

course of four weeks of SA access, while yoked mates receiving non-contingent i.v.

heroin showed a smaller elevation in NAc [DA]e and only on day one of injections

(Lecca et al., 2007). Electrochemical detection via in vivo chronoamperometry

performed in rodents during i.v. heroin SA showed slow but long-lasting elevations in

[DA]e (Kiyatkin et al., 1993; Kiyatkin, 1994). Fewer in vivo voltammetry studies

involving heroin administration have been performed, as compared to cocaine and other

compounds acting directly on DAT. Non-stimulated FSCV in rodents during heroin SA

showed differential biphasic patterns of [DA]e levels in the NAc (Xi et al., 1998). The

current FSCV studies using electrical stimulation on i.v. heroin administration have been

unable to detect elevations in [DA]e, likely due to the high stimulation parameters

necessary to monitor DA release and reuptake events, which may mask a modest increase

in [DA]e due to heroin (Pattison et al., 2011; Pattison et al., 2012). In chronic self-

administering animals, a single bolus i.v. injection of heroin showed no change in

electrically evoked DA but in fact resulted in significantly decreased evoked DA after

one hour (Pattison et al., 2012), which may be explained in part by a reduction in VTA

DA neuron population activity following repeated drug exposure (Shen et al., 2007).

Though in vivo FSCV may not be suitable to confer the observed elevations in NAc

35

[DA]e from microdialysis, because it is a suitable technique to measure cocaine-DAT

interactions, these studies still provide an important framework for examining the

alterations in DAT kinetics following polydrug administration of cocaine-heroin

combinations.

Because heroin SA has shown only moderately increased NAc [DA]e as compared

to cocaine (Hemby et al., 1995; Wise et al., 1995a; Hemby et al., 1997; Leri et al.,

2003b), less work has been done on the effect of heroin on expression of striatal DA

receptors and DAT. One study showed significantly decreased D2R expression in the

NAc of human heroin users (Worsley et al., 2000). Correspondingly, positron emission

tomography (PET) imaging in heroin-dependent subjects has shown decreased striatal

D2/3R availability and attenuated stimulant-induced DA release (Martinez et al., 2012).

Another showed significantly decreased DAT mRNA expression in the VTA and

corresponding decreased DAT protein expression in the NAc, but not dorsal striatum, of

human heroin overdose victims (Horvath et al., 2007). Kish et al. reported increased DAT

affinity and decreased VMAT2 affinity in NAc membranes from human heroin users, but

VMAT2 protein was unchanged, suggesting that the density of DA terminals was not

reduced. They also showed decreased DA and HVA levels in the NAc, and well as

deceased TH protein in the caudate and NAc (Kish et al., 2001). Likewise, in rodent

models of human heroin abuse, heroin SA in rats resulted in decreased TH in the NAc,

but increased TH in the VTA. PKA activity was increased in the NAc of these animals,

while G protein Gi immunoreactivity was decreased, suggesting an upregulation of the

NAc cAMP system (Self et al., 1995). PKA is activated by D1Rs (Monsma et al., 1990)

and is a modulator of DAT functional activity (Gorentla et al., 2009), so it is likely that

36

there are additional unreported alterations of NAc DA receptors as well as DAT resulting

from chronic heroin exposure. Though not as abundantly reported as the effects of

chronic cocaine on the striatal DA system, these results taken together likely represent a

compensatory downregulation in DA neurotransmission in response to prolonged

dopaminergic stimulation by heroin.

Involvement of the dopaminergic system with heroin abuse has also been inferred

from behavioral studies examining the effects of DA receptor antagonists on opioid SA.

D1R antagonist SCH 23390 has been shown to attenuate heroin SA and reinstatement in

rodents (Gerrits et al., 1994; Bossert et al., 2009; Tobin et al., 2013). Equally, D1R

agonists SKF 82958 and SKF 81297 increased heroin SA in rhesus monkeys, but D2R

agonists quinpirole and NPA decreased heroin SA at certain doses (Rowlett et al., 2007).

D2R partial agonist terguride, thought to act as a functional antagonist, dose-dependently

decreased heroin SA under fixed-ratio (FR) and progressive-ratio (PR) schedules in

rodents (Zhang et al., 2010). Another DA receptor antagonist levo-tetrahydropalmatine,

thought to have greater affinity for D1Rs but also for D2/3Rs, also reduced heroin SA

and reinstatement in rodents (Yue et al., 2012). Though these compounds are not as

efficacious as the currently prescribed synthetic opioid treatments for heroin abuse and

dependence, because many other drugs of abuse do directly affect the DA system, such

reports will provide invaluable support for developing improved treatments for

polysubstance abusers that co-abuse heroin.

37

Effects of speedball administration on the mesolimbic dopamine system

It is proposed that the combined mechanisms of heroin-induced increases in VTA

DA cell firing caused by disinhibition of GABA interneurons, along with cocaine-

induced DAT blockade at the presynaptic dopaminergic terminals in the NAc, results in a

supra-additive increase in synaptic and extrasynaptic DA levels in the NAc (Hemby et

al., 1999). Garrido et al. have utilized nuclear magnetic resonance spectroscopy to

suggest a pharmacological alternative of the speedball combination, showing unique

interactions of the compounds that are suggestive of a cocaine-morphine chemical adduct

in solution and solid states (Garrido et al., 2007). Another study that supports formation

of a cocaine-heroin chemical adduct reported decreased viability in cells treated with a

simultaneous speedball mixture as compared to treatment with heroin and subsequently

with cocaine, showing increased [Ca2+

]i and mitochondrial dysfunction and other shifts in

cell death mechanisms from apoptosis toward necrosis (Cunha-Oliveira et al., 2010).

Whether a novel compound is formed by cocaine-heroin/morphine chemical interactions

is yet to be fully determined, but this is only a minor constituent to the profound

behavioral and neurochemical effects of speedball administration demonstrated in

animals and humans alike.

Behavioral evaluations of speedball administration have been fairly widely

reported and, using differential modifications of SA paradigms and lower doses that are

usually non-reinforcing alone, these studies consistently show that speedball

combinations are more reinforcing in mice, rats and nonhuman primates. Speedball SA

compared to either cocaine or heroin alone has demonstrated significant alterations in:

increased FR responses and shifts in dose-response curves (David et al., 2001) in mice;

38

increased responses in PR (Duvauchelle et al., 1998; Ward et al., 2005) and drug choice

(Ward et al., 2005) paradigms, SA escalation (Cruz et al., 2011) and upward shifts in FR-

10 dose response curves (Smith et al., 2006) in rats; and increased discriminative

stimulus effects (Negus et al., 1998; Platt et al., 2010) and increased chained FR

responses (Mattox et al., 1997) in nonhuman primates. Additionally, a clinical study in

methadone-maintained, i.v. cocaine-using humans described increased subjective ratings

including euphoria with methadone and cocaine sequential administration, and

recommended that higher doses of methadone could have a negative impact on cocaine

abuse (Foltin and Fischman, 1992). It is suspected that the incorporation of both

mechanisms of action of cocaine and heroin converging on elevations in NAc [DA]e

trigger the aforementioned behavioral responses. Indeed, several in vivo microdialysis

studies in rats have examined the effects of speedball in the NAc and found that the

combination of cocaine and heroin results in synergistic increases (from 250-1000%) in

[DA]e when acutely administered (s.c. or i.p.) by the experimenter (Zernig et al., 1997;

Gerasimov and Dewey, 1999), and upwards of 1000% increase in baseline concentrations

during SA, even after a prolonged period of training (Hemby et al., 1999; Smith et al.,

2006).

Besides these synergistic increases in NAc [DA]e during speedball SA , very little

has been published on the neurochemical effects of cocaine/heroin combinations on the

DA system. Though in vivo FSCV could not differentiate between cocaine and speedball

electrically evoked DA release in rats with SA history, these data showed a significant

upregulation in basal levels of maximal DAT reuptake rate, or Vmax, following chronic

speedball SA (Pattison et al., 2012). This is likely a compensatory mechanism for DA

39

overflow during repeated SA and may result in reduced basal DA levels in the NAc. It is

currently unknown what the alterations in striatal DA receptor and DAT expression may

be following chronic speedball. Similar to heroin, there have been some behavioral

experiments performed by several groups to investigate the contribution of DA and

opioid receptor systems on speedball SA behaviors in rodents and nonhuman primates;

these results are summarized in Table 2. Overall, these data confirm involvement of

D1Rs (Cornish et al., 2005), D2Rs (Hemby et al., 1996; Cornish et al., 2005), MORs

(Mello and Negus, 1998, 2001; Cornish et al., 2005; Mello and Negus, 2007; Martin et

al., 2008) and most likely decreased basal DA levels (Mello and Negus, 2001, 2007).

The body of work presented here will focus on the alterations in DAT kinetic and

binding parameters following chronic cocaine, heroin and speedball SA in rodents. DAT

is the primary objective of investigation as it is the target of cocaine’s mechanism of

action and sole transporter responsible for the clearance of extracellular DA in the NAc.

Due to the proposed mechanism of action of speedball and observed increases in NAc

[DA]e, it follows that the speedball combination must have unreported deleterious effects

on DAT function. There are currently no approved pharmacotherapies to aid cocaine

addicts in the prevention of relapse and because many methadone-maintained patients

still report co-abuse of cocaine, the DAT also presents itself as an option for novel

relapse-preventing therapy development. Perhaps not even as a direct pharmacotherapy

target, but if hypo-dopaminergic states result from lasting alterations in DAT function,

these must be determined in order to effectively approach treatment for the

cocaine/heroin polysubstance abusers.

40

Table 2: Compounds used to elucidate mechanisms of cocaine/heroin combinations

Compound Treatment Species Cocaine/Heroin SA Paradigm General Effect Reference

Single Compounds

Naltrexone

(opioid R antag)

1.0 - 10 mg/kg i.p.;

10 min prior

R FR10 SA: 125, 250, 500 µg/inf coc

+ 5.4 or 18 µg/inf her*

INC SA (Hemby et al.,

1996)

Eticlopride

(D2R antag)

0.03 – 0.3 mg/kg i.p.;

30 min prior

R FR10 SA: 125, 250, 500 µg/inf coc

+ 5.4 or 18 µg/inf her*

INC SA (Hemby et al.,

1996)

Β-FNA (MOR

alkylating agent)

2.5 nmol i.c. VP &

NAc; 1 nmol i.c.

VTA; 24 hr prior

R FR10 SA: 167/4.5, 83.5/2.3,

41.7/1.1 µg/inf coc/her*

INC SA (VP &

VTA only)

(Martin et al.,

2008)

Β-FNA (MOR

alkylating agent)

2.5 nmol i.c. VP &

NAc; 1 nmol i.c.

VTA; 24 hr prior

R FR10 SA: 167/4.5, 83.5/2.3,

41.7/1.1 µg/inf coc/her*

INC SA (VP &

VTA only)

(Martin et al.,

2008)

Lofexidine (α-2

adrenoreceptor

agonist)

0.1 – 0.2 mg/kg i.p.;

1 hr prior, everyday

R FR1 TO20 s: 250/25 µg/kg/inf

coc/her; extinction; tested stress- &

cue-induced reinstatement

DEC stress-

induced

reinstatement

(Highfield et al.,

2001)

SCH 23390 (D1R

antag)

1 & 6 nmol/side i.c.

NAc; 2 min prior

R PR: 600/4.5 µg/kg/inf coc/her

DEC BP (Cornish et al.,

2005)

Raclopride (D2R

antag)

3 & 10 nmol/side i.c.

NAc; 2 min prior



2005)

40

41


CTOP (MOR

antag)

0.1 – 1.0 nmol/side

i.c. NAc; 2 min prior



2005)

Naltrindole (DOR

antag)

1.0 – 10 nmol/side i.c.

NAc; 2 min prior


NC (Cornish et al.,

2005)

Naltrexone (opioid

R antag)

6.4 – 50 µg/kg i.m.;

10 min prior

NHP PR: 0.4 - 50 µg/kg/inf her + highest

dose coc to not maintain SA

DEC BP at peak

of DRC

(Rowlett et al.,

1998)

Quadazocine

(opioid R antag)

0.1 mg/kg i.m. NHP FR4(VR 16:S): 1 – 100 µg/kg/inf

coc + 0.1 – 32 µg/kg/inf her

downward shift

in DRC

(Mello et al.,

1995)

Buprenorphine

(MOR agonist)

0.075 – 0.75

mg/kg/day i.v.

NHP FR4(VR 16:S): 1 – 100 µg/kg/inf

coc + 0.1 – 10 µg/kg/inf her

downward shift

in DRC

(Mello and

Negus, 1998)

Multiple Compounds

SCH 23390 (D1R

antag) + CTOP

(MOR antag)

1 nmol SCH 23390 +

0.1 nmol CTOP per

side; NAc; 2 min prior

R PR: 600/4.5 µg/kg/inf coc/her DEC BP (Cornish et al.,

2005)

Flupenthixol (DA

antag) +

Quadazocine

(opioid R antag)

0.01/0.1 & 0.032/0.32

mg/kg Flu/Quad; Flu

3 hr prior, Quad 30

min prior

NHP FR30: 10:1 coc:her

(130 – 1300/1.3 - 130 µg/kg/inf

coc/her)

DEC DRC; DEC

FR response

rates

(Negus et al.,

1998)

41

42


Indatraline (DAT

inhibitor) +

Buprenorphine

(MOR agonist)

0.56 mg/kg/day Int +

0.18 mg/kg/day Bup;

10 d

NHP FR4(VR 16:S): 1 - 10 µg/kg/inf coc

+ 0.32 – 3.2 µg/kg/inf her

DEC inf &

response rates;

downward shift

in DRC

(Mello and

Negus, 2001)

d-Amphetamine

(DA releaser) +

Buprenorphine

(MOR agonist)

0.01 mg/kg/hr Amph

+ 0.237 mg/kg/day

Bup; 10 d

NHP FR2(VR 16:S): 1 - 10 µg/kg/inf coc

+ 0.32 – 3.2 µg/kg/inf her

Downward &

rightward shift in

DRC

(Mello and

Negus, 2007)

*doses for 300 g rat

Abbreviations:

R, rat

NHP, nonhuman primate

antag, antagonist

D1R, dopamine D1 receptor

D2R, dopamine D2 receptor

DAT, dopamine transporter

MOR, mu opioid receptor

DOR, delta opioid receptor

her, heroin

coc, cocaine

NAc, nucleus accumbens

VTA, ventral tegmental area

VP, ventral Pallidum

SA, self-administration

i.v., intravenous

i.c., intracranial

i.p., intraperitoneal

inf, infusion

FR, fixed-ratio

VR, variable ratio

PR, progressive-ratio

BP, breakpoint

DRC, dose response curve

INC, significantly increased

DEC, significantly decreased

NC, no change

42

43

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61

CHAPTER II

SPEEDBALL-INDUCED CHANGES IN ELECTRICALLY STIMULATED

DOPAMINE OVERFLOW IN RAT NUCLEUS ACCUMBENS

Lindsey P. Pattison, Keith D. Bonin, Scott E. Hemby, Evgeny A. Budygin

The following manuscript was accepted for publication in Neuropharmacology on

September 14, 2010. Stylistic variations are due to the requirements of the journal.

Lindsey P. Pattison, Evgeny A. Budygin and Scott E. Hemby were responsible for the

study concept and design. Lindsey P. Pattison and Evgeny A. Budygin performed the

experiments and completed the data analyses. Lindsey P. Pattison prepared the

manuscript. Keith D. Bonin manufactured essential materials to perform the experiments.

Lindsey P. Pattison, Evgeny A. Budygin and Scott E. Hemby provided critical revisions

of the manuscript. Reprinted with permission from Elsevier Limited.

62

Abstract

Cocaine/heroin combinations (speedball) induce a synergistic elevation in

extracellular dopamine concentrations ([DA]e) in the nucleus accumbens (NAc) that can

explain the increased abuse liability of speedball. To further delineate the mechanism of

this neurochemical synergism, in vivo fast-scan cyclic voltammetry (FSCV) was used to

compare NAc DA release and reuptake kinetic parameters following acute administration

of cocaine, heroin and speedball in drug-naïve rats. These parameters were extracted

from accumbal DA overflow induced by electrical stimulation of the ventral tegmental

area. Evoked DA efflux was increased following both cocaine and speedball delivery,

whereas heroin did not significantly change evoked DA release from baseline. DA efflux

was significantly greater following cocaine compared to speedball. However, DA

transporter (DAT) apparent affinity (Km) values were similarly elevated following

cocaine and speedball administration, but unaffected by heroin. Neither drug induced

substantial changes in the maximal reuptake rate (Vmax). These data, combined with

published microdialysis and electrophysiological results, indicate that the combination of

cocaine-induced competitive inhibition of DAT and the increase in the DA release

elicited by heroin is responsible for the synergistic increase in [DA]e induced by

speedball.

Keywords: cocaine, heroin, speedball, dopamine, uptake, release, voltammetry

63

1. Introduction

The co-use of cocaine and opiates, termed speedball (Leri et al., 2003a), has been

on the rise since the 1970s and represents a growing subpopulation of drug abusers

(Kosten et al., 1987; Greberman and Wada, 1994; Craddock et al., 1997). The prevalence

of cocaine use among heroin addicts ranges from 30% to 80% (Schutz et al., 1994; Leri et

al., 2003a). Negative health and social consequences of such combinations are severe,

particularly when cocaine is used intravenously (Schutz et al., 1994). Specifically, the

likelihood of positive treatment outcomes for opiate addicts decreases severely when high

amounts of cocaine are co-abused (Downey et al., 2000). Several hypotheses have been

proposed to explain the combined use of cocaine and heroin in humans, including an

enhancement of the positive effects of either drug, reduction in the magnitude or duration

of undesired side effects, induction of euphoria beyond what either drug alone provides,

or independent, non-additive effects even though the drugs are administered concurrently

(Kosten et al., 1987; Foltin and Fischman, 1992; Hemby et al., 1996). The hypothesis of

enhanced euphorigenic effects in humans is paralleled in preclinical studies, which

demonstrate that cocaine and heroin potentiate the reinforcing effects of one another in

the self-administration paradigm (Mattox et al., 1997; Rowlett and Woolverton, 1997);

however, the specific biological contributors which mediate the amplified reinforcing

effects of speedball have not been confirmed.

The potentiated euphorigenic effects in humans and the corresponding

enhancement of reinforcing effects in animal models may be based on an augmented

neurochemical response in brain pathways underlying reinforcement processes. The

mesolimbic dopamine (DA) system is recognized as a critical substrate for the reinforcing

64

effects of drugs of abuse. In general, drugs of abuse activate this pathway and stimulate

DA neurotransmission in the nucleus accumbens (NAc) in humans, nonhuman primates

and rodents (Porrino, 1993; Lyons et al., 1996; Volkow et al., 1997), which is an effect

associated with the abuse liability of such substances. Previous studies have shown that

extracellular DA concentrations ([DA]e) in the NAc are significantly elevated (300-400%

baseline) during cocaine self-administration (Pettit and Justice, 1989; Wise et al., 1995b;

Hemby et al., 1997) and moderately increased (120-150% baseline) by heroin self-

administration (Wise et al., 1995a; Leri et al., 2003b) in rats as measured by in vivo

microdialysis (but see (Hemby et al., 1995; Hemby et al., 1999; Smith et al., 2006) where

heroin self-administration did not significantly alter NAc [DA]e). Interestingly, we have

demonstrated that intravenous speedball self-administration resulted in synergistic

elevations in NAc [DA]e (~1000% of baseline) compared with cocaine (400%) or heroin

alone (no change) (Hemby et al., 1999). Subsequent studies examining self-

administration of lower dose combinations, as well as experimenter-administered

speedball combinations, have reported similar synergistic elevations (Zernig et al., 1997;

Hemby et al., 1999; Smith et al., 2006). While in vivo microdialysis studies provide

information on the extent to which NAc [DA]e is increased during speedball self-

administration, the mechanisms by which heroin potentiates the effects of cocaine on

NAc [DA]e are not fully understood.

In vivo voltammetry is an electrochemical technique that is commonly used in

conjunction with in vivo microdialysis to further characterize DA dynamics (Stamford et

al., 1989; Ng et al., 1991; Wu et al., 2001b; Mateo et al., 2004; Budygin et al., 2007;

Oleson et al., 2009a; Oleson et al., 2009b). Voltammetry facilitates analysis of DA uptake

65

kinetics aside from DA release or metabolism contributions (Zimmerman and Wightman,

1991; Wu et al., 2001a). Importantly also, the higher temporal resolution of this

technique allows the time course of DA uptake inhibition to be determined more

precisely (Budygin et al., 2000; Budygin and Jones, 2008). This is principally beneficial

when drugs are administered intravenously and therefore begin to inhibit dopamine

uptake within several seconds (Mateo et al., 2004). In the present study, we utilized in

vivo fast-scan cyclic voltammetry (FSCV) to assess electrically evoked DA release and

DAT kinetic parameters in rat NAc in pursuit of unveiling the mechanisms of

neurochemical synergy of speedball administration.

