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The Involvement of Ventral Tegmental Area Dopamine and CRF Activity in Mediating the Opponent Motivational
Effects of Acute and Chronic Nicotine
by
Taryn Elizabeth Grieder
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Institute of Medical Science University of Toronto
© Copyright by Taryn Elizabeth Grieder 2012
ii
The Involvement of Ventral Tegmental Area Dopamine and CRF
Activity in Mediating the Opponent Motivational Effects of Acute
and Chronic Nicotine
Taryn Elizabeth Grieder
Doctor of Philosophy
Institute of Medical Science University of Toronto
2012
Abstract
A fundamental question in the neurobiological study of drug addiction concerns the
mechanisms mediating the motivational effects of chronic drug withdrawal. According to one
theory, drugs of abuse activate opposing motivational processes after both acute and chronic
drug use. The negative experience of withdrawal is the opponent process of chronic drug use that
drives relapse to drug-seeking and -taking, making the identification of the neurobiological
substrates mediating withdrawal an issue of central importance in addiction research. In this
thesis, I identify the involvement of the neurotransmitters dopamine (DA) and corticotropin-
releasing factor (CRF) in the opponent motivational a- and b-processes occurring after acute and
chronic nicotine administration.
I report that acute nicotine stimulates an initial aversive a-process followed by a
rewarding opponent b-process, and chronic nicotine stimulates a rewarding a-process followed
iii
by an aversive opponent b-process (withdrawal). These responses can be modeled using a place
conditioning paradigm. I demonstrate that the acute nicotine a-process is mediated by phasic
dopaminergic activity and the DA receptor subtype-1 (D1R) but not by tonic dopaminergic
activity and the DA receptor subtype-2 (D2R) or CRF activity, and the opponent b-process is
neither DA- nor CRF-mediated. I also demonstrate that the chronic nicotine a-process is DA- but
not CRF-mediated, and that withdrawal from chronic nicotine (the b-process) decreases tonic but
not phasic DA activity in the ventral tegmental area (VTA), an effect that is D2R- but not D1R-
mediated. I show that a specific pattern of signaling at D1Rs and D2Rs mediates the motivational
responses to acute nicotine and chronic nicotine withdrawal, respectively, by demonstrating that
both increasing or decreasing signaling at these receptors prevents the expression of the
conditioned motivational response. Furthermore, I report that the induction of nicotine
dependence increases CRF mRNA in VTA DA neurons, and that blocking either the
upregulation of CRF mRNA or the activation of VTA CRF receptors prevents the anxiogenic
and aversive motivational responses to withdrawal from chronic nicotine.
The results described in this thesis provide novel evidence of a VTA DA/CRF system,
and demonstrate that both CRF and a specific pattern of tonic DA activity in the VTA are
necessary for the aversive motivational experience of nicotine withdrawal.
iv
Acknowledgments
It’s pretty shocking that a full seven years have passed since I began the amazing
experience that is graduate school. There are so many people who have influenced my life
throughout this time that it’s hard to decide where to begin. However, as we all know, everything
ultimately began with my parents, Sherri and Gord, who have been the most loving, supportive,
and motivating people throughout my studies and life as a whole. I couldn’t have asked for better
friends and fans.
A huge thanks to my supervisor and mentor, Derek van der Kooy, aka the most
knowledgeable man I’ve ever met. Without the excellent guidance and gentle (ok, sometimes
harsh) constructive criticism I received along the way, I would not have learned and grown into
the scientist I pride myself in being today. Thanks as well to my program advisory committee
members Rachel Tyndale, Bernard Le Foll and Larry Grupp for all their advice and help, and
especially the final push to graduate.
To my love, Oleg, thanks for the support and for putting up with me (and Mini-Me)! A
special thanks to my collaborator, co-author, occasional partner in crime, and always beloved
friend, Olivier George. Our many hours spent dreaming up mad scientific genius ideas have
actually paid off! Thanks as well to my best friends Michelle and Katie, my sisters Jenna and
Holly, Kyle, Lauren, Susan and my lacrosse teammates (FTG!), and of course my unstoppable
dog Bender. Although none of you really understand what I do except that it involves nicotine
and killing mice for their brains, the love and support you’ve all given me has been simply
amazing.
