functional alterations in the dopamine transporter of rodents following self-administration of ...

248 Pages·2013·3.58 MB·English

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.
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.

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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

iii
LIST OF ABBREVIATIONS
[125I]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+] intracellular calcium concentration
I
[DA] dopamine concentration
[DA] extracellular dopamine concentration
e
[DA] concentration of dopamine release per stimulation pulse
p
[Na+] extracellular sodium concentration
e
[Na+] intracellular sodium concentration
i
3-MT 3-methoxytyramine
5-HT serotonin
6-MAM 6-monoacetylmorphine
AC adenylyl cyclase
ANOVA analysis of variance
B binding density
max
B 1 high-affinity binding density
max
B 2 low-affinity binding density
max
B total high- and low-binding densities combined
max
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
iv
COMT catechol-O-methyl transferase
CPE carboxypeptidase
CPu caudate putamen
D1R dopamine D1 receptor subtype
D2L dopamine D2 receptor long isoform
D2R dopamine D2 receptor subtype
D2S dopamine D2 receptor short isoform
D3R dopamine D3 receptor subtype
D4R dopamine D4 receptor subtype
D5R dopamine D5 receptor subtype
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
v
Gα inhibitory G protein alpha subunit
i/o
Gα stimulatory G protein alpha subunit
s/olf
glu glutamate
GP globus pallidus
GPCR G protein-coupled receptor
GP globus pallidus external
e
GP globus pallidus internal
i
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
K dissociation constant
d
K 1 high-affinity dissociation constant
d
K 2 low-affinity dissociation constant
d
K Michaelis-Menten constant, representing apparent affinity
m
L-DOPA L-3,4-dihydroxyphenylalanine
LeuT leucine transporter purified from Aquifex aeolicus
Aa
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
vi
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
vii
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
V maximal reuptake rate
max
VP ventral pallidum
VTA ventral tegmental area
WF-23 2β-propanoyl-3β-(2-naphthyl) tropane

viii
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 (K , or Michaelis-Menten rate constant) in the NAc
m
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 (V ) in the NAc of anesthetized
max
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

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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,
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) 102
Figure 4. DAT apparent affinity (K ) in the NAc of anesthetized rats (n = 5/group)
m
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) 105
Figure 5. (A) DAT-mediated maximal reuptake rate (V ) in the NAc of
max
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, V calculated at three time points prior to
max
bolus drug infusions as compared to the baseline V values of drug-
max
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 K , in NAc following i.v. challenge infusions
m
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

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