2. Methods

2.1. Animals

Male Fisher F-344 rats (120-150 days; 270-320 g; Charles River, Wilmington,

MA) were housed in acrylic cages in a temperature-controlled vivarium on a 12 h

reversed light/dark cycle (lights on at 6:00 PM). Rats were group-housed before surgery

and singly housed after catheterization. Food was restricted to maintain starting body

weight and water was available ad libitum, except during experimental sessions which

were conducted during the dark phase. All procedures were performed in accordance

with the National Institutes of Health Guide for the Care and Use of Laboratory Animals

(NIH Publication No. 80-23) revised in 1996.

66

2.2. Intravenous catheterization

Drug-naïve rats (n = 15) were anesthetized with 1.5 g/kg urethane (i.p.) and

implanted with chronic indwelling venous catheters as described previously (Hemby et

al., 1996; Hemby et al., 1997; Hemby et al., 1999). Catheters were inserted into the right

jugular vein, terminating just outside the right atrium and anchored to muscle near the

point of entry into the vein. The distal end of the catheter was attached to a syringe

containing heparinized saline. Immediately following catheterization, while still under

urethane anesthesia, in vivo fast-scan cyclic voltammetry experiments were performed.

2.3. In vivo fast-scan cyclic voltammetry

While under urethane anesthesia, rats were secured in a stereotaxic frame in a flat

skull position. Holes were drilled into the skull above the NAc and ventral tegmental area

(VTA) (AP: +1.3, L: +1.3 and AP: –5.2, L: +1.0 in mm relative to bregma, respectively).

An additional hole was drilled into the skull of the contralateral hemisphere into which an

Ag/AgCl reference electrode was implanted just below the surface of the skull and

connected to a voltammetric amplifier. A carbon fiber microelectrode (approximately 80

to 200 µm in length beyond the glass capillary in which it was contained) was secured to

the stereotaxic frame arm and also connected to the voltage amplifier. The carbon fiber

electrode was placed into the hole above the NAc, and lowered approximately 5 mm

from the surface into the striatum. A bipolar stimulating electrode was connected to a

voltage output box and lowered into the hole above the VTA approximately 7.5 mm.

Voltammetric recordings were made at the recording electrode every 100 ms for a 15 s

67

duration by applying a triangular waveform (-0.4 to +1.2 V, 400 V/s). The biphasic

stimulation applied by the stimulating electrode consisted of 60 rectangular pulses at 60

Hz, 300 µA and was activated at 5 s into each recording. Recorded signals showed an

oxidation peak at +0.6 V and a reduction peak at -0.2 V (vs. Ag/AgCl reference),

ensuring that the released chemical was indeed DA. The depths of the carbon fiber

microelectrode and bipolar stimulating electrode were adjusted from –6.2 to -7.2 and -7.5

to 8.3 mm, respectively, in order to optimize electrically evoked DA release in the NAc

core.

Once evoked DA recordings were optimized, stable readings were collected every

5 min for at least 50 min. When baseline recordings were within 10% of each other for at

least 5 measurements, drug (1 mg/kg cocaine, 0.03 mg/kg heroin or 1 mg/kg cocaine/0.03

mg/kg heroin combination; n = 5 per drug) was injected i.v. over a 6 sec period,

immediately followed by 0.2 ml of heparinized saline i.v. over another 6 sec. The time

when the 12 sec infusion (of drug then saline) ended constituted the beginning of the

experiment at time zero. Stimulated recordings were then made at 1, 5 and 10 min and

thereafter every 10 min up to 120 min following drug injection.

Carbon fiber microelectrodes were post-calibrated in vitro with known

concentrations of DA (2-5 µM). Calibrations were performed in triplicate and the average

value for the current at the peak oxidation potential was used to normalize recorded in

vivo current signals to DA concentration. The kinetic parameters of DAT (DAT apparent

affinity or the Michaelis-Menten constant, Km, and maximal velocity of uptake rate,

Vmax) were calculated using LVIT software (UNC, Chapel Hill, NC). DA reuptake by

68

DAT was assumed to follow Michaelis-Menten kinetics and the change in DA

concentration during and after stimulated release was fit using the equation:

d[DA]/dt = (f)[DA]p – (Vmax/((Km/[DA]) + 1)),

where f is the stimulation frequency (Hz), and [DA]p is the concentration of DA released

per stimulus pulse. Vmax is the Michaelis-Menten parameter for maximal uptake rate of a

first-order enzymatic reaction, such as DA reuptake by DAT. The Michaelis-Menten

constant, or DAT reuptake apparent affinity, Km, is the [DA]e required for DAT-mediated

reuptake rate to reach one half of Vmax. Baseline Km value was previously determined for

DAT in rat brain synaptosomes to be 0.16 µM (Near et al., 1988). The integral form of

this equation was used to model DA response for individual rat at all time points before

and after drug injections (Wu et al., 2001a). Under current condition, there are two

substrates competing for DAT binding, which are cocaine and endogenous DA.

Therefore, Km is actually apparent Km under this circumstance, but for convenience will

be referred to as just Km. The Km was fixed and the other variables were determined,

when pre-drug parameters were calculated. The calculation of Km requires knowledge of

Vmax and [DA]p (Wu et al., 2001a). Therefore, when a drug effect on the DA efflux was

modeled, Km became the main subject of manipulation, whereas Vmax was kept close to

the pre-drug value. Finally, [DA]p was adjusted to obtain a best fit. A detailed description

regarding a model characterizing changes in electrically evoked DA concentrations was

provided in previous publications (Wightman et al., 1988b; Wightman et al., 1988a;

Wightman and Zimmerman, 1990; Wu et al., 2001a; Oleson et al., 2009b).

69

To perform an alternative analysis of DA uptake changes induced by cocaine and

speedball administration, we fit a decaying exponential to the descending part of the

electrically evoked DA efflux. The numbers reported in this study were obtained by

fitting an exponential function of the form

y = yo + Ae-(x-xo)/t

,

to the DA concentration versus time before and 1 min after cocaine and heroin, when the

maximal effects of drugs on DA efflux were observed. Here y represents the DA

concentration, x represents the time, and y0, x0, A, and t are fitting parameters. The

quantity y0 is the background level of the DA concentration in the absence of any

electrical stimulation, x0 is the time at which the DA concentration begins to decline after

an electrical stimulation, A is the amplitude (peak) of the DA concentration at time x0,

and t represents the decay time for the decrease of DA concentration due to the reuptake

mechanism. The portion of the curves that are fit are the points corresponding to about

70% of the peak to where the concentration is less than 5% above the background

concentration value. The fitting was carried out in OriginLab’s OriginPro8 data analysis

and fitting program.

2.4. Data analysis

Statistical analyses were performed using SigmaStat 3.1 software. One-way

repeated-measures analyses of variance (ANOVA) were used to compare effects after

acute i.v. drug administration to pre-drug baseline values for evoked peak DA, DAT

apparent affinity (Km), and maximal uptake rate (Vmax) values within each drug group.

70

These data were also analyzed using two-way ANOVA with drug group (cocaine, heroin

or speedball) and time as the factors. Evoked DA values were analyzed as a percent of

baseline values (baseline defined as the average of five measurements taken at 25, 20, 15,

10 and 5 min before drug injection). All post hoc analyses were performed using

Bonferroni t-tests to compare post-drug injection values to baseline (for one-way

repeated-measures ANOVAs) and for pairwise comparisons between drugs.

3. Results

3.1. Electrically evoked peak DA

The amplitude of current as detected by FSCV in rat NAc markedly increased

after cocaine (1.0 mg/kg, i.v.) and speedball (1.0 mg/kg cocaine + 0.03 mg/kg heroin,

i.v.), but not after heroin (0.03 mg/kg, i.v.) injection (Fig. 1). As determined by one-way

repeated-measures ANOVA, cocaine significantly increased electrically evoked peak DA

values from baseline (p < 0.001) at 1, 5, 10, 20, 30, 40 and 50 min after injection (p <

0.01), and speedball also significantly increased baseline electrically evoked DA levels (p

< 0.001) at 1, 5, 10, 20, and 30 min (p < 0.005) after drug injection, as determined by

Bonferroni post hoc analyses. Acute i.v. heroin did not significantly change evoked DA

from baseline in drug-naïve animals (p = 0.529) at any time after injection. Two-way

ANOVA revealed significant differences in evoked peak DA values between drug groups

(F(2, 204) = 64.24; p < 0.001). Cocaine elicited an overall greater increase (p < 0.001) in

electrically evoked DA levels than speedball in drug-naïve animals, significantly at 1 min

after drug injections (p < 0.05; Fig. 2). Both speedball and cocaine provoked significant

increases in evoked peak DA compared to heroin from the time of injection until 30 and

71

40 min after drug injections, respectively, as determined by post hoc analyses. A

significant group × time interaction was also observed (F(16, 204) = 16.92; p < 0.001).

Figure 1.

Figure 1. Representative traces of electrically evoked DA signals detected by FSCV in

rat NAc before (solid lines) and 1 min after (dashed lines) cocaine (1.0 mg/kg, i.v.),

heroin (0.03 mg/kg, i.v.) and speedball (1.0 mg/kg cocaine + 0.03 mg/kg heroin, i.v.)

injection in drug-naïve animals (upper panel). These signals had an oxidation peak at

+0.6 V and a reduction peak at -0.2 V vs. Ag/AgCl reference, identifying the released

species as DA. Representative color plots topographically depict the voltammetric data

before drug administration, with time on the x-axis, applied scan potential on the y-axis

and background-subtracted faradaic current shown on the z-axis in pseudo-color (lower

panel).

72

Figure 2.

Figure 2. Electrically evoked DA efflux in the NAc of anesthetized drug-naïve rats

following a single i.v. injection of cocaine (1.0 mg/kg), heroin (0.03 mg/kg) and

speedball (1.0 mg/kg cocaine + 0.03 mg/kg heroin). Injections were given at time = 0

min (↑). Evoked DA efflux values are represented as a percent of the pre-drug baseline

DA concentration. All data are means ± SEM of five rats per group. *p < 0.05 for

cocaine versus speedball (two-way ANOVA followed by Bonferroni tests). Values

significantly different from baseline readings and from the heroin group are not indicated

on plots (see 3.1).

73

3.2. DAT apparent affinity

Cocaine induced a significant increase in Km from baseline (p < 0.001) with post

hoc analyses showing a significant effect (p < 0.05) at time points 1, 5, 10, 20, 30, 40, 50,

60 and 70 min following injection. Speedball resulted in a similar increase in Km values

(p < 0.001) after drug injection, with significant increases from baseline at time points

from 1 to 90 min after drug injection (p < 0.05). Heroin had no effect on baseline Km

values (p = 0.841). Two-way ANOVA showed no difference in Km between cocaine and

speedball following drug injection (p = 1.000) (Fig. 3). Both speedball and cocaine

significantly increased Km compared to heroin (F(2, 204) = 277.0; p < 0.001). A significant

group × time interaction was also observed (F(16, 204) = 64.88; p < 0.001).

74

Figure 3.

Figure 3. DAT apparent affinity (Km, or Michaelis-Menten rate constant) in the NAc of

anesthetized drug-naïve rats following a single i.v. injection of cocaine (1.0 mg/kg),

heroin (0.03 mg/kg) and speedball (1.0 mg/kg cocaine + 0.03 mg/kg heroin). Injections

were given at time = 0 min (↑). All data are means ± SEM of five rats per group. Values

significantly different from baseline readings and from the heroin group are not indicated

on plots (see 3.2).

75

3.3. DAT-mediated reuptake rate

No significant changes in Vmax were seen from baseline values after cocaine (p =

0.880). Heroin also had no effect on baseline Vmax values (p = 1.000). Speedball injection

did produce a modest (~ 10%) but significant increase (p < 0.05) in Vmax (Fig. 4). Post

hoc analyses revealed that the increase in Vmax from baseline (1816.4 ± 478.6 nM/s) was

significant at time points from 1 to 100 min (2023.6 ± 732.7 nM/s) (p < 0.005). However,

two-way ANOVA comparing average Vmax values between drugs showed no statistical

difference between the average Vmax following cocaine (1817.4 ± 662.4 nM/s) and

speedball or heroin (F(2, 204) = 3.883; p > 0.05). To avoid any confusion regarding drug-

induced uptake changes, we performed alternative analysis fitting a single exponential

decay function to the descending phase of electrically evoked DA signal. No significant

difference (t = 1.749, df = 8; p = 0.1184, unpaired t test) was revealed between effects of

cocaine and speedball on the decay time (t) for the decrease of DA concentration due to

reuptake (1.28 ± 0.28 vs. 1.85 ± 0.18 s). The same analysis demonstrated a significant

increase in the t after cocaine (0.95 ± 0.13 s for baseline value vs. 1.85 ± 0.18 s after

cocaine, t = 5.621, df = 4; p < 0.005; paired t test) and speedball (0.68 ± 0.10 s for

baseline value vs. 1.28 ± 0.28 s after speedball, t = 3.263, df = 4; p < 0.05; paired t test).

Taken together, these results indicate the marked decrease in the accumbal DA reuptake

following acute cocaine and speedball administration.

76

Figure 4.

Figure 4. DAT-mediated maximal reuptake rate (Vmax) in the NAc of anesthetized drug-

naïve rats following a single i.v. injection of cocaine (1.0 mg/kg), heroin (0.03 mg/kg)

and speedball (1.0 mg/kg cocaine + 0.03 mg/kg heroin). Injections were given at time = 0

min (↑). All data are means ± SEM of five rats per group. Values significantly different

from baseline readings and from the heroin group are not indicated on plots (see 3.3).

77

4. Discussion

The proposed mechanism of speedball’s actions on the mesolimbic DA system is

a result of combinatory effects of cocaine and heroin on NAc [DA]e. Cocaine blocks

DAT on dopaminergic terminals as well as cell bodies to increase extracellular DA

concentrations (Ritz et al., 1987; Koob and Bloom, 1988; Pettit and Justice, 1989;

Volkow et al., 1997). The DA uptake inhibition leads to several different

pharmacological consequences. The inhibition of soma firing rate due to the activation of

DA autoreceptors is one of the well-described outcomes that should diminish basal DA

release in the NAc. However, the DA release induced by electrical stimulation and

measured by FSCV is increased, since cocaine inhibits DA reuptake between multiple

electrical pulses (Mateo et al., 2004; Oleson et al., 2009a; Oleson et al., 2009b). Heroin

binds µ-opioid receptors to inhibit VTA GABAergic interneurons and remove a tonic

inhibition of DA neurons, thus upregulating cell firing rates and consequently DA release

at NAc terminals (Leone et al., 1991; Johnson and North, 1992; Self et al., 1995). We

evaluated the changes in kinetic parameters of DA release and uptake in order to better

understand the neurobiological substrates involved in the potentiating effects of speedball

on NAc [DA]e.

A significant increase in the magnitude of evoked DA release was observed

following both cocaine and speedball injections. The maximal effect of intravenously

administrated drugs on evoked DA release in the NAc was found to occur one minute

after administration, which is consistent with previous findings in our lab (Mateo et al.,

2004; Oleson et al., 2009a; Oleson et al., 2009b) and correlates with the time it takes for

cocaine to reach peak brain levels (Fowler et al., 1998). Kinetic analyses of the

78

electrically evoked DA signals recorded during cocaine and speedball time courses

indicated that both effects are associated with an increase in the apparent Km for DA

uptake. Importantly, it was previously indicated that the cocaine-induced increase in the

electrically evoked DA release measured by FSCV in vivo is preferentially due to the

decrease in DA reuptake (increase in the apparent Km) (Mateo et al., 2004; Oleson et al.,

2009a; Oleson et al., 2009b). Since the Km changes were indistinguishable between

cocaine and speedball groups, the possibility that heroin makes cocaine more efficient in

inhibiting DA uptake when these two drugs are combined can be ruled out. Because Km

was unaffected by heroin, the elevation in Km due to speedball is likely attributable to the

cocaine component alone. Therefore, changes in the affinity of the DAT cannot be

involved in the potentiating effect of speedball on extracellular DA concentrations.

The magnitude of the increase in evoked DA concentrations was significantly

greater following cocaine in comparison with speedball administration. At first glance,

these voltammetric results may appear contradictory to previously published

microdialysis data (Zernig et al., 1997; Hemby et al., 1999; Smith et al., 2006), but the

findings are in fact compatible. Microdialysis provides an assessment of the steady state

extracellular levels of DA in the region of interest, but with relatively poor temporal

resolution (minutes) (Jones et al., 1999). In contrast, FSCV does not allow assessment of

basal DA concentrations, but detects evoked DA concentrations with subsecond

resolution, allowing DA dynamics to be measured in real time (Budygin and Jones,

2008). Therefore, prompt adaptive changes in DA release and uptake can be detected

with this approach. Taking this into account, the attenuated amplification in electrically

evoked DA effluxes observed following speedball administration compared to cocaine

79

alone is likely due to feedback induced by greater accumulation of DA in NAc

extracellular space after the speedball administration.

Several mechanisms can be considered in this regard. Post-synaptic D2 DA

receptors in the NAc provide indirect feedback to DA neurons via long pathways

(Hommer and Bunney, 1980), while pre-synaptic D2 DA receptors on dopaminergic

terminals and somatodendrites function as autoreceptors (Starke et al., 1989; Wolf and

Roth, 1990; Bunney et al., 1991). Pre-synaptic D2 autoreceptors maintain dopaminergic

homeostasis via feedback mechanisms, which include regulation of DA release

(Uchimura et al., 1986; Herdon et al., 1987; Cardozo and Bean, 1995; Wu et al., 2002).

For example, microdialysis assessment of DA in wild type and D2 receptor knockout

mice (D2R-/-

) following acute injections of cocaine or morphine showed that D2R-/-

mice

exhibited a significantly greater increase in [DA]e in the striatum than wild type mice

(Rouge-Pont et al., 2002). In vitro voltammetric assessment found an increase in

electrically evoked DA release when amphetamine was applied in the presence of the D2

antagonist sulpiride compared to amphetamine alone. Moreover, a greater peak DA

amplitude from amphetamine was observed in striatal slices from D2R-/-

mice in

comparison with wild type mice (Schmitz et al., 2002). Even though amphetamine is

known to elicit DA overflow in the NAc (Fischer and Cho, 1979; Raiteri et al., 1979;

Sulzer et al., 1993; Sulzer et al., 1995; Jones et al., 1998), stimulation-dependent DA

release is decreased by amphetamine (Jones et al., 1998), mostly due to release-regulating

D2 autoreceptor activation which inhibits electrically evoked DA release (Schmitz et al.,

2002). Similarly, markedly increased extracellular DA induced by cocaine (~400 %) and

speedball (~1000%) likely activates release-regulating autoreceptors, decreasing

80

electrically evoked DA release. However, post-synaptic D2 receptors which provide

release inhibition via long, indirect feedback loops can be also involved in this effect.

Since the magnitude of NAc [DA]e is greater following speedball compared to cocaine

administration, D2 feedback initiated by DA overflow from cocaine would be of lesser

magnitude compared to speedball. Therefore, electrically evoked DA release detected by

voltammetry is reduced following speedball administration compared to cocaine alone.

In agreement with this speculation, the dose of heroin used in this study did not

significantly affect electrically evoked DA release. As mentioned previously, heroin

administration increases dopaminergic cell firing rates via activation of mu opiate

receptors on VTA GABAergic neurons causing hyperpolarization and removal of tonic

inhibition on dopaminergic cells (Leone et al., 1991; Johnson and North, 1992; Self et al.,

1995). In vivo extracellular electrophysiological recordings have shown that acute heroin

decreases VTA GABAergic cell firing rates (Steffensen et al., 2006) and microdialysis

studies have shown modest increases in NAc [DA]e following heroin administration

(Wise et al., 1995a; Zernig et al., 1997; Leri et al., 2003b). Perhaps a higher dose of

heroin would better facilitate detection of heroin-induced increases in extracellular DA

levels, which would likely be observable as a decrease in electrically evoked DA release.

In agreement with such a suggestion, we observed decreased electrically evoked DA

release following high doses of ethanol (Budygin et al., 2001; Jones et al., 2006), which

reliably increased basal DA levels measured by microdialysis (Di Chiara and Imperato,

1988; Yoshimoto et al., 1992; Weiss et al., 1993; Weiss et al., 1996).