No acknowledgement would be complete without mention of the amazing people I’ve
worked with in the van der Kooy lab throughout the years. My desk buddy Jessica, fellow motis
Ryan and Drew, laboids Mary Rose, Simon, Rachel and Brenda, and of course my strangest and
dearest friend and roomie, Hector Vargas-Perez. I love you, d__che. Without all of you to
complain, sympathize, and party with, these years would not have flown by so quickly.
I dedicate this thesis to my family: Luka, Oleg, and the Grieders. I love you more than
words could ever say.
v
Contributions
For Chapter 2: Dopaminergic Signaling Mediates the Motivational Response Underlying the
Opponent Process to Chronic but Not Acute Nicotine.
Authors: Taryn E. Grieder, Laurie H. Sellings, Hector Vargas-Perez, Ryan Ting-A-Kee, Eric C.
Siu, Rachel F. Tyndale and Derek van der Kooy.
Author contributions: T.E.G., O.G., B.L.F. and D.V.D.K. designed the experiments. S.G.
provided D1KO mice. T.E.G. performed the minipump surgeries and the place conditioning
experiments, and H.T. and S.R.L. performed the electrophysiology. T.E.G. and O.G. analyzed
the data. T.E.G., O.G. and D.V.D.K. wrote the paper. All authors discussed the results and
commented on the manuscript.
For Chapter 3: Phasic D1 and Tonic D2 Dopamine Receptor Signaling Double Dissociate the
Motivational Effects of Acute Nicotine and Chronic Nicotine Withdrawal.
Authors: Taryn E. Grieder, Olivier George, Huibing Tan, Susan R. George, Bernard Le Foll,
Steven R. Laviolette and Derek van der Kooy
Author contributions: T.E.G., O.G., B.L.F. and D.V.D.K. designed the experiments. S.G.
provided D1KO mice. T.E.G. performed the minipump surgeries and the place conditioning
experiments, and H.T. and S.R.L. performed the electrophysiology. T.E.G. and O.G. analyzed
the data. T.E.G., O.G. and D.V.D.K. wrote the paper. All authors discussed the results and
commented on the manuscript.
For Chapter 4: Recruitment of a VTA CRF system mediates the aversive effects of nicotine
withdrawal.
Authors: Taryn E. Grieder, Hector Vargas-Perez, Candice Contet, Laura A. Tan, John Freiling,
vi
Laura Clarke, Elena Crawford, Pascale Koebel, Brigitte L. Kieffer, Paul E. Sawchenko, George
F. Koob, Derek van der Kooy and Olivier George.
Author Contributions: TEG and OG designed the experiments. TEG and HVP performed
minipump, cannulation and viral vector surgeries. TEG performed place conditioning and open
field testing. CC, LAT and PES performed in situ hybridization. CC performed double in situ
hybridization and immunohistochemistry. TEG and LC performed rtPCR. JF and EC performed
immunohistochemistry. CC, PK and BLK supplied viral vectors. TEG analyzed the data. TEG,
CC, GFK, DVDK and OG wrote the paper. All authors discussed the results and read the paper.