DA D2 receptor activation not only inhibits release but can also upregulate uptake

through DAT (Cass and Gerhardt, 1994; Hoffman and Donovan, 1994; Wu et al., 2002).

81

The attenuated increase in electrically evoked DA efflux observed after speedball

administration could be a consequence of other feedback mechanisms resulting from D2

autoreceptor induced upregulation of DAT. However, no evidence was obtained in our

experiments to support this hypothesis. Indeed, the maximal uptake rate was not notably

increased following cocaine and its combination with heroin.

Although these data revealed significant changes in electrically evoked DA

overflow in response to the enhanced NAc [DA]e elicited by speedball, mechanistically

they cannot explain the potentiation of this drug combination, since the ultimate outcome

of D2 DA autoreceptor-mediated feedback is a decrease in extracellular DA

concentration. At the same time, we have clearly demonstrated that alterations in DAT

affinity cannot contribute to the potentiation of [DA]e observed with speedball.

Therefore, our voltammetric results, when combined with published microdialysis and

electrophysiological data, indicate that the combination of cocaine-induced competitive

inhibition of the DAT (apparent Km increase, but not Vmax changes) and the modest

increase in the DA release elicited by heroin via the enhancement of firing rate, explains

the synergistic increase in extracellular DA concentrations induced by speedball in the

NAc.

Acknowledgements

The research for this study was funded by DA012498 (SEH) and DA021634 (EAB).

82

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87

CHAPTER III

DIFFERENTIAL REGULATION OF ACCUMBAL DOPAMINE

TRANSMISSION IN RATS FOLLOWING COCAINE, HEROIN AND

SPEEDBALL SELF-ADMINISTRATION

Lindsey P. Pattison, Scot McIntosh, Evgeny A. Budygin, Scott E. Hemby

The following manuscript was accepted for publication in Journal of Neurochemistry on

March 19, 2012. Stylistic variations are due to the requirements of the journal. Lindsey P.

Pattison, Evgeny A. Budygin and Scott E. Hemby were responsible for the study concept

and design. Lindsey P. Pattison and Scot McIntosh were responsible for the rodent

behavior. Lindsey P. Pattison performed the in vivo voltammetry experiments. Evgeny E.

Budygin and Lindsey P. Pattison performed the data analyses. Lindsey P. Pattison and

Scott E. Hemby prepared the manuscript. Lindsey P. Pattison, Evgeny A. Budygin and

Scott E. Hemby provided critical revisions of the manuscript. Reprinted with permission

provided by the Copyright Clearance Center, as a License Agreement between Lindsey P.

Pattison and John Wiley and Sons.

88

Abstract

Cocaine/heroin combinations (speedball) exert synergistic neurochemical and

behavioral effects that are thought to contribute to the increased abuse potential and

subjective effects reported by polydrug users. In vivo fast-scan cyclic voltammetry

(FSCV) was used to examine the effects of chronic intravenous self-administration (SA;

25 consecutive sessions) of cocaine (250 µg/inf), heroin (4.95 µg/inf) and speedball

(250/4.95 µg/inf cocaine/heroin) on changes in electrically evoked dopamine (DA)

efflux, maximal rate of DA uptake (Vmax) and the apparent affinity (Km) of the dopamine

transporter (DAT) in the nucleus accumbens (NAc). The increase in electrically evoked

DA was comparable following cocaine and speedball injection; however, heroin did not

increase evoked DA. DAT Km values were similarly elevated following cocaine and

speedball, but unaffected by heroin. However, speedball self-administration significantly

increased baseline Vmax, while heroin and cocaine did not change baseline Vmax,

compared to the baseline Vmax values of drug-naïve animals. Overall, elevated DA

clearance is likely the consequence of synergistic elevations of NAc extracellular DA

concentrations by chronic speedball SA, as reported previously in microdialysis studies.

The present results indicate neuroadaptive processes that are unique to cocaine/heroin

combinations and cannot be readily explained by simple additivity of changes observed

with cocaine and heroin alone.

Keywords: cocaine, dopamine, heroin, speedball, self-administration, voltammetry

89

Introduction

Polysubstance abuse remains an issue of rising importance when treating addicts,

especially the increasing population who co-abuse stimulants and opioids. The combined

use of cocaine and opiates, termed “speedball” (Leri et al., 2003), represents a growing

subpopulation of drug abusers (Kosten et al., 1987; Greberman and Wada, 1994;

Craddock et al., 1997). The prevalence of cocaine use among heroin addicts ranges from

30% to 80% (Schutz et al., 1994; Leri et al., 2003). In 2009, the most frequently reported

drug combination among European patients entering treatment was heroin and cocaine

(Addiction, 2009). A controlled clinical study indicated that the co-administration of

cocaine and morphine produced subjective effects unique to the combination and distinct

from the effects of either drug alone (Foltin and Fischman, 1992). Preclinical studies

support the concept of pharmacological uniqueness of the combination (Garrido et al.,

2007), suggesting that current pharmacotherapeutic treatment approaches for cocaine or

heroin abuse may not be as effective for individuals abusing cocaine/heroin

combinations. Consequently, a detailed understanding of speedball-induced

neurochemical changes in the brain is a prerequisite to discovering effective treatments.

Dopamine (DA) neurotransmission in the nucleus accumbens (NAc) plays an

important role in the neuropharmacological effects of drugs of abuse. The manner in

which cocaine and opiates impact the mesolimbic DA system is of particular interest,

when considering the unique neurochemical profiles reported with speedball

combinations. Numerous studies using rodent models of self-administration (SA) and

experimenter-administered delivery in combination with in vivo microdialysis have

shown that speedball induces a synergistic elevation in extracellular DA concentrations

90

([DA]e) in the NAc compared to cocaine or heroin alone (Zernig et al., 1997; Hemby et

al., 1999; Smith et al., 2006). In order to further evaluate the contribution of DAT with

regard to the neurochemical effects of speedball administration, in vivo fast-scan cyclic

voltammetry (FSCV) can be used to examine DAT reuptake kinetics following drug

administration. To this end, analysis of acute speedball effects on DA transmission in the

NAc of drug-naïve rats revealed that DAT apparent affinity values were similarly

elevated following cocaine and speedball intravenous administration, but unaffected by

heroin. Furthermore, neither cocaine, heroin nor speedball induced significant changes in

the maximal reuptake rate (Vmax) when administered acutely (Pattison et al., 2011).

Unlike a single injection, chronic cocaine administration, including drug SA

procedures, significantly affects baseline DA uptake parameters (Oleson et al., 2009).

Enhanced DA uptake rate, as measured by Vmax, has been reported in the rat NAc after a

history of cocaine exposure (Oleson et al., 2009; Addy et al., 2010) and binge (Mateo et

al., 2005) cocaine SA. Moreover, the ability of cocaine to inhibit DA uptake, measured

by an increase in apparent Km, was severely attenuated following a cocaine binge (Mateo

et al., 2005). In contrast, apparent Km was potentiated in the NAc following a cocaine

challenge in rats that repeatedly received cocaine injections (15 mg/kg, i.p., seven daily

injections) compared to those with only one pre-exposure (Addy et al., 2010). Alterations

in DA uptake parameters that take place following chronic speedball administration and

how these neuroadaptations influence subsequent acute effects of the drug combination is

unknown. In this in vivo voltammetric study, the effects were examined of intravenously

injected cocaine, heroin and the speedball combination on evoked DA release and DAT-

mediated reuptake parameters in the NAc of rats with a chronic SA history.

91

Materials and Methods

Subjects

Male Fisher F-344 rats (n=15; 120-150 days; 270-320 g; Charles River,

Wilmington, MA) were housed in acrylic cages in a temperature-controlled vivarium on a

12 hour reversed light/dark cycle (lights on at 6:00 PM). Rats were group housed before

surgery and individually housed after catheterization. Food was restricted to maintain

starting body weight and water was available ad libitum, except during experimental

sessions that were conducted during the dark phase. All procedures were performed in

accordance with the National Institutes of Health Guide for the Care and Use of

Laboratory Animals (NIH Publication No. 80-23) revised in 1996.

Drugs

Cocaine hydrochloride and heroin hydrochloride were obtained from the Drug

Supply Program of the National Institute on Drug Abuse. Pentobarbital and sodium

thiopental were purchased from the pharmacy at Wake Forest Baptist Hospital. Sodium

heparin was from Elkin-Sinn (Cherry Hill, NJ), methyl atropine nitrate and urethane were

from Sigma Chemical Co. (St Louis, MO).

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Intravenous self-administration

Animals were pretreated with methyl atropine nitrate (10 mg/kg; i.p.) and

anesthesia was induced by administration of pentobarbital (40 mg/kg; i.p.). While

anesthetized, rats were implanted with chronic indwelling venous catheters as described

previously (Hemby et al., 1995; Hemby et al., 1996; Hemby et al., 1997; Hemby et al.,

1999). Briefly, catheters were inserted into the right jugular vein, terminating just outside

the right atrium and anchored to muscle near the point of entry into the vein. The distal

end of the catheter was guided subcutaneously to exit above the scapulae through a

Teflon shoulder harness. The harness provided a point of attachment for a spring leash

connected to a single channel swivel at the opposing end. The catheter was threaded

through the leash for attachment to a swivel. The fixed end of the swivel was connected

to a syringe (for saline and drug delivery) by polyethylene tubing. Infusions of sodium

thiopental (150 µl; 15 mg/kg; i.v.) were manually administered as needed to assess

catheter patency. Health of the rats was monitored daily by the experimenter and weekly

by institutional veterinarians according to the guidelines issued by the Wake Forest

University Animal Care and Use Committee and the National Institute of Health.

Rats were returned to their home cages and monitored for two to three days before

initiating the intravenous (i.v.) drug SA procedure. Following surgery, rats received

hourly infusions of heparinized 0.9% bacteriostatic saline (1.7 U/ml; 200 µl/hour) using a

computer controlled motor-driven syringe pump in the home cage vivarium. For SA

sessions, rats were transferred to operant conditioning cages (24.5 x 23.5 x 21 cm) that

were enclosed in sound-attenuating chambers and contained a retractable lever positioned

2.5 cm above the floor (requiring approximately 0.25 N to operate), an exhaust fan, an 8

93

ohm speaker, a tone source, a house light and a red stimulus light directly above the

retractable lever. A counterbalanced arm was mounted to the rear corner of the operant

chamber onto which the single channel swivel at the end of the rat’s leash was attached.

A motor-driven 20 ml syringe pump was attached outside of the sound-attenuating

chamber and polyethylene tubing was fed from the drug syringe into the operant chamber

through a small hole in the outer chamber.

Rats were assigned randomly into groups to self-administer cocaine (n = 5; 250

µg/inf), heroin (n = 5; 4.95 µg/inf) or speedball (n = 5; 250/4.95 µg/inf cocaine/heroin).

Responding was engendered under a fixed-ratio (FR) 1 schedule and the daily session

terminated after 15 infusions or 3 hours. Once rats consistently self-administered 15

infusions per session, the schedule was increased to FR2 for 5 training days, followed by

25 consecutive days of 15 infusions per day where stable responding was maintained (as

monitored by average inter-infusion intervals within 10%). Under these experimental

parameters, rats self-administered 3.75 mg/day of cocaine, 74.25 µg/day of heroin or 3.75

mg/day of cocaine and 74.25 µg/day of heroin (speedball).

In vivo fast-scan cyclic voltammetry

Approximately 24 hours after the last SA session, rats were anesthetized with

urethane (1.2-1.5 g/kg, i.p.) and secured in a stereotaxic frame in a flat skull position.

Trephinations were made above the NAc and ventral tegmental area (VTA) (AP: +1.3,

ML: ±1.3, and AP: –5.2, ML: ±1.0, in mm relative to bregma, respectively). An

additional trephination was made in the contralateral hemisphere into which an Ag/AgCl

94

reference electrode was implanted just below the surface of the skull and connected to a

voltammetric amplifier. A carbon fiber microelectrode (approximately 80 to 200 µm in

length beyond the glass capillary in which it was contained) was secured to the

stereotaxic frame arm and connected to the voltage amplifier. The carbon fiber electrode

was secured above the NAc trephination and lowered approximately 5 mm below the

dura matter and was lowered further by 0.2 mm increments. A bipolar stimulating

electrode was connected to a voltage output box, secured above the VTA trephination and

lowered approximately 7.5 mm below dura terminating within the medial forebrain

bundle. Voltammetric recordings were collected every 100 ms over a 15 second duration

by applying a triangular waveform (-0.4 to +1.2 V, 400 V/s). The biphasic stimulation

applied by the stimulating electrode consisted of 60 rectangular pulses at 60 Hz, 300 µA

and was activated at 5 seconds into each recording. Recorded signals exhibited an

oxidation peak at +0.6 V and a reduction peak at -0.2 V (vs. Ag/AgCl reference), the

signature cyclic voltammogram indicative of DA. The average DAT uptake rate (Vmax)

was between 1.5 and 2.5 µM/s, indicative of electrode placements in the NAc. The depths

of the carbon fiber microelectrode and bipolar stimulating electrode were adjusted from –

6.2 to -7.2 and -7.5 to 8.3 mm, respectively, in order to optimize electrically evoked DA

release in the NAc.

Once evoked DA recordings were optimized, stable readings were collected every

5 minutes for at least 50 minutes. When baseline recordings were within 10% of each

other for at least 5 measurements, drug (1.0 mg/kg cocaine, 0.03 mg/kg heroin or 1.0/0.03

mg/kg cocaine/heroin) that was previously self-administered was infused through the

previously implanted intravenous catheter over a six second period, immediately

95

followed by an infusion of 0.2 ml of heparinized saline over an additional six seconds.

The conclusion of the infusion constituted the beginning of the experiment at time zero.

Stimulated recordings were then made at 1, 5 and 10 minutes and thereafter every 10

minutes up to 120 minutes following drug injection.

Carbon fiber microelectrodes were post-calibrated in vitro with known

concentrations of DA (2-5 µM). Calibrations were performed in triplicate and the average

value for the current at the peak oxidation potential was used to normalize recorded in

vivo current signals to DA concentration. The kinetic parameters of DAT (DAT apparent

affinity or the Michaelis-Menten constant, Km, and maximal velocity of dopamine

reuptake, Vmax) were calculated using LVIT software (UNC, Chapel Hill, NC). DA

reuptake by DAT was assumed to follow Michaelis-Menten kinetics and the change in

DA concentration ([DA]) during and after stimulated release was fit using the equation:

d[DA]/dt = (f)[DA]p – (Vmax/((Km/[DA]) + 1))

where f is the stimulation frequency (Hz), and [DA]p is the concentration of DA released

per stimulus pulse. Vmax is the Michaelis-Menten parameter for maximal uptake rate of a

first-order enzymatic reaction, such as DA reuptake by DAT. Km is defined as the

dopamine concentration required for DAT-mediated reuptake rate to reach one half of

Vmax. The baseline Km value for DAT was reported previously to be 0.16 µM (Near et al.,

1988). The integral form of this equation was used to model the DA response for

individual rats at all time points before and after drug injections (Wu et al., 2001). Under

the current conditions, there are two substrates competing for DAT binding, which are

cocaine and endogenous DA. Therefore, Km is actually apparent Km under this

96

circumstance, but for convenience will be referred to as just Km. A detailed description

regarding a model characterizing changes in electrically evoked DA concentrations was

provided in previous publications (Wightman et al., 1988; Wightman and Zimmerman,

1990; Wu et al., 2001; Oleson et al., 2009).

Data analysis

One-way repeated-measures analyses of variance (ANOVAs) were used to

compare effects after acute i.v. drug administration to pre-drug baseline values for

evoked DA efflux, DAT apparent affinity (Km), and maximal velocity of dopamine

uptake (Vmax) values within each drug group. These data were also analyzed using two-

way ANOVA with drug group (cocaine, heroin or speedball) and time as the factors.

Evoked DA values were analyzed as a percent of baseline values (baseline defined as the

average of five measurements taken at 25, 20, 15, 10 and 5 minutes prior to drug

injection). All post hoc analyses were performed using Bonferroni t-tests to compare

post-drug injection values to baseline (for one-way repeated-measures ANOVAs) and for

pairwise comparisons between drugs.

97

Results

Self-administration

All animals self-administered cocaine (250 µg/infusion), heroin (4.95

µg/infusion) or speedball (250/4.95 µg/infusion cocaine/heroin) under an FR2 for 25

consecutive days. Overall, animals had as close to the same drug SA history as possible,

and no group had a total number of SA days that was significantly more than another

group. The only slight differences are that heroin self-administering animals required on

average three fewer training days under an FR1 and showed shorter interinfusion

intervals throughout the 25 consecutive days of SA under an FR2 (Fig. 1). Those rats

self-administering heroin obtained significantly more infusions (P < 0.05) than speedball

rats on days 1 and 2, and on days 1 through 4 compared to cocaine rats (Fig. 1a). One-

way ANOVA revealed that animals self-administering heroin also showed an overall

significantly shorter average interinfusion interval (2.72 ± 0.34 min) as compared to

cocaine (3.66 ± 0.092 min) or speedball (4.25 ± 0.17 min) self-administering animals (P

< 0.05) over the 25 day period (Fig. 1b). However, these variables do not contribute to

the overall amount of drug intake and enforcing a threshold of 15 infusions per session

ensured that speedball animals did not have an overall different amount of cocaine intake

than those self-administering cocaine alone, and also did not have an overall difference in

heroin intake than those self-administering heroin alone.

98

Figure 1A.

Figure 1. (A) Acquisition of SA during the first 14 days of training rats under an FR1 to

self-administer cocaine (250 µg/infusion), heroin (4.95 µg/infusion) or speedball

(250/4.95 µg/infusion cocaine/heroin). Once rats reached the maximum number of

infusions (15) for two days in a row, they began to self-administer under an FR2 for five

training days and continued to self-administer for 20 consecutive days thereafter until in

vivo FSCV was performed 24 hours following the last SA sessions. Data are mean

number of infusions for each group (n = 5) ± SEM. Rats trained to self-administer heroin

responded for more infusions on days 1 and 2 compared to speedball rats, and on days 1

through 4 compared to cocaine rats (*P < 0.05).

99

Figure 1B.

Figure 1. (B) Mean interinfusion intervals for each group (n = 5) ± SEM throughout

daily SA sessions over the 25 day period of SA under an FR2. Animals self-

administering heroin showed an overall significantly shorter average interinfusion

interval as compared to cocaine or speedball (P < 0.05) over the 25-day period. However,

enforcing a threshold of 15 infusions per session ensured that speedball animals did not

have an overall different amount of cocaine intake than those self-administering cocaine

alone, and also did not have an overall difference in heroin intake than those self-

administering heroin alone.

100

Electrically evoked DA efflux

Baseline values of electrically evoked DA efflux, ranging from 0.85-2.20 μM

(Fig. 2), were not statistically different between the SA groups (P = 0.343). Cocaine,

heroin and speedball intravenous infusions significantly altered the levels of electrically

evoked DA in the NAc during FSCV (Fig. 3). Cocaine and speedball infusions resulted in

significant increases in evoked DA compared to baseline values (P < 0.001); however,

there was no significant difference between the cocaine and speedball groups (P = 0.291).

Post hoc analyses revealed significant elevations in evoked DA efflux following cocaine

administration at 1, 5, 10, and 20 minutes, and at 1, 5, and 10 minutes following

speedball administration (P < 0.05). In contrast, heroin administration resulted in a

significant decrease in evoked DA efflux compared to baseline (P < 0.001) and compared

to cocaine and speedball infusions (P < 0.001). Post hoc analyses identified significant

reductions from baseline at 60, 70, 80, 90, 100, 110, and 120 minutes after the heroin

infusion (P < 0.05). A possible explanation is that heroin-induced decreases in VTA

GABAergic cell firing rate (Steffensen et al. 2006) may disinhibit dopaminergic

projections to the NAc, resulting in increased basal DA release. An increase in

extracellular DA concentration in the absence of DAT inhibition should result in a

decrease in electrically evoked DA efflux (Budygin et al. 2001). However, this

mechanism is not in agreement with the striking delay of the effect induced by

intravenously injected drug. Therefore, the changes in electrically evoked DA efflux at

one hour after heroin administration are more likely due to secondary alterations. For

example, they could be associated with a higher susceptibility of DA storage to depletion

following heroin SA.

101

Figure 2.

Figure 2. DA changes detected by FSCV in the NAc of anesthetized rats following i.v.

cocaine, speedball and heroin infusion. Upper panel: Background-subtracted cyclic

voltammograms taken from the peak response to the stimulation, which provide chemical

information on the analyte. Every signal has an oxidation peak at +0.65 V and reduction

peak at -0.2 V vs. Ag/AgCl reference, identifying the detected species as DA. Lower

panel: Representative concentration-time plots of electrically evoked DA release

measured before (solid lines) and 10 min after (dashed lines) drug challenges.

102

Figure 3.

Figure 3. The influence of chronic SA on electrically evoked DA release in NAc

following single i.v. challenge infusions of cocaine (1.0 mg/kg), heroin (0.03 mg/kg) and

speedball (1.0/0.03 mg/kg cocaine/heroin). Data are expressed as percent of pre-drug

baseline DA levels and are represented as mean ± SEM (n = 5/group). Baseline values

were not significantly different between groups. Cocaine and speedball infusions

significantly increased evoked DA levels above baseline levels from 1-20 minutes and 1-

10 minutes following drug injection, respectively (*P < 0.05). Heroin administration

significantly reduced evoked DA levels below baseline levels from 60-120 minutes

following injection (‡P < 0.05). No statistically significant difference in peak DA levels

between the cocaine and speedball groups were observed.

103

DAT apparent affinity (Km)

Cocaine and speedball administration significantly increased DAT apparent

affinity (Km), whereas heroin exerted no effect (P < 0.001; Fig. 4). There was no

significant difference in apparent Km between cocaine and speedball (P = 1.000) but Km

values were found to be significantly lower following heroin infusion compared to

speedball or cocaine throughout the experiment (P < 0.001) following drug injections.

Post hoc analyses revealed that cocaine induced a significant increase in Km from

baseline from 1 to 90 minutes (P < 0.05) and speedball injection resulted in a significant

increase in Km from baseline from 1 to 80 minutes (P < 0.05) following drug infusion.

Heroin did not alter Km from baseline values (P = 0.994).

DAT-mediated reuptake rate (Vmax)

No significant differences in Vmax values were observed between baseline and the

post-infusion periods within the cocaine, heroin or speedball groups (Fig. 5a).

Comparison of baseline Vmax values revealed significant differences between the three

groups (P < 0.005). Post hoc analyses revealed elevated baseline Vmax values for the

speedball group (2456 ± 336 nM/s) compared to heroin (1440 ± 80 nM/s; P < 0.05), but

not cocaine (1802 ± 100 nM/s). Significant differences in Vmax were also observed

between groups following drug infusion. Vmax values were significantly greater in the

speedball group compared to the cocaine or heroin groups (P < 0.001) and also greater in

the cocaine group when compared to heroin (P < 0.05).

104

In order to assess the effect of chronic drug exposure on Vmax, we compared the

baseline Vmax values from drug-naïve subjects (Pattison et al., 2011) with values from

subjects following chronic SA of cocaine, heroin or speedball. The analysis revealed

significant increases in baseline Vmax values in subjects with a history of chronic

speedball SA compared to drug-naïve subjects (P < 0.005; Fig. 5b). Interestingly, no

significant differences were observed in baseline Vmax between the drug-naïve and

cocaine or heroin self-administering groups.

105

Figure 4.

Figure 4. Effects of i.v. challenge infusions of cocaine (1.0 mg/kg), heroin (0.03 mg/kg)

and speedball (1.0/0.03 mg/kg cocaine/heroin) on the inhibition of DA uptake, or

apparent Km, in NAc following chronic SA. Data are expressed as nM concentrations and

represented as mean ± SEM (n = 5/group). Baseline values were not significantly

different between groups. Cocaine and speedball increased DAT inhibition above

baseline levels in a time-dependent manner for 90 and 80 minutes following infusions,

respectively (*P < 0.05). As anticipated, heroin did not affect DAT inhibition.

106

Figure 5A.

Figure 5. (A) Effects of i.v. challenge infusions of cocaine (1.0 mg/kg), heroin (0.03

mg/kg) and speedball (1.0/0.03 mg/kg cocaine/heroin) on maximal velocity of dopamine

uptake (Vmax) in NAc following chronic SA. The maximal uptake rate is expressed as

nM/s and represented as mean ± SEM (n = 5/group). Baseline Vmax values were

significantly greater in the speedball group compared to heroin (*P < 0.05). Following

drug infusions, Vmax values were significantly increased after speedball administration

compared to cocaine and heroin alone (‡P < 0.05). Challenge infusions did not

significantly alter Vmax values from baseline values within any of the groups.

107

Figure 5B.

Figure 5. (B) Effects of chronic SA of cocaine (grey bar), speedball (black bar) and

heroin (white bar) on Vmax compared to drug-naïve animals (n = 15). Data represent

average Vmax values (± SEM) calculated at three time points prior to drug infusions (n =

5/group). The solid and dashed lines (1732 ± 87 nM/s) represent the average ± SEM Vmax

for drug-naïve subjects. Chronic SA of speedball significantly increased the baseline Vmax

value in NAc compared to drug-naïve controls (**P < 0.005). Baseline Vmax values were

not significantly different between drug-naïve and self-administering subjects following

cocaine or heroin administration.

108

Discussion

The present study revealed that chronic speedball SA resulted in significantly

elevated rates of DA reuptake (Vmax) in the NAc compared to cocaine and heroin SA.

Furthermore, comparison of the present results with the previously published effects

following acute speedball, cocaine and heroin administration in drug-naïve subjects

(Pattison et al., 2011) revealed significant neuroadaptive responses in DA release and

uptake rate, which are attributable to chronic drug SA. The most substantial observation

between these data sets is that chronic speedball SA significantly increased the baseline

rate of Vmax compared to drug-naïve subjects. In the present study, a significant increase

in the magnitude of electrically evoked DA efflux was observed following both cocaine

and speedball injections in animals with a chronic SA history. Evoked DA efflux was

reduced following chronic SA of cocaine, heroin or speedball compared to acute

administration of each respective drug in naïve animals (Fig. 6a-c), which may be

explained in part by a reduction in VTA DA neuron population activity following

repeated drug exposure (Shen et al., 2007).

In contrast to the differences in evoked DA efflux between acute cocaine and

speedball administration (Pattison et al., 2011), no differences were observed between the

cocaine and speedball groups following chronic SA. The lack of difference may be due to

enhanced rate of DA reuptake following speedball SA, as has been observed following

chronic alcohol and cocaine administration (Budygin et al., 2003; Mateo et al., 2005;

Budygin, 2007; Oleson et al., 2009). In the present study, baseline Vmax was increased in

the speedball group 24 hours following the previous SA session.

109

Fig

ure

6.

110

Figure 6. Upper Panel: Comparison of electrically evoked DA efflux in NAc of drug-

naïve rats (open shapes) and chronically self-administering rats (filled shapes) following

a single challenge i.v. infusion (↑) of (A) cocaine (1.0 mg/kg), (B) speedball (1.0/0.03

mg/kg cocaine/heroin), and (C) heroin (0.03 mg/kg). Data are expressed as percent of

pre-drug baseline DA levels and are represented as mean ± SEM (n = 5/group). (A)

Cocaine increased the level of evoked DA efflux from baseline in both acute and chronic

groups; however, cocaine infusion in the SA group resulted in significantly lower levels

of evoked DA efflux compared to the acute group (P < 0.001) at 1, 5, 10, 20, 30, 60 and

70 minutes following cocaine challenge (*P < 0.05). (B) Speedball increased the level of

evoked peak DA form baseline in both acute and chronic groups, with a significantly

greater increase observed in the acute group (P < 0.010). However, post hoc analyses

showed that this was an overall difference and not significant at any specific time points.

(C) Evoked DA efflux was not significantly altered from baseline following heroin

administration in drug-naïve animals; however, chronic heroin SA significantly decreased

evoked DA compared to the naïve animals (P < 0.001) at 60 to 120 minutes following

heroin challenge (*P < 0.05). Lower Panel: Effects of i.v. challenge infusions of (D)

cocaine (1.0 mg/kg), (E) speedball (1.0/0.03 mg/kg cocaine/heroin) and (F) heroin (0.03

mg/kg) on the inhibition of DA uptake, or apparent Km, in NAc following chronic SA

(filled shapes) and in drug-naïve animals (open shapes). Data are expressed as nM

concentrations and represented as mean ± SEM (n = 5/group). There were no significant

differences in apparent Km between acute and chronic animals within any of the drug

groups. (Data of acute administration has been published previously (Pattison et al.,

2011) and reprinted with permission.)

111

This consequence of chronic speedball SA likely contributes to a lack of observed

difference in evoked DA efflux between the cocaine and speedball groups in the present

study.

The elevated maximal rate of uptake observed in the speedball group is

potentially related to the abundance of DATs in the NAc, alterations in membrane

polarity, and/or changes in mitogen-activated protein (MAP) kinase and/or protein kinase

C signaling cascades (Lin et al., 2003; Moron et al., 2003; Copeland et al., 2005; Zhen et

al., 2005). Increases in DAT availability have been observed previously following

chronic cocaine SA in rats and non-human primates (Tella et al., 1996; Letchworth et al.,

2001), as well as in human post-mortem tissue from cocaine addicts (Little et al., 1993).

Elevated DAT activity would serve to decrease [DA]e below baseline levels (Kahlig and

Galli, 2003) leading to a state of dopaminergic hypofunction during SA withdrawal

periods. Speedball SA may exert similar effects on DAT expression but would be

expected to be greater in magnitude given the substantial elevations in [DA]e compared

with cocaine alone, as observed in previous studies (Zernig et al., 1997; Hemby et al.,

1999; Smith et al., 2006). Further studies are warranted to determine DAT expression in

the NAc following speedball SA.

Alternatively, the rate of DAT reuptake may be influenced in a DAT density-

independent manner. The sodium gradient, which is tightly coupled to membrane

potential, is a critical driving force for DA reuptake by the DAT. Increases in Na+/K

+

pump activity can increase the rate of DA reuptake. In addition, Na+/K

+ ATPase also

activates MAP kinase signaling pathways, which appear to be involved in the regulation

of DAT transport capacity (Moron et al., 2003). DAT uptake rate is also modulated by

112

both protein kinase A and C signaling pathways (Pristupa et al., 1998). Future studies are

necessary to examine the influence of these pathways on the increased rate of DA

reuptake kinetics observed in the speedball SA group. Particular interest should be paid to

examining chronic effects of speedball SA on DAT-mediated Vmax, as there appear to be

no neuroadaptations apparent in the measure of apparent Km between acute and chronic

animals within each drug group (Fig. 6d-f).

Neuroadaptations in DAT function triggered by chronic drug exposure are not

necessarily limited to changes in DAT number, but rather to the functional capacity of the

rate of uptake (Vmax). Studies report that repeated cocaine administration alters the

affinity of DAT (Km) (Mateo et al., 2005; Addy et al., 2010; Ferris et al., 2011). For

example, the voltammetric analysis of DA uptake parameters in brain slices from rats

with binge cocaine SA history and withdrawn for one or seven days have shown that

Vmax was increased while Km (cocaine-induced inhibition of DAT) was diminished

(Mateo et al., 2005). The reduced efficacy of cocaine to inhibit DAT appears to be

independent of the ability of DAT to transport DA and other substrates (Ferris et al.,

2011). In contrast, administration of cocaine for seven days (i.p., 15 mg/kg) revealed

increases in apparent Km of cocaine-exposed animals following one day of withdrawal

(Addy et al., 2010). These data suggest a rapid DAT sensitization in the NAc after a short

withdrawal period. However, Oleson et al. (2009) did not find significant changes in the

efficacy of intravenous cocaine (0.75 mg/kg) to inhibit DAT in rats that exhibited an

escalation in the rate of cocaine intake, 24 hours following the final SA session. Possible

explanations for these discrepancies include differences in experimental preparations (in

vitro versus in vivo), dose and route of drug administration, duration of cocaine exposure

113

and withdrawal, presence of drug during testing and differences in the parameters of

electrical stimulation. In the present study, the main goal was to compare the effects on

accumbal DA kinetics between drug groups (cocaine, heroin and speedball) following

chronic (25 days) SA and acute (24 hours) withdrawal based on the measures of evoked

DA efflux, apparent affinity (Km) and maximal reuptake rate (Vmax), before and after an

i.v. drug challenge.

Previous studies have indicated that cocaine-induced increases in electrically

evoked DA dynamics as measured by in vivo FSCV are primarily due to DA reuptake

inhibition (i.e. increase in the apparent Km) (Mateo et al., 2004; Oleson et al., 2009).

Given that increases in Km and evoked DA release following drug challenges were

indistinguishable between cocaine and speedball groups in the present study, changes in

the apparent affinity of DAT are not likely a neuroadaptive response contributing to the

observed differences in Vmax. This contention is supported by a previous finding

demonstrating that increased inhibition of DAT by chronic cocaine SA is not sufficient to

modify the apparent Km of DAT for cocaine (Oleson et al., 2009). Additionally, no

differences were found in Km values between drug-naïve and SA subjects within drug

treatment groups over the time course of the experiments. However, since several

voltammetric studies have demonstrated a rapid sensitization (Addy et al., 2010) or

desensitization of the DAT (Mateo et al., 2005; Ferris et al., 2011) to repeated cocaine

administration, additional experiments are needed to further delineate the alterations in

Vmax and Km as a function of chronic cocaine and speedball SA.

The present study revealed significant differences in DA release and uptake

kinetics between cocaine, heroin and speedball following chronic SA. Additional efforts

114

utilizing various techniques should be directed toward the significant public health

concern of polysubstance abuse. Results from our lab and others have clearly

demonstrated that the biochemical and behavioral effects of drug combinations cannot be

predicted from individual components alone. The present results in combination with a

previous study (Pattison et al., 2011) indicate neuroadaptive processes unique to

cocaine/heroin combinations that cannot be readily explained by simple additivity of

changes observed with cocaine and heroin alone.

Author contributions

LPP, EAB and SEH were responsible for the study concept and design. LPP and SM

contributed to the acquisition of animal data. LPP and EAB performed the data analysis.

LPP, EAB and SEH interpreted the findings. LPP and SEH drafted the manuscript. LPP,

EAB, SEH provided critical revision of the manuscript for important intellectual content.

All authors critically reviewed content and approved the final version for submission.

Acknowledgements

The study was supported in part by R01DA012498 (SEH) and K08DA021634 (EAB).

115

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(2009) Dopamine uptake changes associated with cocaine self-administration.


Pattison LP, Bonin KD, Hemby SE, Budygin EA (2011) Speedball induced changes in



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118

CHAPTER IV

EFFECTS OF COCAINE, HEROIN AND SPEEDBALL SELF-

ADMINISTRATION ON DOPAMINE TRANSPORTER BINDING SITES

Lindsey P. Pattison, Scot McIntosh, Tammy Sexton, Royuo Xiao, Amanda Grigg,

Steven R. Childers, Scott E. Hemby

The following manuscript is in preparation for publication submission in June 2013.

Lindsey P. Pattison, Scott E. Hemby and Steven R. Childers designed the experiments,

and Lindsey P. Pattison and Scot McIntosh performed the data collection and analyses.

Tammy Sexton, Royuo Xiao and Amanda L. Grigg assisted Lindsey P. Pattison in

performing data collection. Lindsey P. Pattison prepared the manuscript, and Steven R.

Childers and Scott E. Hemby acted in advisory and editorial capacities.

119

Abstract

Concurrent use of cocaine and heroin (speedball) has been shown to exert

synergistic effects on DA neurotransmission in the nucleus accumbens (NAc), as

observed by significant increases in extracellular DA levels ([DA]e) and compensatory

elevations in DAT-mediated baseline maximal reuptake rate (Vmax). Utilizing cocaine-

like ligands, homologous competition binding and quantitative autoradiography were

carried out for DAT in the NAc of rats after chronic self-administration (SA) of cocaine,

heroin and speedball combinations. Saturation binding of iodinated 3β-(4-

iodophenyl)tropan-2β-carboxylic acid methyl ester ([125

I]RTI-55) in rat NAc membranes

resulted in binding curves that were best fit to two-site binding models. This allowed for

calculation of Kd and Bmax values corresponding to high- and low-affinity DAT binding

sites. Chronic speedball SA resulted in significantly higher affinity of the low-affinity

DAT binding site. There were no significant changes in high- or low-affinity [125

I]RTI-55

binding densities, as confirmed by quantitative densitometry of tritiated 3β-(4-

fluorophenyl)tropan-2β-carboxylic acid methyl ester ([3H]CFT) binding in NAc sections.

However, a shift in the ratio of high- and low-affinity DAT binding sites, towards an

increased percent of high-affinity binding sites and concomitant decrease in the percent

of low-affinity binding sites, was observed, even though there was no absolute change in

binding site densities. These modulations in binding site parameters may indicate

physical alterations in DAT binding sites following speedball SA, which contribute to

previously observed increases in baseline Vmax following speedball SA.

Keywords: cocaine, heroin, polydrug, nucleus accumbens, dopamine transporter,

binding sites

120

Introduction

Polysubstance abuse is the more common form of abuse among addicts in the US,

and over the past few decades, clinical and experimental consideration have been given to

a phenomenon in which addicts co-abuse cocaine and opioids (commonly heroin) either

simultaneously or within a short period of time. This form of polysubstance abuse is

referred to as “speedballing” and the cocaine/opioid combination as “speedball” (Hunt et

al., 1984; Leri et al., 2003b). The number of Americans that received treatment for

cocaine abuse in 2010 alone was 699,000 and 417,000 Americans also received treatment

for heroin abuse (SAMHSA, 2011a). Patients entering a substance abuse clinic for

intravenous (i.v.) drug use were surveyed on their patterns of administration and heroin

was reported the most commonly injected drug with cocaine as the second-most injected.

Fifteen percent of patients indicated i.v. injection as the route of administration for their

secondary and tertiary drugs of choice (SAMHSA, 2011b). Compared to heroin alone,

patients with co-dependence on cocaine and opioids have been shown to engage in more

risky sexual behaviors (Hudgins et al., 1995), exhibit antisocial personality disorders

(King et al., 2001), engage in more income-generating crimes (Cross et al., 2001), and

most importantly, have worse treatment outcomes than those dependent on heroin only

(Perez de los Cobos et al., 1997; Preston et al., 1998; DeMaria et al., 2000; Downey et

al., 2000b; Sofuoglu et al., 2003; Tzilos et al., 2009). Describing the neurobiological

correlates that underlie chronic speedball use will aid in developing more efficacious

treatment options for many polysubstance abusers.

The reported subjective effects following speedball use in humans are enhanced

euphoria and decreased aversive side effects of cocaine or heroin alone (Foltin and

121

Fischman, 1992b; Foltin et al., 1995). These accounts have been linked to observations of

significantly increased levels of dopamine (DA) in the nucleus accumbens (NAc) (Wise

and Bozarth, 1987; Pettit and Justice, 1989; Hemby et al., 1999), which lies at the core of

the mesolimbic “reward” pathway. Virtually all drugs of abuse acutely increase DA

neurotransmission in the mesolimbic system (Koob and Volkow, 2010), but the specific

combination of cocaine and heroin seems to have a profound effect on extracellular DA

concentrations ([DA]e). Several in vivo microdialysis studies in rats have reported

significant increases in accumbal [DA]e following speedball as compared to cocaine or

heroin alone, in both experimenter-delivered (250-1000% of baseline) (Zernig et al.,

1997a; Gerasimov and Dewey, 1999) and i.v. self-administration (SA) experiments (800-

1000% of baseline), even after a prolonged period of training (Hemby et al., 1999; Smith

et al., 2006b). The effects of such substantial increases in NAc [DA]e following speedball

exposure have not been widely explored. We have, however, previously reported a

significant increase in baseline levels of maximal reuptake rate (Vmax) for the dopamine

transporter (DAT) using in vivo fast-scan cyclic voltammetry (FSCV) following chronic

i.v. speedball SA in rats (Pattison et al., 2012). Given the increased DA reuptake rate

following chronic speedball, the current study was undertaken in order to determine if

DAT function is altered by possible changes in affinity and/or density.

Several previous studies have reported increased DAT density in the NAc

following cocaine exposure (Letchworth et al., 1997; Little et al., 1999; Letchworth et al.,

2001a; Ben-Shahar et al., 2006; Bailey et al., 2008; Miguens et al., 2008; Beveridge et

al., 2009) [but see (Wilson et al., 1996; Calipari et al., 2012)]. In addition, cocaine

exposure has been shown to increase DAT uptake rate in synaptosomal preparations

122

(Daws et al., 2002; Mash et al., 2002; Samuvel et al., 2008), but divergent results have

been shown with FSCV [increased (Mateo et al., 2005; Oleson et al., 2009b), decreased

(Calipari et al., 2012) and unaltered (Pattison et al., 2012) baseline Vmax has been

observed following cocaine], likely due to variations in cocaine SA paradigms, as shown

with biphasic changes in uptake rate over different durations of SA (Ferris et al., 2011).