vii
Table of Contents
Abstract ........................................................................................................................................... ii
Acknowledgments .......................................................................................................................... iv
List of Figures ................................................................................................................................ ix
List of Abbreviations .................................................................................................................... xii
Chapter 1 ....................................................................................................................................... 1
General Introduction .................................................................................................................... 2
1.1 What is Motivation? ............................................................................................................... 2
1.2 The Study and Measurement of Motivation .......................................................................... 3
Operant conditioning procedures ................................................................................................................................. 4
Classical conditioning procedures ................................................................................................................................ 7
1.3 Models of Drug Motivation ................................................................................................. 13
The Mesolimbic Dopamine Reward Hypothesis .................................................................................................. 13
The error prediction model ........................................................................................................................................... 16
The incentive-‐sensitization theory ............................................................................................................................ 18
The non-‐deprived/deprived hypothesis ................................................................................................................. 20
The opponent process theory ...................................................................................................................................... 22
1.4 The Anatomy of the Ventral Tegmental Area ..................................................................... 27
Neurons and Projections ................................................................................................................................................ 28
Nicotinic Receptors ........................................................................................................................................................... 29
1.5 The Neurobiology of Nicotine Motivation: DA and CRF ................................................... 31
The Use and Abuse of Nicotine .................................................................................................................................... 31
VTA Dopamine .................................................................................................................................................................... 33
1.6 Research Aims and Hypotheses ........................................................................................... 37
Chapter 2 ..................................................................................................................................... 42
Dopaminergic Signaling Mediates the Motivational Response Underlying the Opponent
Process to Chronic but Not Acute Nicotine .............................................................................. 42
Abstract .................................................................................................................................................................................. 43
Introduction ......................................................................................................................................................................... 44
viii
Materials and Methods .................................................................................................................................................... 45
Results .................................................................................................................................................................................... 52
Discussion ............................................................................................................................................................................. 66
Chapter 3 ..................................................................................................................................... 71
Phasic D1 and Tonic D2 Dopamine Receptor Signaling Double Dissociate the Motivational
Effects of Acute Nicotine and Chronic Nicotine Withdrawal ................................................. 71
Abstract .................................................................................................................................................................................. 72
Introduction ......................................................................................................................................................................... 73
Materials and methods .................................................................................................................................................... 74
Results .................................................................................................................................................................................... 77
Discussion ............................................................................................................................................................................. 93
Chapter 4 ..................................................................................................................................... 98
Recruitment of a VTA CRF system mediates the aversive effects of nicotine withdrawal .. 98
Abstract .................................................................................................................................................................................. 99
Introduction ....................................................................................................................................................................... 100
Materials and methods .................................................................................................................................................. 100
Results .................................................................................................................................................................................. 107