Besides elevated density and reuptake rates, DAT function could also be increased by

alterations in ligand binding affinities. Determining how the affinity of cocaine-like

ligands for DAT may be altered following chronic speedball SA as compared to cocaine

or heroin alone will provide molecular insights that may aid in developing novel targets

for polysubstance abuse. We utilized membrane binding with a potent cocaine-like DAT

inhibitor to assess the affinities and densities of high- and low-affinity DAT binding sites,

and subsequently performed quantitative autoradiography with a lower-affinity cocaine-

like DAT inhibitor, in attempt to assess the density of high-affinity DAT binding sites in

the NAc of rats following chronic SA of cocaine, heroin and the speedball combination.

Materials and methods

Subjects

Male Fisher F-344 rats (120-150 days; 270-320 g; Charles River, Wilmington,

MA) were housed in acrylic cages in a temperature-controlled vivarium on a 12 hour

reversed light/dark cycle (lights on at 6:00 PM). Rats were group housed before surgery

and individually housed after catheterization. Food was restricted to maintain starting

body weight and water was available ad libitum, except during experimental sessions that

123

were conducted during the dark phase. All procedures were performed in accordance

with the National Institutes of Health Guide for the Care and Use of Laboratory Animals

(NIH Publication No. 80-23) revised in 1996.

Drugs

Cocaine hydrochloride and heroin hydrochloride were obtained from the Drug

Supply Program of the National Institute on Drug Abuse (Bethesda, MD). Pentobarbital

and sodium thiopental were purchased from the pharmacy at Wake Forest Baptist

Hospital (Winston-Salem, NC). Sodium heparin was from Elkin-Sinn (Cherry Hill, NJ),

methyl atropine nitrate was from Sigma-Aldrich (St Louis, MO) and penicillin G was

purchased from Webster Vet Supply (Devens, MA). Iodinated 3β-(4-iodophenyl)tropan-

2β-carboxylic acid methyl ester ([125

I]RTI-55; 2200 Ci/mmol) and tritiated 3β-(4-

fluorophenyl)tropan-2β-carboxylic acid methyl ester ([3H]WIN 35,428, or [

3H]CFT; 82.0

Ci/mmol) were purchased from PerkinElmer (Waltham, MA). Unlabeled CFT was

purchased from Sigma-Aldrich (St Louis, MO) and unlabeled RTI-55 was supplied by

the National Institute on Drug Abuse Drug Supply Program (Bethesda, MD). WF-23 [2β-

propanoyl-3β-(2-naphthyl) tropane] was previously synthesized and dissolved in

phosphate-buffered saline (Davies et al., 1994). TR tritium-sensitive phosphorimaging

screens were purchased from PerkinElmer (Waltham, MA).

124

Intravenous self-administration

Animals were pretreated with methyl atropine nitrate (10 mg/kg; i.p.) and

anesthesia was induced by administration of pentobarbital (40 mg/kg; i.p.). While

anesthetized, rats were implanted with chronic indwelling venous catheters as described

previously (Hemby et al., 1995; Hemby et al., 1996; Hemby et al., 1997; Hemby et al.,

1999). Briefly, catheters were inserted into the right jugular vein, terminating just outside

the right atrium and anchored to muscle near the point of entry into the vein. The distal

end of the catheter was guided subcutaneously to exit above the scapulae through a

Teflon shoulder harness. The harness provided a point of attachment for a spring leash

connected to a single channel swivel at the opposing end. The catheter was threaded

through the leash for attachment to a swivel. The fixed end of the swivel was connected

to a syringe (for saline and drug delivery) by polyethylene tubing. Infusions of sodium

thiopental (150 µl; 15 mg/kg; i.v.) were manually administered as needed to assess

catheter patency. Rats were administered penicillin G procaine (75,000 units in 0.25 ml,

i.m.) at the termination of catheter implantation. Health of the rats was monitored daily

by the experimenters and weekly by institutional veterinarians according to the guidelines

issued by the Wake Forest University Animal Care and Use Committee and the National

Institute of Health.

Rats were returned to their home cages and monitored for several days before

initiating the i.v. drug SA procedures. Following surgery, rats received hourly infusions

of heparinized 0.9% bacteriostatic saline (1.7 U/ml; 200 µl/hour) using a computer

controlled motor-driven syringe pump in the home cage vivarium. For SA sessions, rats

were transferred to operant conditioning cages (24.5 x 23.5 x 21 cm) that were enclosed

125

in sound-attenuating chambers and contained a retractable lever positioned 2.5 cm above

the floor (requiring approximately 0.25 N to operate), an exhaust fan, an 8 ohm speaker, a

tone source, a house light and a red stimulus light directly above the retractable lever. A

counterbalanced arm was mounted to the rear corner of the operant chamber onto which

the single channel swivel at the end of the rat’s leash was attached. A motor-driven 20 ml

syringe pump was attached outside of the sound-attenuating chamber and polyethylene

tubing was fed from the drug syringe into the operant chamber through a small hole in the

outer chamber.

Rats were assigned randomly into groups (n = 8-10) to self-administer cocaine

(250 µg/inf), heroin (10 µg/inf) or speedball (250/10 µg/inf cocaine/heroin). Responding

was engendered under a fixed-ratio (FR) 1 schedule and the daily session terminated after

25 infusions or 4 hours. Once rats consistently obtained 25 infusions per session, the

schedule was increased to FR2 and then FR5 for several training days, followed by 25

consecutive days of 25 infusions per day where stable responding was maintained under

an FR5 reinforcement schedule (as monitored by average inter-infusion intervals within

10%). Under these experimental parameters, rats self-administered 6.25 mg/day of

cocaine, 250 µg/day of heroin or 6.25 mg/250 µg/day of a cocaine/heroin combination.

Approximately 24 hours following the final SA session, animals were sacrificed by rapid

decapitation and brains were promptly removed and hemisected, then each hemisphere

was flash-frozen in isopentane at -35°C to -50°C and stored at -80°C until further use.

Left and right hemispheres were randomly assigned to DAT radioligand binding

membrane binding assay or quantitative autoradiography in brain sections.

126

DAT radioligand binding in membranes

Brain hemispheres used in membrane binding assays were brought up to -20°C in

a modified cryostat, where the NAc was dissected under a 10X microscope. The entire

NAc from one hemisphere of each animal from approximately bregma +3.00 mm to

bregma +0.45 mm was microdissected and stored in an eppendorf tube at -80°C. NAc

tissue was thawed on ice for several minutes and homogenized in 10 ml ice-cold assay

buffer (10 mM sodium phosphate buffer, pH 7.4, with 0.32 M sucrose). Membrane

proteins were precipitated by high-speed centrifugation (10 min at 48,000 × g) and

resuspended in cold buffer and kept ice-cold during assay preparation. Protein

concentration of membrane tissue from each sample was determined using the

colorimetric bicinchoninic acid (BCA) protein assay reagents (Thermo Scientific) by

absorbance at 562 nm.

To determine Kd and Bmax values of [125

I]RTI-55 binding to DAT, saturation

binding of [125

I]RTI-55 was accomplished using a constant concentration of [125

I]RTI-55

with a range of concentrations of unlabeled RTI-55 (0.001–30 nM) in rat NAc

membranes. Each NAc membrane sample was assayed in triplicate (carried out in ice-

cold assay buffer, total volume 2 ml) using 0.1 nM [125

I]RTI-55 and fourteen increasing

concentrations of unlabeled RTI-55. Assays were incubated at 25°C for 50 min and

reactions were terminated by rapid filtration with 3 × 5 ml of cold 50 mM Tris-HCl, pH

7.4, through Whatman GF/B glass fiber filters pre-soaked in Tris buffer containing 0.1%

bovine serum albumin. Nonspecific binding was determined with 1 µM WF-23 (Davies

et al., 1994). Radioactivity was determined by liquid scintillation spectrometry in filters

127

eluted for 8 hours in 5 ml of Ecolite scintillation fluid. Binding curves were fit to one-site

and two-site models by iterative non-linear curve fitting (Prism).

Quantitative autoradiography in rat brain sections

The opposite brain hemispheres for each subject used in the membrane binding

assay were used in quantitative autoradiography. Tissue blocks were mounted on a chuck

using cryostat mounting medium and brought up to -20°C in a Leica CM3050 S cryostat.

Coronal forebrain sections (20 µm thickness) were thaw-mounted on plain gelatin-coated

microscope slides with six sections per slide. Slide-mounted sections were stored in slide

boxes at -80°C until time of the quantitative autoradiography assay. Preliminary

saturation binding experiments with [3H]CFT in rat striatal membranes (data not shown)

provided high- and low-affinity Kd values for [3H]CFT that were similar to those

previously reported in the literature (Madras et al., 1989a; Pristupa et al., 1994; Katz et

al., 2000). Thus, the concentration of [3H]CFT chosen for quantitative autoradiography

(20 nM) was approximately the same as the affinity of CFT for the DAT high affinity

binding site, and therefore occupied approximately 50% of those sites.

Slide-mounted tissue sections were thawed under a cool stream of air for 2 hours

then pre-incubated in ice-cold assay buffer (50 mM Tris, pH 7.4, containing 120 mM

NaCl and 0.32 M sucrose) for 20 min at 0-4°C. Sections were then incubated for 120 min

with 20 nM [3H]CFT at 0-4°C to determine specific binding (5-7 subjects per SA group).

Nonspecific binding was determined in the presence of 1 µM WF-23 (3 subjects per SA

group). All sections were rinsed twice briefly in fresh, ice-cold Tris buffer (50 mM, pH

128

7.4, containing 120 mM NaCl) and once in ice-cold distilled water. Slides were dried

under a cool stream of air for at least 2 hours before being placed in autoradiography

cassettes against uncoated, tritium-sensitive phosphor storage screens along with

calibrated [3H] autoradiographic standards for 12 days at RT. Screens were scanned using

the Cyclone Storage Phosphor System with OptiQuant software (version 3.10;

PerkinElmer). Images were then imported into and analyzed with ImageJ64 (version

1.45s for Macintosh; National Institutes of Health), by quantitative densitometry. Regions

of interest were comprised of the NAc at two levels: approximately bregma +2.20 mm

(medial NAc) and bregma +1.20 mm (caudal NAc). Optical density values were

converted to pmol/mg (of wet-weight tissue) by reference to the calibrated [3H]

standards. Nonspecific binding was determined by subtraction of each respective group’s

mean of standardized optical densities for the sections incubated with the cocaine analog

WF-23 (n = 3/group).

Data analysis

Saturation binding data were analyzed using GraphPad Prism 4.0 for nonlinear

regression curve fitting to determine the Kd and Bmax from data fit to one- and two-site

binding hyperbolae. Statistical analyses using an F test were performed to determine

whether those data could be best described by a single- or two-site model, and P < 0.05

determined the best-fit model. For two-site modeling results, ratios of high- and low-

affinity binding sites were assessed by defining Bmaxtotal = Bmax1 + Bmax2, and percent

high-affinity sites = (Bmax1 / Bmaxtotal) × 100 and percent low-affinity binding sites =

129

(Bmax2 / Bmaxtotal) × 100. For quantitative autoradiography, densitometry of phosphor

screen images was carried out using NIH ImageJ and intensities of background-

standardized radioactivity in regions of interest (medial and caudal NAc) were used to

calculate differences in DAT binding density between SA groups. Group differences for

both membrane binding and section autoradiography studies were determined using one-

way analysis of variance (ANOVA) with pairwise Bonferroni multiple comparison post-

hoc tests (P < 0.05).

Results

Self-administration

Rats self-administered i.v. cocaine (250 µg/inf), heroin (10 µg/inf) or speedball

(250/10 µg/inf cocaine/heroin) under an FR5 schedule for approximately 25 consecutive

days following roughly two weeks of SA training, first under an FR1 then FR2, for 4

hours per day or until the maximum 25 infusions were acquired. Control animals self-

administered saline under an FR1 schedule for approximately 30 days. Cumulative drug

intake was monitored for each animal throughout the study to ensure that the total intake

of cocaine was within 10% between the cocaine and speedball groups, and likewise for

total heroin intake. To ensure consistent total drug intake levels for speedball SA animals

with their single-drug counterparts, the total number of consecutive SA sessions varied

slightly. This was also due to the length of time needed for animals in different drug

groups to acquire 25 infusions per session while responding under an FR5 schedule of

reinforcement. Rats in the cocaine SA group achieved 25 infusions per session in about

130

one week of training, while heroin and speedball animals required roughly three weeks of

training to reach 25 daily infusions (Fig. 1A). The inter-infusion interval (time between

subsequent infusions) also varied between groups. The average (mean ± SEM) inter-

infusion interval for rats self-administering cocaine was 5.3 ± 0.97 min, heroin was 7.4 ±

0.27 min and speedball was 6.9 ± 0.18 min. Rats self-administering cocaine did so at a

significantly faster rate (interinfusion interval) than speedball or heroin rats (P < 0.001,

Fig. 1B) as determined by Bonferroni’s correction for pairwise comparisons following

one-way repeated measures ANOVA.

131

Figure 1A.

Figure 1. (A) SA behaviors of rats responding under an FR5 for i.v. cocaine (250 µg/inf),

heroin (10 µg/inf) or speedball (250/10 µg/inf cocaine/heroin). Responding was initially

engendered under an FR1 (and remained FR1 for saline controls) then increased to FR2

and FR5 (n = 8/group). The number of days required to reach the maximum number of 25

infusions per day varied between drug groups. On average, rats took about 8 days to

reach the maximum number of infusions per session for cocaine, about 20 days for

speedball and about 25 days for heroin. Saline control rats never averaged more than 10

infusions per 4-hour daily session.

132

Figure 1B.

Figure 1. (B) SA behaviors of rats responding under an FR5 for i.v. cocaine (250 µg/inf),

heroin (10 µg/inf) or speedball (250/10 µg/inf cocaine/heroin). Responding was initially

engendered under an FR1 (and remained FR1 for saline controls) then increased to FR2

and FR5 (n = 8/group). The average (mean ± SEM) interinfusion intervals for rats self-

administering cocaine was faster than speedball or heroin rats (P < 0.001) as determined

by Bonferroni’s correction for pairwise comparisons following one-way repeated

measures ANOVA.

133

DAT membrane binding

Radiolabeled RTI-55 was used for membrane binding studies because its high

specific activity allows for assaying low amounts of tissue, as in a single hemisphere of

NAc membranes. Using this radioligand, membranes from one hemisphere of a single rat

could be used for full biphasic saturation analysis. Both one- and two-site binding fits

were applied to the specific binding data for each subject, and F tests were used to

determine which fit best described the data. In agreement with previous published results

(refs), fits were significantly better to two-site than one-site models; therefore, all groups

were compared using one-way ANOVA tests on two-site binding results. These data

revealed differences in both the high- and low-affinity DAT binding parameters for RTI-

55 between the groups. There were no significant differences in the dissociation constant

of the high-affinity binding site (Kd1) for RTI-55 between the groups, but there was a

trend toward increased Kd1 following chronic cocaine SA (P = 0.08, Fig. 2A). The

dissociation constant of the low-affinity binding site (Kd2) in speedball SA animals (4.99

± 1.3 nM) was significantly decreased (P < 0.01, Fig. 2B) compared to saline controls

(16.9 ± 2.9 nM); therefore, speedball SA significantly increased the affinity of RTI-55 at

the low-affinity DAT binding site.

Two-site modeling also revealed a trend toward increased density of high-affinity

binding sites (Bmax1) (P = 0.07, Fig. 3A) but no significant changes (P = 0.17, Fig. 3B) in

DAT low-affinity binding density (Bmax2) for RTI-55. Table 1 shows the group averages

(mean ± SEM; n = 5/group) for all two-site binding parameters, as plotted in Fig. 2 and 3.

Although there were no significant changes in the absolute Bmax values, the ratio of high-

affinity to low affinity sites was significantly increased following speedball SA (where

134

high affinity sites represented 16 ± 3% of total sites) as compared to saline (5 ± 2%, P <

0.01), cocaine alone (4 ± 2%, P < 0.01) and heroin alone (6 ± 1%, P < 0.05, Fig. 4A).

There was also a concomitant decrease in the percent of low-affinity binding sites in the

speedball SA group (84 ± 3%) as compared to saline (95 ± 2%, P < 0.01), cocaine alone

(96 ± 2%, P < 0.01) and heroin alone (94 ± 1, P < 0.05, Fig. 4B).

Figure 2.

Figure 2. High- and low-affinity DAT binding site dissociation constants (Kd) were

determined by membrane binding assays of [125

I]RTI-55 and unlabeled RTI-55 at varying

concentrations. Saturation curves were statistically better fit by a two-site binding model

(F value range, 4.47-802.9; P < 0.05). Data shown are mean ± SEM for each SA group (n

= 5/group). (A) There was no statistically significant difference between groups in Kd

values determined for the high-affinity binding site, but one-way ANOVA suggests a

trend for group differences (P = 0.0803). (B) Speedball SA resulted in significantly

decreased Kd for the low-affinity binding site (**P < 0.01) as compared to saline controls,

as determined by Bonferroni’s correction for pairwise comparisons following one-way

ANOVA.

A B

135

Figure 3.

Figure 3. High- and low-affinity DAT binding site densities (Bmax) were determined by

membrane binding assays of [125

I]RTI-55 and unlabeled RTI-55 at varying

concentrations. Saturation curves were statistically better fit by a two-site binding model

(F value range, 4.47-802.9; P < 0.05). Data shown are mean ± SEM for each SA group (n

= 5/group). (A) Speedball SA resulted in a trend for increased Bmax of the high-affinity

binding site (P = 0.0663), but this was not significant as determined by one-way

ANOVA. (B) There was also no statistically significant difference between groups in

Bmax values determined for the low-affinity binding site.

Table 1. Effects of chronic cocaine, heroin and speedball SA on the binding parameters

of high- and low-affinity DAT binding sites.

High Kd (pM) Low Kd (pM)

High Bmax

(pmol/mg)

Low Bmax

(pmol/mg)

Saline 132 ± 46 16900 ± 2900 0.286 ± 0.14 4.56 ± 1.3

Cocaine 410 ± 98 7960 ± 2200 0.284 ± 0.095 3.04 ± 0.47

Speedball 256 ± 82 4990 ± 1300** 0.591 ± 0.15 5.89 ± 0.81

Heroin 237 ± 23 11400 ± 1800 0.131 ± 0.028 3.01 ± 1.2

Mean (± SEM) data determined by nonlinear regression for two-site modeling of

membrane binding assays with [125

I]RTI-55 and unlabeled RTI-55 in rat NAc

membranes. **P < 0.01 vs. saline (one-way ANOVA with Bonferroni post hoc)

A B

136

Figure 4.

Figure 4. Percents of high- and low-affinity DAT binding sites were determined using

the Bmax values by defining Bmaxtotal = Bmax1 + Bmax2, and percent high-affinity sites =

(Bmax1 / Bmaxtotal) × 100 and percent low-affinity binding sites = (Bmax2 / Bmaxtotal) ×

100. Data shown are means ± SEM for each SA group (n = 5/group). (A) Speedball SA

resulted in significantly increased percent of high-affinity binding sites as compared to

saline, cocaine and heroin groups (*P < 0.05, **P < 0.01), as determined by Bonferroni’s

correction for pairwise comparisons following one-way ANOVA. (B) There was also a

corresponding decrease in the percent of low-affinity binding sites following speedball

SA as compared to saline, cocaine and heroin groups (*P < 0.05, **P < 0.01), as

determined by Bonferroni’s correction for pairwise comparisons following one-way

ANOVA.

A B

137

DAT quantitative autoradiography

Quantitative autoradiography for DAT was performed in rat brain sections to

determine whether there were any neuroanatomical differences in DAT binding between

these groups of rats. Radiolabeled CFT was chosen for DAT binding in sections because

it has a lower specific activity for DAT, allowing a greater percentage of the high-affinity

binding sites to be occupied than would be with [125

I]RTI-55. The cocaine analog

[3H]CFT was used at a concentration (20 nM) that occupied 52% of high affinity sites,

and 13% of low affinity sites, as calculated based on our preliminary membrane binding

in rat striatal membranes. This compares to only 7% of high-affinity and 0.05% of low-

affinity DAT sites that would have been occupied using a concentration of [125

I]RTI-55

that is standard for section autoradiography (10 pM). Density of [3H]CFT binding was

quantified at the medial and caudal levels of the NAc to include both the core and shell

(Fig. 5). Specific [3H]CFT binding was observed in both caudate and NAc (see

representative autoradiograms in Fig. 5). Unlike the membrane binding assay, in which

multiple concentrations of radioligand were used to calculate Kd and Bmax values, in the

autoradiography experiments using only one concentration of [3H]CFT, there were no

significant differences between any SA groups or saline controls in specific [3H]CFT

binding density at either the medial (P = 0.4585) or caudal (P = 0.780) levels of the NAc

(Table 2).

138

Figure 5.

Figure 5. Representative images from DAT quantitative autoradiography are shown with

(A) schematics of the medial NAc (bregma+2.20mm, left) and caudal NAc

(bregma+1.20mm, right) regions of interest that determined the size and location of

densitometry, as outlined in black. (B) Sample autoradiographs of sections at the medial

NAc (left) and caudal NAc (right) levels from saline control animals with regions of

interest outlined in black.

Table 2. Effects of chronic cocaine, heroin and speedball SA on densities of [3H]CFT

binding to DAT in rat NAc.

SA Group Medial NAc Density Caudal NAc Density

Saline 15.6 ± 0.61 13.1 ± 0.42

Cocaine 14.8 ± 0.76 13.1 ± 0.39

Speedball 15.1 ± 0.83 13.0 ± 0.43

Heroin 13.9 ± 0.59 12.6 ± 0.35

Mean (± SEM) quantitative autoradiography data quantified at the medial and caudal

levels of the NAc are presented as specific binding in pmol/mg of wet-weight tissue (n =

5-7/group). There were no significant differences in [3H]CFT binding density at either

level of the NAc.

A B

139

Discussion

The present study revealed alterations in the binding parameters for DAT high-

and low-affinity binding sites in rat NAc following chronic SA of cocaine/heroin

combinations. Speedball SA resulted in a significant increase in the affinity of the DAT

low-affinity biding site for RTI-55, as well as a shift in the composition of binding sites

toward a greater percentage of high-affinity binding sites. Previous studies have also

reported that DAT contains both high- and low-affinities for certain ligands (Madras et

al., 1989a; Madras et al., 1989b; Boja et al., 1992; Pristupa et al., 1994; Staley et al.,

1994; Gracz and Madras, 1995; Rothman et al., 1995; Letchworth et al., 1999; Katz et al.,

2000; Mash et al., 2002). The physical nature of these high- and low-affinities,

specifically whether they represent binding sites for multiple ligands, and whether they

are distinct, overlapping, or one and the same in physical location on the DAT protein,

remains debated. The possible correlation between physical binding locations of DAT

substrates and observed high- and low-affinity binding characteristics would provide

valuable insight to the functional alterations in DAT following chronic drug abuse.

Computational modeling studies of DAT binding sites have recently utilized the

crystal structure of a homologue to Na+/Cl

--dependent neurotransmitter transporters,

bacterial leucine transporter from Aquifex aeolicus (LeuTAa) (Yamashita et al., 2005), and

reported divergent sites of DAT protein interactions with different ligands, namely DA

and cocaine. Several groups have reported a secondary pocket as the binding site for

cocaine distinct from the primary substrate binding site for DA (Singh et al., 2007;

Indarte et al., 2008; Shi et al., 2008; Huang et al., 2009; Quick et al., 2009; Gedeon et al.,

2010; Shan et al., 2011; Merchant and Madura, 2012). The binding sites are located at the

140

center of the 12 transmembrane (TM) segments of the DAT protein, within the alpha

helical structures of TMs 1, 3, 6 and 8 (Yamashita et al., 2005). In LeuTAa and DAT, the

primary binding site (S1) is located closer to the intracellular space, below the secondary

binding site (S2) which is located above S1 in the extracellular vestibule (Singh et al.,

2007; Shi et al., 2008; Huang et al., 2009; Quick et al., 2009). Computational modeling

employing minimum free energy profiles and atomic distances between DAT and its

ligands suggest that two Na+ ions and one DA molecule bind to S1 and that cocaine binds

to S2 (Merchant and Madura, 2012), at least at first (Huang et al., 2009). Huang et al.

report that the binding site at S2 can naturally accommodate cocaine, and that a

conformational change likely occurs between the TM segments surround the binding

pocket to allow cocaine to move into the S1 binding site. The authors also concluded that

cocaine can bind to DAT alone or DAT with DA substrate already bound in order to

inhibit the transport of DA, through both blocking the initial binding of DA to DAT and

by reducing the kinetic turnover of DAT once DA is bound, by inhibiting the

conformational change necessary to translocate DA internally (Huang et al., 2009). The

probability of cocaine binding to the S2 site is also supported by LeuTAa modeling with

the tricyclic antidepressant clomipramine which was reported to inhibit substrate uptake

non-competitively by binding to S2 and inducing a conformational change to stabilize the

extracellular gate in a closed conformation (Singh et al., 2007). However, when leucine is

modeled to bind to both S1 and S2 of LeuTAa, substrate binding at S2 acts as a symport

effector to allosterically trigger intracellular release of Na+ and substrate from S1 (Shi et

al., 2008). Insight into these physical characteristics of DAT binding sites and

mechanisms of substrate translocation lend support for our current results of increased

141

affinity at S2 and increased percentage of high-affinity DAT binding sites to provide a

mechanism by which we have previously observed a significant increase in baseline DAT

reuptake rate following chronic speedball SA (Pattison et al., 2012).

Integration of these modeled binding mechanisms with the reported membrane

binding calculations for DAT of one- versus two-site modeling suggests it is possible that

some ligands can accommodate a two-site fit by initially binding to the entryway site of

the binding pocket at S2 and also fit into the S1 binding site, possibly following an

allosteric change of the TMs surrounding the binding sites. Low-affinity DAT binding

that is described by two-site modeling of membrane binding data may in fact be

physically represented by the S2 site located in the substrate entryway, whereas high-

affinity DAT binding would be expressed as the primary binding site, S1. Our results of

RTI-55 binding to DAT conform to a two-site binding model, as others have also

reported in the past (Boja et al., 1992; Rothman et al., 1995; Letchworth et al., 1999).

Homologous competition assays with [3H]cocaine and unlabeled cocaine has also been

reported to best fit two-site binding hyperbolae (Madras et al., 1989a; Katz et al., 2000),

further supporting the unification of two-site membrane binding with the computational

models of cocaine binding to DAT at S2 and potentially relocating to S1 to block DA

binding and reduce the kinetic turnover (Huang et al., 2009). The binding characteristics

of RTI-55 can be directly applied to cocaine because of their close structural similarity,

and presumably applied to the smaller endogenous substrate DA.

Our results indicate that the Kd value for RTI-55 associated with the low-affinity

DAT binding site (Kd2) is significantly decreased following chronic speedball SA, which

is perhaps physically represented by the DAT S2 binding site, corresponding to an

142

increased affinity of DAT for cocaine-like substrates at the S2 binding site. The present

binding data also imply a shift in the composition of binding sites, by a significant

increase in the percent of high-affinity binding sites. Due to the concomitant decrease in

low-affinity binding sites, it is possible that there is no overall increase in DAT

expression, as supported by quantitative autoradiography with [3H]CFT. It is likely that

quantitative autoradiography did not show any significant differences between groups

because only one concentration of radioligand was used and could not detect the subtle

differences in affinity as were observed in the membrane experiments. The shift in

binding site composition toward increased high-affinity binding sites could be directly

related to the previously discussed observation of increased DAT reuptake rate following

speedball SA. In fact, an increase in the affinity at S2 also confers greater DAT reuptake

rate. The increased affinity of DAT at S2 may physically represent a relaxation in the

binding pocket in order to permit its substrate DA and other ligands to enter the

extracellular vestibule more easily and efficiently. A more flexible binding pocket

already in place would represent an outward facing conformation that is potentially ready

to complete its conformational change if two molecules of substrate bind to execute

symport of DA and Na+. Basal alterations in the DAT binding conformations to allow

greater outward facing states ready for substrate binding and transport would directly lead

to increased Vmax, as we have previously reported.

We hypothesize that this observation is indicative of the flexibility of the DAT

binding pocket including S1 and S2 following chronic speedball SA, and that substrates

like [125

I]RTI-55 are able to occupy more of the S1 binding sites as compared to the

saline controls, as observed in the shift in binding site composition toward more high-

143

affinity binding sites. This does not necessarily mean a dramatic alteration in chemical

composition of the binding pocket (e.g. via post-translational modifications); rather, the

mechanism of relaxation in binding sites may occur simply via faster or greater

sequestration of Na+ ions into the binding pocket. The sequential binding of two Na

+, one

Cl- and one DA has been previously described (Torres et al., 2003); however, these

previous methods would have been unable to predict the binding of a second substrate

molecule to induce an allosteric change and complete the reuptake on DA by

translocation through the plasma membrane (Shi et al., 2008). The dynamics of LeuT in

the region of the extracellular entryway associated with binding of Na+ and substrate

have been preliminarily captured with the use of site-directed spin labeling and electron

paramagnetic resonance analysis. The authors showed that Na+ increases the accessibility

of the extracellular entryway, specifically at TM1, TM3 and ECL4 that project into the

entryway above the binding site S1, as noted by an increase in motility and looser

packing of the vestibule in the presence of Na+ (Claxton et al., 2010). One mechanism by

which an Na+-induced outward facing conformational state could occur is by an increase

in extracellular levels of Na+ ([Na

+]e). Future studies are necessary to assess NAc [Na

+]e

following speedball SA, but it has been shown with repeated cocaine or amphetamine

injection that NAc core MSNs exhibit increased intrinsic excitability (Kourrich and

Thomas, 2009), which could also be a correlate of increased [Na+]e. If it can be shown

that the converse is true, as in cultured hippocampal neurons where elevated [Na+]e

directly increased intrinsic excitability, then elevations in [Na+]e may result from

administration of psychostimulants that significantly increase NAc [DA]e and

subsequently lead to alterations in DAT reuptake function.

144

Future experiments are necessary to deduce the physical correlates to the present

observations of increased affinity of DAT at the secondary binding site and increased

percentage of high-affinity binding without an overall increase in total DAT binding

sites, which contribute to an increase in baseline reuptake rate after prolonged speedball

SA. Identifying this structural information associated with high- and low-affinity binding

would provide valuable insight to drug-induced functional alterations in DAT and could

provide framework for designing novel allosteric modulators of DAT to aid in stabilizing

DA neurotransmission during withdrawal from DA-enhancing drugs of abuse.

Acknowledgements

The study was supported in part by R01 DA012498 (SEH) and P50 DA006634 (SRC).

145

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169

CHAPTER V

COMPARING IN VIVO AND IN VITRO ALTERATIONS IN DAT KINETICS

AND BINDING TO ELUCIDATE FUNCTIONAL ADAPTATIONS TO CHRONIC

SPEEDBALL SELF-ADMINISTRATION

Lindsey P. Pattison and Scott E. Hemby

Lindsey P. Pattison prepared the chapter and Scott E. Hemby acted in an advisory and

editorial capacity.

170

Introduction

The mechanism of action of cocaine/heroin combinations (speedball) to induce

supra-additive elevations in extracellular dopamine (DA) levels ([DA]e) in the nucleus

accumbens (NAc) (Hemby et al., 1999; Smith et al., 2006a) is the result of the combined

effects of cocaine-induced inhibition of DA transporter (DAT)-mediated reuptake (Pettit

and Justice, 1989) and heroin-induced disinhibition of dopaminergic neurons leading to

increased firing rates and DA release in the NAc (Johnson and North, 1992). The supra-

additive increases in NAc [DA]e observed with in vivo microdialysis in rats (Zernig et al.,

1997a; Hemby et al., 1999; Smith et al., 2006b) which underlie the potentiated

reinforcing effects of speedball observed with SA paradigms in rats (Ward et al., 2005)

and nonhuman primates (Mattox et al., 1997a; Rowlett and Woolverton, 1997b; Rowlett

et al., 2005) likely contribute to the continued patterns of speedball abuse in human drug

users. Currently, there are no approved pharmacotherapies for cocaine abuse.

Furthermore, many polysubstance abusers undergoing treatment for heroin addiction

admit to the concurrent use of cocaine (Hartel et al., 1995). These polydrug users exhibit

severely blunted rates of success for treatment (Downey et al., 2000b; Sofuoglu et al.,

2003; Tzilos et al., 2009). Because DAT is the sole transporter responsible for the

clearance of extracellular DA in the NAc, understanding the neurochemical adaptations

in DAT function will provide important insights to the state of dopaminergic

neurotransmission following long-term speedball use. Therefore, the studies discussed

here were undertaken to address the effects of chronic speedball self-administration (SA)

in rats on DAT functional alterations in the NAc.

171

Combining in vivo microdialysis and in vivo voltammetry results

To date, most of the published studies on the effects of cocaine/heroin

combinations have been of a behavioral nature, showing that dopamine D1 and D2

receptors (D1Rs and D2Rs), DAT and mu opioid receptors (MORs) are implicated in the

reinforcing effects of speedball SA (Mello and Negus, 1998, 2001; Cornish et al., 2005;

Mello and Negus, 2007). We began by investigating the neurochemical adaptations by

examining the effects of speedball administration on the kinetic parameters of DAT-

mediated reuptake in the NAc using in vivo fast-scan cyclic voltammetry (FSCV) in

anesthetized rats. FSCV can provide information about the dynamics of DAT kinetics

and how they adapt to modulate DA reuptake on a subsecond timescale, which gives us

insight to what molecular modifications may be occurring. When these data are examined

in comparison with our previous in vivo microdialysis data, we can gain a wealth of

information about speedball-induced alterations in DA neurotransmission. In order to

effectively compare and contrast these data sets, it is important to first understand the

differences between these techniques.

Microdialysis can provide assessments of in vivo concentrations of

neurotransmitters, metabolites and drug concentrations. These data have provided

important knowledge of in vivo concentrations of neurotransmitters of interest, such as

DA, which can offer accurate assessments of experimental protocols and allow one to ask

questions about the relevancy of different types of data, such as what receptor binding

constants mean relative to in vivo DA concentration ([DA]), for example (Justice, 1993).

Microdialysis has been particularly useful in the assessment of changes in

neurotransmitter levels in awake, behaving animals engaged in behaviors such as SA

172

(Willuhn et al., 2010). In vivo voltammetry is an electrochemical technique that is

commonly used in conjunction with microdialysis to further characterize the dynamics of

DA neurotransmission (Stamford et al., 1989; Ng et al., 1991; Wu et al., 2001a). In vivo

voltammetry affords the advantages of a microscopic (μm) spatial scale and subsecond

temporal scale of detection as compared to the larger spatial (mm) and temporal (several

minutes) scales necessary for microdialysis techniques (Greco and Garris, 2003). Indeed,

the temporally resolved measurements are suitable for monitoring the rapid clearance of

DA and resolving the DAT kinetic parameters. FSCV allows for the analysis of DA

uptake kinetics separately from the contributions of DA release or metabolism, which

cannot be discerned from microdialysis studies (Wightman et al., 1988b; Wu et al.,

2001a). Although FSCV techniques are not without disadvantages, such as necessity of

artificial stimulation and lack of account for basal neurotransmitter levels, to be discussed

later.

In these voltammetric techniques, a high frequency electrical stimulation of

ventral tegmental area (VTA) dopaminergic cell bodies or the median forebrain bundle

projecting to the NAc elicits a spike in extracellular DA. The observed increase in [DA]e

during a pulse train is consequence of combined DA release due to electrical stimulation

and immediate DA reuptake by DAT. Following termination of the stimulus, the

voltammetric recording shows only DA uptake working to return DA to pre-stimulation

levels (Wightman et al., 1988b). Taking the first derivative of the DA decay curve and

plotting it versus [DA] yields a hyperbola following Michaelis-Menten kinetics. Setting

the derivative of [DA] equal to rate of stimulated DA release (stimulation frequency ×

[DA] release per pulse) minus the clearance rate defined by Michaels-Menten kinetics

173

allows the equation to be solved for the uptake parameters of maximal reuptake velocity

(Vmax) and the Michaelis-Menten constant representing apparent affinity (apparent Km)

(Wu et al., 2001a). Apparent Km refers to the observed affinity of DA for DAT

(Wightman et al., 1988b), and higher Km values correspond to lower DA uptake rates or

increased DA uptake inhibition. As such, we employed electrically stimulated FSCV in

anesthetized rats to assess the effects of cocaine, heroin and speedball on DAT uptake

kinetics in drug-naïve (Pattison et al., 2011b) and chronic self-administering animals

(Pattison et al., 2012).

The voltammetric study in Chapter II was undertaken to assess the acute effects of

a single bolus intravenous (i.v.) infusion of cocaine, heroin or speedball on electrically

stimulated DA release and DAT-mediated reuptake kinetics. The main findings provide

insight into reuptake inhibition by cocaine and the potential involvement of DA release-

mediating feedback mechanisms induced by speedball. Infusion of i.v. cocaine (1.0

mg/kg) and speedball (1.0 mg/kg cocaine + 0.03 mg/kg heroin) both significantly

potentiated evoked DA efflux as compared to pre-drug baseline levels. However, in

contrast to previous in vivo microdialysis data that showed speedball eliciting a

significantly greater level of [DA]e as compared to cocaine, FSCV showed that cocaine

infusion produced a significantly greater increase in evoked DA as compared to speedball

(Pattison et al., 2011b). The attenuated amplification in electrically evoked DA efflux

observed following speedball is likely due to feedback mechanisms activated by greater

accumulation of DA in the NAc extracellular space due to speedball administration.

Specifically, it is hypothesized that the supra-additive overflow of accumbal DA

known to occur based on the aforementioned microdialysis studies may lead to ancillary

174

activation of presynaptic D2R autoreceptors, beyond the potential activation attributable

to cocaine-induced DA overflow. Activation of the presynaptic D2R short isoform (D2S)

by extracellular DA overflow leads to subsequent attenuation of DA synthesis and release

from dopaminergic terminals (De Mei et al., 2009), which might only be detected at a

smaller timescale, as with FSCV. It logically follows that the speedball combination

which elicits greater non-stimulated DA overflow would then activate D2S to a greater

extent than cocaine alone in order to further attenuate NAc DA release. Thus the

synergistic increase in [DA]e as observed with in vivo microdialysis would not be

detected at this small timescale following immediate D2R autoreceptor feedback to

reduce further DA efflux by electrical stimulation. When considering this mechanism,

one must remember that the microdialysis observations of speedball-induced supra-

additive [DA]e in the NAc are over a large time scale (ten minute bins of dialysate

collection per data point). Thus the proposed D2R-mediated attenuation of DA release

occurring simultaneously with drug-mediated potentiation of DA release will result in the

net effect of increased DA release over such a large timeframe as in microdialysis

collections, especially considering that a rat will self-administer multiple i.v. drug

infusions over a single ten minute period. To further support the theory of D2

autoreceptor-mediated attenuation in electrically evoked DA release following i.v.

speedball, several studies have directly examined the effects of D2Rs on electrically-

evoked and drug-induced DA release.

175

D2R modulation of DA release and DAT-mediated reuptake

Both pre- and postsynaptic D2Rs are involved in the regulation of DA release.

Postsynaptic D2Rs located in the NAc are able to regulate the release of glutamate

(Bamford et al., 2004), GABA (Centonze et al., 2002) and acetylcholine (Zhang et al.,

2009b), which in turn modulate DA release. Moreover, presynaptic D2Rs, or

autoreceptors, have been suspected as the primary controllers of D2-induced attenuation

in DA release (De Mei et al., 2009). Activation of autoreceptors by the D2R agonist

quinpirole has been shown to reduce evoked DA efflux with FSCV (Palij et al., 1990;

Bull and Sheehan, 1991; Wieczorek and Kruk, 1995; Jones et al., 1996; Mateo et al.,

2005). Conversely, blockade of autoreceptors enhanced evoked DA release, and

synergistically enhanced cocaine-induced DA overflow (Aragona et al., 2008).

Additionally, in the presence of the D2R antagonist sulpiride, electrically evoked DA

release was abolished by subsequent application of amphetamine, due to its

pharmacological activation of enhancing DA release (Jones et al., 1998a). Likewise, in

vivo microdialysis conducted in mice lacking D2R expression (D2R-/-

) following i.p.

injection of cocaine or s.c. injection of morphine (delivered to separate groups, not in

combination) that showed significantly increased [DA]e in D2R-/-

as compared to wild

type littermates (Rougé-Pont et al., 2002). One can therefore reason that speedball-

induced DA overflow will activate D2 autoreceptors more so than cocaine alone, and this

pharmacological effect would reduce further DA release by electrical stimulation; thus

leading to attenuation in evoked DA following i.v. speedball as compared to cocaine

alone as we observed with in vivo FSCV.