Discussion ........................................................................................................................................................................... 127
Chapter 5 ................................................................................................................................... 129
General Discussion .................................................................................................................... 129
5.1 Overview ............................................................................................................................ 130
5.2 Overall conclusion ............................................................................................................. 143
5.3 Future directions ................................................................................................................. 144
References .................................................................................................................................. 149
Copyright Acknowledgements ................................................................................................. 166
ix
List of Figures
Figure 1.1. The place conditioning paradigm…………………………………………………. 9
Figure 1.2. The opponent process theory of motivation…………………………………….. 24
Figure 1.3. The allostatic state of drug addiction…………………………………………..... 26
Figure 1.4. The VTA: Neurons, receptors, inputs and projections………………………… 30
Figure 2.1. The opponent process theory of motivation and its modeling by use of the place
conditioning paradigm………………………………………………………………………… 46
Figure 2.2. The time course of spontaneous nicotine somatic and motivational
withdrawal……………………………………………………………………………………... 54
Figure 2.3. The opponent processes of chronic and acute nicotine and the effect of DA
antagonism…………………………………………………………………………………….. 57
Figure 2.4. Dopaminergic signaling differentially mediates the opponent motivational
process after acute and chronic nicotine……………………………………………………... 62
Figure 2.5. The D2R mediates the aversive response to chronic nicotine
withdrawal……………………………………………………………………………………... 65
Figure 3.1. Phasic DA activity mediates aversions to acute nicotine while the specific
pattern of tonic DA activity mediates aversions to withdrawal from chronic nicotine.…... 79
Figure 3.2. The DAR agonist and antagonist have no motivational effects on their own.... 81
Figure 3.3. The cannabinoid-1 receptor inverse agonist rimonabant significantly decreases
phasic VTA DA activity but does not affect tonic DA activity…..…………………………. 83
x
Figure 3.4. A specific pattern of signaling at D1Rs is required for aversions to acute
nicotine in nondependent mice, while a specific pattern of D2R activity is required for
aversions to nicotine withdrawal in dependent mice..……………………………..………... 85
Figure 3.5. D1R and D2R agonists and antagonists have no motivational effects on their
own at the doses used in this study, however a high dose of the D1R agonist prevents
learning………………..……………………………………………………………………….. 88
Figure 3.6. Manipulations of the A2AR block the aversive response to withdrawal from
chronic nicotine but not acute nicotine..……………………………………………………... 91
Figure 4.1. Nicotine dependence increases CRF mRNA levels in the VTA………………. 108
Figure 4.2. Nicotine dependence and withdrawal recruits and activates the CRF system in
the VTA……………………………………………………………………………………..… 110
Figure 4.3. Nicotine dependence increased the number of cells that contain CRF mRNA in
the pVTA but not aVTA……………………………………………………………………... 112
Figure 4.4. Double labeling of DA neurons and CRF mRNA using CRF in situ
hybridization and TH immunohistochemistry……………………………………………... 113
Figure 4.5. Withdrawal from chronic nicotine depletes CRF peptide in the pVTA……... 115
Figure 4.6. Nicotine dependence and withdrawal decreases CRF peptide density in the CeA
but not the PVN………………………………………………………………………………. 117
Figure 4.7. Downregulation of CRF mRNA in the VTA by a viral vector prevents the
aversive motivational response to withdrawal from chronic nicotine……………………. 119
Figure 4.8. The silencing vector must be injected in the VTA to block withdrawal
aversions………………………………………………………………………………………. 122
Figure 4.9. CRF1R antagonism prevents the aversive motivational response to withdrawal
from chronic nicotine………………………………………………………………………… 124
xi
Figure 4.10. The opponent motivational process occurring after acute nicotine is not
blocked by CRF1R antagonism……………………………………………………………... 126
Figure 5.1. Summary of the involvement of DA and CRF in the opponent motivational
responses occurring after acute and chronic nicotine……………………………………... 141
xii
List of Abbreviations
α-flu α-flupenthixol
ANOVA analysis of variance
AP anterior-posterior
aVTA anterior ventral tegmental area
A2AR adenosine receptor subtype-2A
CeA central nucleus of the amygdala
CRF corticotropin-releasing factor
CRF-BP corticotropin-releasing factor-binding protein
CRF1R corticotropin-releasing factor receptor subtype-1
CRF2R corticotropin-releasing factor receptor subtype-2
CMV cytomegalovirus
DA dopamine
DNA deoxyribonucleic acid
DV dorsal-ventral
D1R dopamine receptor subtype-1
D2R dopamine receptor subtype-2
EGFP enhanced green fluorescent protein
ELISA enzyme-linked immunosorbent assay
GABA gamma-aminobutyric acid
xiii
GAD glutamic acid decarboxylase
Hz hertz
i.p. intraperitoneal
KO knockout
ML medial-lateral
MΩ milliohm
mRNA messenger ribonucleic acid
ms millisecond
NAc nucleus accumbens
nAChR nicotinic acetylcholine receptor
NMDA N-methyl-D-aspartic acid
PBS phosphate buffered saline
PFC prefrontal cortex
PVN paraventricular nucleus
pVTA posterior ventral tegmental area
RNA ribonucleic acid
rtPCR real-time polymerasechain reaction
s.c. subcutaneous
siRNA small interfering ribonucleic acid
SEM standard error of the mean
xiv
TH tyrosine hydroxylase
TPP tegmental pedunculopontine nucleus
VP ventral pallidum
VTA ventral tegmental area
WT wild-type
1
Chapter 1
General Introduction
2
General Introduction 1
1.1 What is Motivation?
Broadly defined, motivation is the internal driving force that initiates, guides and
maintains goal-directed behaviour. Motivation is what causes an organism to act, whether it is
simply getting out of bed in the morning, finding water or food to reduce thirst or hunger,
reading a book to gain knowledge, or taking a drug to feel its pleasurable effects. These are
examples of conditioned motivation, because the organism learns to associate the benefits
obtained from the action, leading to future motivated responses. However, motivation is not
simply appetitive or approach behaviour, as organisms may also be motivated to avoid
experiences, stimuli, or environments that are perceived to be bad or aversive.
Motivation involves the biological, emotional and social forces that activate behaviour. In
everyday usage, the term motivation is frequently used to describe why a person or animal does
something. Whether the organism approaches, avoids, or is neutral toward a particular stimulus,
their action depends on the motivational context within which the stimulus has been perceived.
This process of attributing motivational value to a particular stimulus allows organisms to make
associations between the cues they encounter and positive or negative outcomes. In this sense,
motivation can be quantified using behavioural paradigms that measure an organisms’ appetitive
or aversive response to a particular stimulus. Motivation is thus defined here as the driving force
responsible for approach or avoidance behaviour.
Motivated behaviour is required for an organism’s survival: In the absence of basic
motivation, an organism would not eat or drink and would thus perish. However, not all
motivated behaviour is beneficial to the organism, as there are instances when motivation drives
the actions that lead to compulsive activities. The best example of this, and the focus of this
thesis, is drug addiction: the habitual psychological and physical dependence on a substance that
is beyond voluntary control and persists in spite of negative consequences. Drugs of abuse such
as nicotine, opiates, cocaine or ethanol are capable of producing robust appetitive responding and
3
subjective feelings of pleasure on the first exposure. This rewarding experience due to drug
exposure is attributed a positive motivational value and leads to subsequent motivated behaviour
to obtain more of the drug, eventually leading to physical dependence and withdrawal when drug
use is discontinued. The aversive experience of withdrawal then leads to further motivated
behaviour to avoid this negative outcome. The studies described in this thesis define the
neurobiological substrates mediating the rewarding and aversive motivational responses to acute
and chronic nicotine, being primarily concerned with the aversive motivational response
occurring after withdrawal from chronic nicotine.
1.2 The Study and Measurement of Motivation
Motivation can be studied at the psychological, physiological, sociological, or
philosophical levels, however, this thesis will focus on the neurophysiological study of
motivation. One of the first neurobiological demonstrations of motivation occurred almost 70
years ago by Olds and Milner after they observed that rats would repeatedly press a lever that led
to electrical stimulation of certain brain regions such as the septal area, mammilothalamic tract,
cingulate cortex and tegmentum (Olds and Milner, 1954). It was proposed that electrical
stimulation in certain areas of the brain produced acquisition and extinction curves that
compared with those produced by a primary reward. Furthermore, they observed that stimulation
of electrodes placed in other areas of the brain appeared to be punishing or aversive (Olds and
Milner, 1954). Subsequent studies reproduced this rewarding self-stimulation phenomena and
showed that the amount of reward increased with the amount of electrical stimulation, especially
in the area corresponding to a group of ascending and descending axon fibers, the medial
forebrain bundle, which passes through the hypothalamic system (Olds et al., 1960).
Furthermore, the rewarding effects of electrical brain stimulation in the medial forebrain bundle,
but not the septal area, could overshadow the appetitive properties of food even while under food
deprivation, with rats preferring to self-stimulate the medial forebrain bundle rather than to eat
and satisfy their hunger (Routtenberg and Lindy, 1965). These experiments and many more that
4
followed were the first neurobiological demonstration of motivation, showing that stimulation of
the medial forebrain bundle and other brain areas was both rewarding and drive inducing, and
stimulation of certain other brain areas was aversive and induced avoidance.
In the study of motivation, especially the motivational responses to abused drugs, it is
most often necessary as well as beneficial to utilize experimental animal subjects. The huge
amount of work and progress made in drug addiction research over recent years can be attributed
to animal models of drug motivation. However, animals cannot verbalize their feelings or
reactions to a drug stimulus, thus their motivation must be inferred by use of an accepted
experimental procedure. Motivation has been defined here as the driving force responsible for
conditioned approach or avoidance behaviour. As such, there have been a variety of behavioural
paradigms developed that can infer and quantify motivation by measuring an animals’ appetitive
or aversive response to a particular stimulus. These paradigms provide a way to assess the
neurobiological and behavioural processes underlying drug motivation, factors which may not be
easily tested in human subjects due to ethical restrictions. Furthermore, various animal models
may be used to investigate the relationship between environmental, developmental, behavioural
or neurobiological influences that are hypothesized to contribute to drug addiction. Most of these
procedures can be categorized as either operant or classical conditioning.