176

Several reports also suggest that that D2 autoreceptor activation may alter the

kinetics of DA uptake by increasing Vmax upon autoreceptor activation (Meiergerd et al.,

1993; Parsons et al., 1993). Wu et al. showed that D2 autoreceptors concurrently

downregulate DA release and upregulate DAT-mediated reuptake in the NAc and caudate

putamen (CPu) of anesthetized rats using in vivo FSCV (Wu et al., 2002). Newer studies

have used site-specific D2R knockout mice to show that both pre- and postsynaptic D2Rs

are able to control evoked DA release, but that only presynaptic autoreceptors control

DAT reuptake (Anzalone et al., 2012). Furthermore, Bolan et al. have elucidated the

mechanism by which D2 autoreceptors mediate DAT reuptake. D2R autoreceptor agonist

activation in live cells co-expressing D2S and DAT increased phosphorylation of

extracellular signal-regulated kinases (ERK1/2) and rapidly increased cell-surface

expression of DAT to enhance DA clearance rate. The study also suggests the possibility

of a direct physical interaction between DAT and presynaptic D2Rs (Bolan et al., 2007).

Our data show a slight increase in Vmax following acute cocaine and speedball

injection in drug-naïve animals (Pattison et al., 2011b) as well as significantly increased

baseline Vmax following chronic speedball SA (Pattison et al., 2012). Overall we surmise

this data to represent compensation for chronic repeated elevations in accumbal [DA]e

during long-term speedball SA. These observations may also be extrapolated to

immediate and prolonged activation of presynaptic D2Rs by drug-induced DA overflow,

respectively, to enhance DAT clearance rate and likely surface expression. The long-term

effect of elevated DA clearance in speedball SA animals likely contributes to a basal

hypodopaminergic state in these animals. Basal [DA]e cannot be accurately measured

using electrically stimulated FSCV techniques (Chen and Budygin, 2007), thus our

177

evoked DA data cannot support this theory. Although there were no significant

differences observed in baseline [DA]e between cocaine and speedball SA groups with in

vivo microdialysis (Hemby et al., 1999; Smith et al., 2006b), the dialysate extraction

methods used were not those that can most accurately measure basal DA levels. In order

to precisely measure basal DA differences following chronic SA, multiple dialysate

collections need to be performed with no net flux methods of varying [DA] added to the

perfusate and/or varying the perfusion flow rates in order to extrapolate basal [DA]e at

zero net flow (Parsons and Justice, 1992). Aside from of the consequence that elevated

DAT reuptake rate may have on basal [DA]e, it is important to understand the physical

mechanism(s) by which Vmax is increased following speedball SA in order to propose

methods for reversing this effect as a possible addiction treatment technique.

Physical correlates of DAT kinetic alterations

DAT trafficking and surface expression

Increased Vmax can be correlated with a few different mechanisms. The first and

most widely accepted is increased DAT surface expression. Cocaine has been shown to

increase Vmax for in vitro [3H]DA uptake assays (Wheeler et al., 1994) with

corresponding increased cell-surface expression of DAT as shown by surface

biotinylation (Daws et al., 2002) or radioligand binding (Little et al., 2002). These studies

show kinetic increases in Vmax with no change in Michaelis-Menten constant Km.

Typically, an inhibitor acting through changes in Vmax and not Km is considered a

noncompetitive inhibitor (Greco and Garris, 2003). However, Samuvel et al. (2008)

178

showed that cocaine SA and withdrawal leads to increased DAT surface expression (with

concomitant decrease in cytosolic DAT expression) in the caudate putamen (CPu) but not

in the NAc, and that increased cell-surface DAT expression in the CPu was linked to

increased DAT association with protein phosphatase 2A catalytic subunit (PP2Ac) and

decreased serine phosphorylation of DAT (Samuvel et al., 2008). Though there was no

increase in DAT surface expression in the NAc, they did, however, show with tritiated

DA ([3H]DA) uptake that cocaine SA increased Vmax in both CPu and NAc

synaptosomes. There was no change observed in Km in the CPu, but a significant

reduction in Km was measured in the in the NAc, relating to increased affinity of DAT for

DA (Samuvel et al., 2008). Thus for the case of increased Vmax in the NAc, enhanced

uptake rate is not due to increased DAT expression, but increased affinity of the

transporter for its substrate. An inhibitor acting through alterations in both Km and Vmax is

said to work as an uncompetitive inhibitor (Greco and Garris, 2003). In Chapter III, we

detected a significant increase in Vmax but no change in apparent Km following chronic

speedball SA (Pattison et al., 2012), also supporting that cocaine is acting as a

noncompetitive inhibitor.

Unpublished observations from our lab of DAT protein quantification in the NAc

using Western blotting have shown an increase in the 80 kDa N-glycosylated form of

DAT in the membrane fraction following chronic speedball SA, but this result was only

significant compared to the heroin SA group, not cocaine or saline. Cocaine SA rats did

not showed increased DAT in NAc membranes, as some other studies have reported.

Similar to the results from Samuvel et al. (2008) previously mentioned, we also saw a

concomitant decrease in the 80 kDa band of DAT in the cytosol fraction of speedball SA

179

animals, which was significantly reduced compared to saline controls (Hemby and

McIntosh, unpublished). Though not conclusive, these data suggest that increased cell

surface expression of DAT via trafficking of transporter to the plasma membrane

following speedball SA may contribute to the observed increase in Vmax with FSCV, at

least partially. It is also possible that there are changes in DAT affinity that we were

unable to detect with electrically stimulated in vivo FSCV, as these techniques are very

different from in vitro [3H]DA uptake. Though FSCV is regarded as a steadfast technique

for measuring cocaine inhibition of DAT uptake, the use of high frequency electrical

stimulation and the methods of voltammetric analysis may not be as ideal to detect the

nature of alterations in DAT affinity that could be occurring following concurrent cocaine

and heroin SA, as they are for DAT alterations due to cocaine alone.

Methods for analyzing DAT reuptake parameters

Michaelis-Menten modeling of voltammetric data relies on [DA] and also on the

affinity of DAT for cocaine to calculate both apparent Km and Vmax (Greco and Garris,

2003; Chen and Budygin, 2007). Conversely, evoked DA release also depends on Km,

Vmax and DA released per pulse, which also directly relates to stimulation frequency.

These relationships in the modeling methods make it difficult to discern uptake kinetics,

especially when discriminating competitive from noncompetitive DAT inhibitors, which

exhibit distinct relationships between evoked DA and frequency (Greco and Garris,

2003). This could prove problematic, as cocaine has been shown to act as a competitive

inhibitor by altering Km (Krueger, 1990), a noncompetitive inhibitor by decreasing Vmax

180

(Missale et al., 1985), and an uncompetitive inhibitor by altering both Km and Vmax

(Wheeler et al., 1994; Samuvel et al., 2008; Ferris et al., 2011; Calipari et al., 2012). In

fact, even within similar experimental techniques employing FSCV the effects of cocaine

SA on the uptake parameters apparent Km and Vmax are not entirely agreed upon. In vitro

FSCV performed in rats following brief cocaine SA (five days) showed significantly

decreased baseline Vmax as well as a significant reduction in dopamine uptake inhibition

by cocaine as shown by decreased apparent Km values with cocaine on board (Ferris et

al., 2011; Calipari et al., 2012). Mateo et al. also performed in vitro FSCV following

binge cocaine SA in rats. In contrast to the previously mentioned studies, baseline Vmax

was significantly increased in cocaine SA rats following one or seven days withdrawal.

However, both withdrawal groups did show attenuated apparent Km with cocaine on

board (Mateo et al., 2005) in agreement with the other in vitro FSCV results, meaning

that cocaine is less effective at inhibiting DAT following cocaine SA. On the other hand,

Oleson et al. employed in vivo FSCV and showed significantly increased baseline Vmax in

cocaine SA animals while no changes were observed in apparent Km at baseline or

following cocaine injections (Oleson et al., 2009b). Our in vivo FSCV results in chronic

cocaine SA rats showed no alterations in Vmax or apparent Km at baseline or following a

single cocaine infusion as compared to drug-naïve animals, but our observations in

speedball SA rats were similar to those in cocaine SA animals by Oleson et al. (Pattison

et al., 2012). In order to further investigate the observation of significantly increased Vmax

with no accompanying changes in apparent Km in chronic speedball SA animals, we

utilized additional methods of radioligand binding in NAc membrane preparations and

quantitative receptor autoradiography in striatal brain sections to potentially identify

181

underlying physical correlates in transporter binding to these DAT reuptake functional

parameters.

Radioligand binding assays in membrane preparations can provide information

about the affinity of a receptor for a certain ligand (related inversely to the dissociation

constant, Kd) and the density of binding sites in the tissue for that ligand (Bmax), while

quantitative autoradiography can show the region-specific binding density of a

radioligand to its receptor. Membrane binding and quantitative autoradiography studies

have been extensively performed in animal and human striatal tissue following cocaine

administration. Most of these studies agree that cocaine increases DAT density in the

NAc (Little et al., 1993b; Staley et al., 1994; Wilson et al., 1994; Hitri et al., 1996;

Letchworth et al., 1997; Little et al., 1998; Little et al., 1999; Letchworth et al., 2001a;

Mash et al., 2002; Ben-Shahar et al., 2006; Bailey et al., 2008; Miguens et al., 2008;

Beveridge et al., 2009). Of these studies that utilized membrane binding methods in

addition to others that reported no changes in DAT density, none of the reports showed

alterations in DAT affinity following cocaine SA (Little et al., 1993b; Staley et al., 1994;

Hitri et al., 1996; Wilson et al., 1996; Letchworth et al., 1997; Little et al., 1998;

Letchworth et al., 1999; Little et al., 1999; Mash et al., 2002). One may infer that with an

abundance of literature supporting increased density of DAT ligand binding that more

agreement would be seen in kinetic analyses of DAT Vmax following cocaine SA, since

the two findings of increased DAT expression and increased reuptake rate should be

associated. Due to the methodological differences between FSCV studies and in vitro

binding assays, and the means by which voltammetric data are fit to simple Michaelis-

Menten enzyme modeling, it is understandable why this discrepancy exists.

182

Membrane binding data can be analyzed using nonlinear regression analyses that

do not necessarily follow Michaelis-Menten parameters and assumptions. In fact, many

binding studies with DAT ligands have found that two-site binding hyperbola models are

best-fit for certain ligands (Madras et al., 1989a; Madras et al., 1989b; Boja et al., 1992;

Staley et al., 1994; Gracz and Madras, 1995; Letchworth et al., 1999; Katz et al., 2000),

which can provide a more detailed description of binding characteristics than one

generalized binding constant or affinity term, as with Michaelis-Menten modeling. Our

investigation of DAT binding parameters using the cocaine-like DAT inhibitor 3β-(4-

iodophenyl)tropan-2β-carboxylic acid methyl ester (RTI-55) in membrane binding assays

in the NAc showed a trend toward increased Kd for the high-affinity DAT binding site

following cocaine SA, as well as a significant decrease in the Kd of DAT for RTI-55 at

the low-affinity binding site following speedball SA (Pattison et al., unpublished, see

Chapter IV). These data imply a significant increase in the affinity of DAT for substrate

binding at the low-affinity binding site following speedball SA. We did not observe any

significant alterations in the density of iodinated RTI-55 ([125

I]RTI-55) binding to the

high-affinity DAT binding site following speedball SA, as confirmed by subsequent

quantitative autoradiography using tritiated 3β-(4-fluorophenyl)tropan-2β-carboxylic acid

methyl ester ([3H]CFT). However, we did observe a shift in the composition of high- and

low-affinity DAT binding sites following speedball SA, as a significant increase in the

percent of high-affinity sites with a concomitant decrease in the percent of low-affinity

binding sites, with no overall change in total Bmax. This shift in binding site composition

may represent a physical alteration in the binding pocket of DAT in order to allow

substrates better access to the high-affinity binding site, which may contribute to the

183

observed increase in reuptake rate following speedball SA. Taken together, these in vitro

binding data imply specific alterations in DAT binding that lend additional support for

the observed alterations in DAT reuptake function that are likely independent from any

possible increases in DAT expression.

DAT protein structure and mechanism of DA translocation

The physical nature of high- and low-affinity DAT binding sites has not been

experimentally described. Researchers have attempted to predict the location of DAT

substrate binding sites since the 1990s when researchers began performing site-directed

amino acid mutations based on primary amino acid sequence from the originally cloned

DAT cDNA (Kilty et al., 1991; Shimada et al., 1991). But the physical protein-substrate

interactions of DAT had not been described until recently when Yamashita et al. reported

the successful crystal structure of a bacterial homologue to sodium/chloride (Na+/Cl

-)-

dependent neurotransmitter transporters, leucine transporter from Aquifex aeolicus

(LeuTAa) (Yamashita et al., 2005). The X-ray diffraction pattern of LeuT revealed a

dimeric protein containing 12 transmembrane (TM) segments in a unique fold with a

substrate binding site for leucine located at the center of the protein. A pseudo-twofold

axis of symmetry is present in the plane of the plasma membrane, which is dictated by a

structural repeat in the first ten TMs that relates TM1-5 with TM6-10 (Yamashita et al.,

2005). The binding pockets for leucine and Na+ are formed by TMs 1, 3, 6 and 8. TM3

and TM8 are long helices related by twofold symmetry along a tilted axis through the

plasma membrane, and TM1 and TM6 are characterized by unwound breaks in their

184

helical structures in the middle of the lipid bilayer (Yamashita et al., 2005). These breaks

expose main carbonyl oxygen and nitrogen atoms for direct interaction with the substrate

and two Na+ ions, which also interact with the unwound portions of TM1 and TM6 to

provide a key stabilizing role in substrate binding. The authors note that the structure is

crystallized in a conformation in which both external and internal gates are closed, but

that the structure predicts substantial conformational movements to perform secondary

active transport (Yamashita et al., 2005). TMs 1, 3, 6 and 8 are predicted to act in the

major conformational change, along with extracellular loop 4 (ECL4), which forms a lid

over the substrate binding site and is thought to act as the extracellular gate. The internal

gate is thought to be comprised of the cytoplasmic end of TM6 that forms a cation-π

interaction with the N-terminus just below TM1, which forms a salt bridge with other

residues at the cytoplasmic end of TM8. These intracellular gate interactions are thought

to be essential in stabilizing the transporter in an outward-facing conformation

(Yamashita et al., 2005).

This atomic-resolution structure has since allowed for three-dimensional

computational modeling of substrate binding interactions for eukaryotic Na+/Cl

--

dependent neurotransmitter transporters, namely DAT. Currently, there are several

different results regarding the substrate binding locations and patterns for DAT. One side

of the debate has suggested that there exists a single binding site into which DA and

inhibitors such as cocaine can bind, and that the amino acid binding sites for DA and

cocaine are overlapping. Using the LeuT structure as a template for DAT modeling,

Beuming et al. reported that the binding site for cocaine and cocaine analogs is also

deeply buried between TM segments 1, 3, 6 and 8, and overlaps with the binding sites for

185

the substrates DA and amphetamine, as well as for benztropine-like DAT inhibitors such

as CFT. This overlapping binding site model suggests that the molecular basis for

inhibition of DA transport by cocaine is by competitive inhibition (Beuming et al., 2008).

The other side of the debate says that there exist two distinct binding sites in LeuT

and DAT that are allosterically coupled, both located at the center of the protein as

predicted, but with the primary site (S1) located deeper toward the intracellular surface

and the secondary binding site (S2) located above the primary, closer to the extracellular

surface (Singh et al., 2007; Indarte et al., 2008; Shi et al., 2008; Huang et al., 2009; Quick

et al., 2009; Gedeon et al., 2010; Shan et al., 2011; Merchant and Madura, 2012). Quick

et al. reported a logical reason for the possible discrepancy between those groups

reporting a single binding site versus those favoring two distinct binding sites within the

pocket. The detergent initially used for LeuT crystallization, n-octyl-β-D-

glucopyranoside (OG), acts as an inhibitor to the LeuT S2. Using a different detergent, n-

dodecyl-β-D-maltopyranoside (DMM), comparison of the two forms of LeuT crystals in

these detergents revealed that the OG-resolved crystals represent functionally blocked

states of LeuT where inhibitors can bind in the S2 site, whereas a substrate bound in S2

would promote a different state essential for the Na+-coupled symport mechanism (Quick

et al., 2009). Other studies that suggest inhibition at the S2 location were performed in

LeuT models with the tricyclic antidepressant, clomipramine. Singh et al. reported that

clomipramine noncompetitively inhibited substrate uptake in LeuT by binding to an

extracellular-facing vestibule (same binding site as S2) located just above the primary

binding pocket (same binding site as S1) where leucine and two Na+ were bound.

Clomipramine binding stabilized ECL4, which acts as the extracellular gate, in a closed

186

conformation to trap the leucine substrate in S1 of LeuT (Singh et al., 2007). Shi et al.

showed that the same residues participating in clomipramine binding to LeuT can also

bind a leucine substrate, and that dual substrate binding to S1 and S2 allosterically

triggers intracellular release of Na+ and the leucine substrate from S1. In this manor,

substrate binding to S2 functions as a symport effector to induce transport, in opposition

to inhibitor binding at S2 which acts as a symport uncoupler to inhibit transport (Shi et

al., 2008).

Much like these studies using LeuT and its substrate leucine to predict S1 and S2

binding sites and transport mechanisms, computational modeling of DAT with DA and

cocaine have shown similar binding and transport mechanisms. Cocaine is shown to bind

to S2 with a better structural fit than S1 using atomic distances and free energy profiles

(Huang et al., 2009; Merchant and Madura, 2012). Huang et al. reported that cocaine

binds to S2, at least initially, since S2 can naturally accommodate cocaine without the

need for a large conformational change in DAT, but that cocaine may move further into

S1 (which would represent overlapping binding mode) after DAT makes some necessary

conformational change to expand the binding site cavity. They also hypothesized that

because of the electrostatic repulsion between positively charged DA and positively

charged cocaine, the affinity of cocaine for the complex of DAT with DA bound (DAT-

DA) is expected to be significantly lower than that of cocaine binding with DAT in the

absence of DA (Huang et al., 2009). The authors proposed three equilibrium binding

processes are present when cocaine and DA coexist with DAT, where DAT complexes

with DA alone (DAT-DA), DAT complexes with cocaine alone (DAT/cocaine) and DAT

complexes with both DA and cocaine (DAT-DA/cocaine). By defining each binding

187

reaction with a dissociation constant and utilizing their observed free energy calculations

from computational modeling, the authors report that the amount of DAT-DA is

dependent on the concentrations of DA and cocaine in the environment, and that when

the concentration of cocaine used in an experimental assay is much less than the

dissociation constant for the formation of the DAT-DA/cocaine complex, cocaine would

preferentially bind to DAT over DAT-DA (Huang et al., 2009). On the contrary, when a

cocaine concentration used in an experimental assay is much greater than the dissociation

constant for DAT-DA/cocaine formation, the experimental observation of cocaine

blocking DAT-DA binding should be close to that of a noncompetitive inhibitor because

cocaine can always bind with DAT regardless if DA is already bound or not (Huang et

al., 2009). Taken together, these binding site models and predictions of bound DAT

complexes show that the initial cocaine binding site on DAT does not overlap with but is

close to the DA binding site, and that DA cannot bind if cocaine is blocking the DA entry

tunnel at S2 but cocaine can bind to DAT regardless if DA is already bound or not. When

both DA and cocaine bind to DAT, the DAT protein is not expected to undergo the

conformational change necessary for transport of DA to the intracellular space, so

cocaine may inhibit the transport of DA through both blocking the initial binding of DA

to DAT and by reducing the kinetic turnover of DAT after DA is bound (Huang et al.,

2009).

Incorporation of these reported binding mechanisms with regard to membrane

binding calculations of one- versus two-site modeling suggests it is possible that some

ligands can accommodate a two-site fit by initially binding to the entryway site of the

binding pocket at S2 and also fit into the S1 binding site, possibly following an

188

appropriate conformational change in DAT that allows induced-fit binding of substrates.

The S2 site located in the substrate entryway may in fact represent the low-affinity DAT

binding site that is elucidated by two-site modeling of membrane binding data, whereas

the primary binding site, S1, would be represented by high-affinity DAT membrane

binding. Our binding data of RTI-55 to DAT in NAc membranes conform to a two-site

binding model, as others have also reported in the past (Boja et al., 1992; Rothman et al.,

1995; Letchworth et al., 1999). Membrane binding assays with tritiated ([3H]) cocaine

and unlabeled cocaine have also been shown to fit two-site binding modeling (Madras et

al., 1989a; Katz et al., 2000), further supporting the unity of two-site membrane binding

with the computational model of cocaine-like ligands binding to DAT at S2 and

potentially relocating to S1 (Huang et al., 2009). Although we did not employ the use of

[3H]cocaine in these binding studies, the chemical structure of RTI-55 is only different

from cocaine by an ester group (on cocaine) and a 4-iodophenyl addition (on RTI-55)

(Davies et al., 1994), so the binding characteristics can be directly applied to cocaine and

generally applied to the smaller endogenous substrate DA.