Operant conditioning procedures
Operant conditioning is a form of learning where an animal’s voluntary (operant)
behaviour is modified and maintained by its consequences. The consequences of the animal’s
behaviour are reinforcement or punishment, which cause subsequent behaviour to occur with
more or less frequency, respectively. When a certain behaviour leads to a reward, such as food
for a hungry animal, this behaviour is reinforced and the organism’s motivation is increased. In
other words, the driving force behind their approach behaviour increases. Conversely, if a
behaviour leads to punishment, the organism’s motivation to repeat that behaviour will be
decreased. Additionally, if a certain behaviour is inconsequential, it will eventually extinguish.
5
Operant conditioning procedures are typified by self-administration paradigms, where the
subject performs an action such as a lever press or nose poke that leads to the delivery of a
stimulus. The stimulus can come in many forms, such as a drug or shock delivery, access to
food, or electrical stimulation of the brain. The behaviour observed by Olds and Milner that rats
would work for stimulation of certain brain areas is an example of self-administration. In the
context of this thesis, I will discuss self-administration in terms of psychostimulant drug
delivery. The method of drug self-administration can be intravenous, through a surgically
implanted catheter, or directly to a specific area of the brain. Different schedules of
reinforcement may also be used in this procedure, with the simplest being a fixed ratio schedule
of continuous reinforcement. On this schedule, a fixed number of lever presses leads to the
delivery of one unit of the drug, which eventually leads to a stable pattern of drug self-
administration (Caine et al., 1993). Other schedules of reinforcement such as a second-order
schedule use a fixed number of lever presses that lead to a brief stimulus, usually visual in
nature, then the next fixed number of lever presses completed after that stimulus produces the
same brief stimulus accompanied by a drug injection (Katz and Goldberg, 1987). The brief
stimulus in this schedule of reinforcement will acquire its own reinforcing properties, termed
secondary reinforcers (Koob and Le Moal, 2006). Another schedule of reinforcement, which is
hypothesized to directly evaluate a drug’s reinforcing efficacy, is a progressive-ratio schedule of
reinforcement. In this schedule, the number of lever presses required for each successive drug
delivery is increased. Drugs that lead to higher numbers of responses are thought to be more
reinforcing. Using this method, a ‘break point’ can be determined where the subject will finally
cease responding, and again a higher break point is thought to be produced in drugs of abuse that
are more reinforcing (Koob and Le Moal, 2006).
In the self-administration paradigm, the rate of responding for a drug delivery is taken as
a measure of the reinforcing efficacy of the abused drug. When a stable rate of responding has
developed, which often requires initial training with food prior to the introduction of an abused
drug, the effects of experimental manipulations can be measured on responding. Common
manipulations are pharmacological pretreatment with agonists or antagonists, lesioning of a
particular brain region of interest, or more recently genetic methods such as ribonucleic acid
6
(RNA) interference or optogenetic approaches (which temporarily activate or silence specific
neurons) (Caille et al., 2012; Cao et al., 2011).
Although there is no animal model that fully mimics the human condition, self-
administration is the closest procedure to human drug-seeking and -taking, as it allows the
animal subjects to regulate their own amount of drug intake. Additionally, the animal may serve
as its own control, which reduces the number of animals required per study. However, there are
many drawbacks of the self-administration paradigm. For example, a number of abused drugs,
such as nicotine, caffeine and marijuana, are not readily self-administered by animals. Some
hallucinogenic compounds abused by humans, such as ecstasy, have never been shown to be
self-administered by animals (Corrigall, 1999). Nicotine and other drug self-administration is
acquired by rats and less often in mice, but usually requires extensive prior training with food
self-administration after deprivation. Furthermore, nicotine self-administration does not mimic
the inhalation route of administration that it is trying to model in humans (Corrigall, 1999).
Self-administration also relies on the animals’ ability to make a motor response (press a
lever or nose poke), thus the use of drugs that decrease motor activity, such as high doses of
abused drugs or dopamine (DA) receptor antagonists, may not provide accurate measures of
drug-taking behaviour. Similarly, if a drug increases motor activity, more lever presses may
occur and an inaccurate measure of drug reward may be obtained. Throughout self-
administration procedures, the animal is under the influence of the drug(s) being studied, making
the determination of whether changes in response rate are indeed due to motivational changes or
to drug-induced changes in motor responses or other unconditioned motivational effects difficult.