Speedball may alter DAT binding sites to increase reuptake rate

Our results indicate that chronic speedball SA significantly decreased the Kd

value associated with the low-affinity binding site, which could potentially be physically

represented by the cocaine binding site S2 on DAT. The decrease in low-affinity Kd

corresponds to an increased affinity of DAT for cocaine-like substrates at the S2 binding

site. We also observed a shift in the composition of binding sites, by a significant

189

increase in the percent of high-affinity binding sites and concomitant decrease in low-

affinity binding sites indicating no overall increase in DAT expression, as supported by

quantitative autoradiography with [3H]CFT. This shift toward increased high-affinity

binding sites could be directly related to the previously discussed observation of

increased DAT reuptake rate following speedball SA (Pattison et al., 2012). In fact, an

increase in the affinity at the low-affinity site, or S2, may also confer greater reuptake

rate. The increased affinity of DAT at S2 may physically represent a relaxation in the

binding pocket in order to allow substrates into the entryway more easily and efficiently.

If this is true, then it would follow that substrates are able to slide from S2 to S1 with

more ease, as the conformational change for flexibility within TMs 1, 3, 6 and 8

surrounding the entire binding pocket containing S1 and S2 may have already begun.

However, we would suspect that increased affinity of DAT for cocaine-like substrates at

S2 would be indicated by a change in apparent Km from voltammetric data, which we did

not observe. This theory could still be possible without an alteration in apparent Km, as

these reaction constants Kd and Km are calculated very differently. As previously

mentioned, the calculation of apparent Km relies upon several factors that are also

changing during FSCV recordings, such as stimulation frequency, amount of DA released

and Vmax (Greco and Garris, 2003), whereas two-site binding modeling only relies on

substrate concentration and the measured output, which in this case is radioactive counts

representing amount of ligand bound. It is possible that the use of high frequency

electrical stimulation in our FSCV experiments as well as potential alterations in basal

[DA] that were not accounted for may have contributed to no observation in apparent Km

even though there appears to be an alteration in DAT affinity at the S2 binding site

190

(decreased Kd2). Additionally, with the Michaelis-Menten model using only one reaction

constant, this may represent an averaging of the affinity contributions from both high-

and low-affinity binding sites depicted from the two-site membrane binding experiments

and the difference observed in the low-affinity site may be lost in averaging.

It is important to note that the shift in binding site composition toward more high-

affinity binding sites does not necessarily mean a dramatic alteration in chemical

composition (e.g. via post-translational modifations) of the binding pocket. Rather, we

hypothesize that this observation is indicative of the flexibility of the whole binding

pocket including S1 and S2 following chronic speedball SA, such that substrates similar

to the radioactive cocaine-like ligand [125

I]RTI-55 was able to occupy more of the S1

binding sites as compared to the saline controls, as indicated by an increased percent of

high-affinity binding sites with [125

I]RTI-55 binding. The mechanism by which a

relaxation in the DAT binding pocket may occur to allow substrates easier access to the

S1 site could include faster or greater sequestration of Na+ ions to open the binding

pocket. Claxton et al. utilized site-directed spin labeling and electron paramagnetic

resonance analysis to capture the dynamics of LeuT in the region of the extracellular

entryway associated with binding of Na+ and substrate. The results showed that Na

+

increases the accessibility of the extracellular entryway, specifically at TM1, TM3 and

ECL4 that project into the entryway above the binding sites (Claxton et al., 2010). The

authors noted an increase in motility and looser packing of the vestibule in the presence

of Na+ and thus greater induction of an outward-facing conformation. Na

+-induced

relaxation of the entryway would indicate greater accessibility to S2, which is located in

the binding pocket entrance above S1. Perhaps long-term speedball SA leads to increased

191

[Na+]e that could contribute to a relaxed entryway into the DAT binding site, which

would be a contributing factor in our observations of increased DAT affinity at the low-

affinity S2 binding site. This loosening of the entryway would also allow substrates to

enter the binding pocket and slide into the high-affinity S1 binding site with greater ease,

contributing also to our observed increase in the percent of high-affinity binding and

overall increased baseline reuptake rate.

Although further studies are necessary to confirm similar findings following

speedball SA, it has been shown with repeated cocaine or amphetamine administration

during the period of early abstinence that NAc core medium spiny neurons (MSNs)

exhibit increased intrinsic excitability, although opposite effects were observed in the

NAc shell (Kourrich and Thomas, 2009). The authors reported an increase in NAc core

MSN firing capacity, decreased inter-spike interval, increased duration of spike trains and

decreased first-spike latency in NAc core MSNs, although there was no observed change

in input resistance, possibly do to the lower input resistance of NAc core neurons as

compared to shell (Kourrich and Thomas, 2009). These alterations in intrinsic excitability

following cocaine or amphetamine administration may be applicable to speedball, as all

three drugs function to increase NAc [DA]e, albeit by slightly different mechanisms.

Even though calcium and glutamate signaling also contribute to increased intrinsic

excitability of NAc core MSNs, it is possible that an increase in extracellular Na+ levels

([Na+]e) contributes to the increased excitability following treatment with drugs that

increase [DA]e. Measuring [Na+]e in vivo is a difficult task due to the rapid speed at

which the sodium gradient is regulated by the Na+/K

+-ATPase, and to date there have

been no reports on the effects of increased DA or administration of drugs of abuse

192

directly on [Na+]e in the NAc. However, if long-term speedball SA did indeed increase

[Na+]e, it is likely that this increase would contribute to the increased excitability of NAc

core MSNs. This idea is inferred from whole cell patch clamp experiments performed in

cultured hippocampal pyramidal cells where increases in [Na+]e directly increased both

action potential frequency and amplitude (Arakaki et al., 2011). These alterations indicate

both a pre- and postsynaptic mechanism of increased intrinsic excitability, but it is

possible that only increased action potential frequency would result from increased [Na+]e

in slices, since the results have only been published in cultured neurons thus far.

Experiments investigating the effects of speedball SA on NAc [Na+]e could lend credence

to our hypothesis that DAT binding site flexibility may be induced by basal increases in

[Na+]e following chronic SA.

Although we have determined that chronic speedball significantly increased the

maximal reuptake rate of DAT and affinity of the low-affinity DAT binding site, and we

have identified a shift toward a greater percentage of high-affinity DAT binding sites

exposed for radioligand binding, we cannot discount the possibility of other transport

mechanisms being involved in these observations. Organic cation transporters (OCTs)

belong to a family of polyspecific transporters expressed throughout various mammalian

tissues including the brain and thought to function in a detoxification capacity (Amphoux

et al., 2006). In vitro uptake assays for monoamines have been conducted with rat and

human OCT1, OCT2 and OCT3 transfected in HEK293 cells. [3H]DA uptake could only

be detected with human OCT2, while rat OCT1-3 all showed affinity for DA, albeit in the

1-2 µM range while DAT affinity for DA is in the nM range (Amphoux et al., 2006).

OCT3 protein has been shown to be expressed in the NAc, CPu and VTA in small cells

193

(5-8 µm in size), hypothesized to be glia because of the much larger dimensions of

typical neurons in those basal ganglia regions (Gasser et al., 2009). Because of the low

affinity of OCTs for DA, it is hypothesized that these proteins may be active during

instances of very high concentrations of monoamines, and as such the role of OCTs

should be examined in the case of supra-additive increases in NAc [DA]e caused by

speedball administration. Even though OCTs have a markedly reduced affinity for DA

than DAT does, the possibility that our membrane binding data for the low-affinity DAT

binding site may actually represent radioligand binding to an OCT protein does exist.

However, this is unlikely due to both the order of magnitude difference between our

observed low-affinity Kd value and the affinities reported for OCT1-3, as well as the

same study that reported no effect on [3H]DA uptake by addition of cocaine to the assay

(Amphoux et al., 2006), implying that cocaine-like compounds will not bind to OCT

proteins.

Future experiments are desired to elucidate the physical mechanisms by which we

observe greater affinity of DAT at the low-affinity or secondary binding site and a shift

toward increased percentage of high-affinity binding without an overall increase in total

DAT binding sites, which contribute to an increase in baseline reuptake rate after

prolonged speedball SA. Logically, we would next determine if speedball administration

could possibly contribute to an increase in NAc [Na+]e and if DAT can maintain an

altered conformation and increased uptake rate in such an environment. We would also

utilize site-directed mutagenesis to determine any possibilities of amino acids that may

contribute to an open-facing DAT conformation and then determine if these mutant

DATs do function at an increased rate of reuptake. Our findings incorporated with the

194

current literature investigating DAT binding sites and DA translocation mechanisms will

hopefully spur the continued research of the effects of chronic elevated [DA]e by drug-

induced mechanisms, particularly those involving multiple substances, as the need for

greater understanding of the neurochemical alterations from polydrug abuse is urgent.

195

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APPENDIX

LICENSE AGREEMENTS TO REPRINT PUBLICATIONS AS

CHAPTERS II AND III FOR THIS DISSERTATION

231

SCHOLASTIC VITAE

Lindsey P. Pattison, Ph.D.

Education

Wake Forest University Graduate School of Arts and Sciences

Aug. 2008 – May 2013

Winston-Salem, NC 27157

Doctor of Philosophy Degree received on May 20, 2013

Graduate Program: Program in Neuroscience

Laboratory of Dr. Scott Hemby in the Department of Physiology and

Pharmacology

James Madison University Aug. 2004 – May 2008

Harrisonburg, VA 22807

Bachelor of Science Degree Received on May 3, 2008

Major: Chemistry (American Chemical Society Certified Biochemistry

Concentration)

Minor: Mathematics

Academic and Professional Activities and Honors

2007 Granted a stipend by the US National Science Foundation to participate in the

Research Experience for Undergraduates Program in the Chemistry

Department at James Madison University

2008 American Institute of Chemists Award from James Madison University

2008 Granted a travel award by the JMU Chemistry Department to attend the 235th

American Chemical Society National Meeting

2008 Invited oral presentation at the James Madison University Undergraduate

Student Research Day

2010 Abstract selected for inclusion as a lay-language summary in the Press Book

for the 40th

Meeting of the Society for Neuroscience

2012 Invited oral presentation at the 8th

Annual Conference of the US Human

Proteomics Organization

2012 Awarded a Career Development Award at the 8th

Annual Conference of the

US Human Proteomics Organisation

2012 Appointed Assistant Editor of an Open Access journal, Neuroproteomics

2012 Awarded a scholarship to attend the 2nd

Latin American School of Advanced

Neurochemistry, “The synapse in health and disease”

2012 Awarded the Best Student Poster Presentation Award at the 5th

Special

Conference of the International Society of Neurochemistry

2012 Granted the Alumni Student Travel Award by the Wake Forest Graduate

School of Arts and Sciences to attend the 42nd

Annual Meeting of the Society

for Neuroscience

2013 Awarded the Runner-Up to Best Poster Award for the Integrative Science

Category at the 13th

Annual Graduate Student and Postdoctoral Fellow

232

Research Day for Wake Forest Graduate School of Arts and Sciences

Professional Societies

2007 – 2008 Member, American Chemical Society

2008 – 2013 Member, Society for Neuroscience

2008 – 2013 Member, Western North Carolina Chapter of the Society for

Neuroscience (WNCSfN)

2008 – 2013 Member, Brain Awareness Council, Wake Forest University Graduate

School of Arts and Sciences

2009 – 2010 Committee Chairperson, Faculty Teaching Award Council, Program in

Neuroscience at Wake Forest University Graduate School

2009 – 2010 Member, Project REACH Tutoring for High School Students, Wake

Forest University

2009 – 2013 Contributing Author for The Neurotransmitter, quarterly newsletter for

the WNCSfN

2009 – 2013 Steering Committee Member, Brain Awareness Council

2010 – 2013 Committee Chairperson of Brain Awareness Week for Brain

Awareness Council

Publications

1. Pattison LP, Bonin KD, Hemby SE, Budygin EA. 2011. Speedball induced

changes in electrically stimulated dopamine overflow in rat nucleus accumbens.

Neuropharmacology, 60(2-3), 312-317. PMID: 20869972.

2. Pattison LP, McIntosh S, Budygin EA, Hemby SE. 2012. Differential regulation

of accumbal dopamine transmission in rats following cocaine, heroin and

speedball self-administration. J Neurochem, 122(1), 138-146. PMID: 22443145.

3. Pattison LP, McIntosh S, Sexton T, Xiao R, Grigg AL, Childers SR, Hemby SE.

In preparation to be submitted. Effects of cocaine, heroin and speedball self-

administration on dopamine transporter binding sites.

Abstracts

1. Pattison LP, East RE, Mariani VL. April 9, 2008. Kinetic analysis of N-

acylethanolamine-hydrolyzing acid amidase toward a novel class of anti-

inflammatory drugs. The 235th

American Chemical Society National Meeting.

2. Pattison LP, Hemby SE, Budygin EA. October 30, 2009. Effects of cocaine,

heroin and speedball on dopamine dynamics in the rat nucleus accumbens: a

voltammetry study. 2009 Western North Carolina Chapter of the Society for

Neuroscience Research Symposium.

3. Pattison LP, McIntosh S, Horman BM, Budygin EA, Hemby SE. November 14,

2010. Comparison of mesolimbic dopaminergic system neurotransmission using

in vivo microdialysis and fast-scan cyclic voltammetry following cocaine, heroin

and “speedball” self-administration. The 40th

Annual Meeting of the Society for

Neuroscience.

4. Pattison LP, McIntosh S, Horman BM, Budygin EA, Hemby SE. November 19,

2010. Comparison of mesolimbic dopaminergic system neurotransmission using

in vivo microdialysis and fast-scan cyclic voltammetry following cocaine, heroin

233

and “speedball” self-administration. 2010 Western North Carolina Chapter of the

Society for Neuroscience Research Symposium.

5. Pattison LP, McIntosh S, Grinevich V, Budygin EA, Hemby SE. June 21, 2011.

Dopamine transmission following acute and chronic “speedball” administration.

The 73rd

Annual Meeting of the College on Problems of Drug Dependence.

6. Pattison LP, Riegle MA, Staniforth ER, Skelly MJ, Dolson EP, Martucci KT,

Willard SL, Wiggins WF, Perret DS, Godwin, DW. November 12, 2011. Brain

awareness season in NC’s triad area. Annual Brain Awareness Campaign Event at

the 41st Annual Meeting of the Society for Neuroscience.

7. Pattison LP, Tannu NS, McIntosh S, Grigg AL, Hemby SE. November 16, 2011.

Membrane proteomic analysis of rat nucleus accumbens following self-

administration of cocaine, heroin and cocaine/heroin combinations. The 41st

Annual Meeting of the Society for Neuroscience.

8. Pattison LP, Tannu NS, McIntosh S, Grigg AL, Hemby SE. November 18, 2011.


administration of cocaine, heroin and cocaine/heroin combinations. 2011 Western

North Carolina Chapter of the Society for Neuroscience Research Symposium.

9. Pattison LP, Tannu NS, McIntosh S, Grigg AL, Hemby SE. March 6, 2012.


administration of cocaine, heroin and cocaine/heroin, “speedball,” combinations.

The 8th

Annual Conference of the US Human Proteomics Organization.

10. Pattison LP, Tannu NS, McIntosh S, Grigg AL, Hemby SE. March 29, 2012.

Membrane proteomic analysis of rat nucleus accumbens following speedball self-

administration. The 12th

Annual Graduate Student and Postdoctoral Fellow

Research Day for Wake Forest Graduate School of Arts and Sciences.

11. Pattison LP, Tannu NS, McIntosh S, Grigg AL, Hemby SE. September 13, 2012.

Membrane proteomic analysis of rat nucleus accumbens following speedball self-

administration. The 5th

Special Conference of the International Society for

Neurochemistry.

12. Pattison LP, Aston ER, Riegle MA, Rose JH, Spence TE, Skelly MJ, Johnston

W, Haynes K, Bach S, Godwin DW. October 14, 2012. Wake Forest University’s

Brain Awareness Council: Illuminating minds, one brain at a time. The 42nd

Annual Meeting of the Society for Neuroscience.

13. Pattison LP, Childers SR, Xiao R, Grigg AL, Hemby SE. October 17, 2012.

Altered dopamine transporter and D2 receptor expression and function in the

nucleus accumbens of rodents following chronic self-administration of

cocaine/heroin combinations. The 42nd

Annual Meeting of the Society for

Neuroscience.

14. Pattison LP, Sexton T, Xiao R, Grigg AL, McIntosh S, Childers S, Hemby SE.

December 6, 2012. Altered dopamine transporter function in the nucleus

accumbens of rodents following chronic self-administration of cocaine/heroin

combinations. 2012 Western North Carolina Chapter of the Society for

Neuroscience Research Symposium.

15. Pattison LP, Sexton T, Xiao R, Grigg AL, McIntosh S, Childers S, Hemby SE.

March 21, 2013. Altered dopamine transporter function in the nucleus accumbens

of rodents following chronic self-administration of cocaine/heroin combinations.

234

The 13th

Annual Graduate Student and Postdoctoral Fellow Research Day for

Wake Forest Graduate School of Arts and Sciences.

Teaching Experience

Biochemistry Lab Teacher’s Assistant 2007 – 2008

James Madison University, Department of Chemistry and Biochemistry

Conducted hands-on demonstrations for small groups in lab

and one-on-one instruction for laboratory protocols and

homework assignments

Course Facilitator: Dr. Victoria Mariani

Brain Awareness Council Activities 2008 – 2013


Attended 20+ visits to K-12 schools in the Winston-Salem area

to run hands-on activity stations while introducing topics in

neuroscience to students

Coordinated the activity stations for field trips to local

museums for 400+ students each spring during Brain

Awareness Week

Faculty Director: Dr. Dwayne Godwin

Course Designer and Facilitator for Scientific Outreach (GRAD 709 & 710)

2012 – 2013


Designed this course for graduate students to receive credit for

participating in scientific outreach and got the course approved

by the graduate school office

Created a syllabus and facilitated the course for two semesters

o GRAD 709 & 710 were added to the Graduate Program

in Neuroscience required curriculum

Faculty Director: Dr. Dwayne Godwin

Guest Lecturer for Anatomy & Physiology II (BIO 2312) August 29 & 31, 2012

Winston-Salem State University, Department of Life Sciences

Adapted and presented lecture presentations from the text on neuroanatomy

and cranial nerves

Used preserved human brains for an interactive demonstration with the

students

Course Professor: Dr. Jeff Overholt

Guest Lecturer for Pharmacology for Doctoral of Physical Therapy Students

(DPT 6206) January 9, 2013

Winston-Salem State University

Developed and presented a lecture on the topic of psychostimulants for the

“Drug Abuse Workshop” in a pharmacology course for Physical Therapy

students

235

Designed an in-class activity and prepared questions for an exam to evaluate

the students

Course director: Dr. Allyn Howlett, Professor of Physiology and

Pharmacology at Wake Forest School of Medicine

Adjunct Laboratory Instructor for Anatomy & Physiology I (BIO 165)

Sept. 2012 – May 2013

Guilford Technical Community College, Department of Biology

Taught one laboratory section of Anatomy & Physiology I

during the fall 2012 semester

Teaching three laboratory sections of Anatomy & Physiology I

during the spring 2013 semester

Used human models and dissected preserved cats to teach

students about the skeletal, muscular, blood vessel, digestive,

urinary, respiratory and reproductive systems

BIO 165 Laboratories Chair: Jeremy Creech

Biology Department Chair: Dr. George Whitesides

Professional Development Activities

Teaching & Learning Center Seminars Fall 2012 & Spring 2013

Wake Forest University Professional Development Center

Attended the following seminars in the “Post Docs and Teaching Assistants

New Instructor Series”

o What are Learning Objectives and How to Write Them (September 20,

2012)

o Interactive Teaching (October 11, 2012)

Attended the following seminars in the “Science, Technology, Engineering

and Mathematics (STEM) Series”

o Introduction to Scientific Teaching (January 29, 2013)

Seminar Facilitator: Catherine Ross, Director of the Teaching and Learning

Center

Topics in College Level Teaching (GRAD 720) Fall 2012 & Spring 2013


Graduate-level course offered to Ph.D. Candidates only

Received credit for completing teaching internships and creating a teaching

portfolio

Course director: Dr. Allyn Howlett, Professor of Physiology and

Pharmacology at Wake Forest School of Medicine

speedball by lindsey p. pattison wake forest … · figure 5. representative images from dat...

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