Additionally, self-administration procedures require extensive and time-consuming surgeries in
addition to the prior training that is often required with food self-administration and deprivation;
therefore a single self-administration experiment often requires many months to complete.
Importantly, self-administration procedures cannot measure the aversive motivational
properties of drugs, simply because animals will not self-administer an aversive substance. This
problem could be overcome by either negatively reinforcing the self-administration behaviour,
where the animal presses a lever to avoid the administration of an aversive substance, or training
7
the animal to self-administer food, then giving the aversive drug and observing if the motivation
or obtain food decreases. However these types of procedures are complicated and not widely
utilized.
Finally, perhaps the most concerning drawback of self-administration paradigms is the
fact that both increases or decreases in the amount of responding can be interpreted as increases
in the reinforcing properties and motivation to take the abused drug being studied. When an
animal increases responding, they are thought to find the outcome pleasurable and therefore want
more of the drug. Likewise, when the animal decreases responding, they are thought to have
responded less because each drug infusion is more rewarding or pleasurable. The use of a
progressive-ratio schedule of reinforcement and the determination of a break point in responding
addresses this problem to some extent, but further problems may then arise involving the relation
of the rate of responding (which can be influenced by motor effects, detailed above) to break
point, and these two factors are not necessarily correlated (Richardson and Roberts, 1996).
In summary, although self-administration more closely models human nicotine intake
(Rose and Corrigall, 1997), separating drug motivation due to its rewarding effects or the
alleviation of withdrawal is more easily performed using a classical conditioning procedure
(Mucha et al., 1982). The self-administration paradigm possesses a number of drawbacks that
classical conditioning procedures can account for, however, these procedures also posses their
own set of limitations which are detailed below.
Classical conditioning procedures
Classical conditioning is a form of learning in which one stimulus is associated with and
becomes a signal or predictor for the occurrence of another stimulus. Behaviours conditioned via
a classical conditioning procedure are not maintained by consequences as in operant
conditioning, but by the ability of one stimulus to successfully predict a second stimulus.
8
Classical conditioning procedures are typified by place conditioning, where animals
experience two similar but distinctly different neutral environments that are paired spatially and
temporally with distinct drug cues. The animal is passively administered a drug by the
investigator and associates the motivational effect of that drug with one of the environments.
During the alternate conditioning treatment, the animal is administered a vehicle control
treatment and placed in the other distinct environment. These drug- and vehicle-pairings are
performed for a fixed amount of time and number of cycles (the conditioning phase), after which
the animals undergo preference testing (Figure 1.1). During testing, the animals are given a
choice with equal opportunity to enter and explore either environment, and the amount of time
spent in the previously drug-paired environment versus the vehicle-paired environment is taken
as a measure of the motivational value of the drug. If the choice is made to spend more time in
the drug-paired environment relative to the vehicle-paired environment, the drug is considered to
be rewarding, and the motivational effect is a conditioned place preference. Conversely, if the
animal chooses to spend less time in the drug-paired environment relative to the vehicle-paired
environment, the drug is considered to have an aversive motivational effect and a conditioned
place aversion is observed. The behaviour observed by Olds and Milner where rats would return
to or avoid a particular area of a chamber where they had received electrical brain stimulation in
certain areas is an example of place conditioning.
The apparatus used in place conditioning experiments typically consists of two or three
distinct environments that may be differentiated from each other based on color, texture, smell
and/or lighting. The environments must be distinct for conditioning to develop and be observed,
as the animal associates the cues experienced in the separate environments with the motivational
effects of the drug in question. The distinct conditioning environments should be selected and
modified in a way such that the animal can differentiate between them, but should not exhibit a
preference for either of them in the absence of any treatment. A procedure with three
environments to choose between on testing adds additional controls for nonspecific effects and
permits easier balancing between the two environments being used for drug- and vehicle-pairings
(Koob and Le Moal, 2006). The pairing of the drug under investigation with a particular
environment is fully counterbalanced, with half the animals receiving drug first, the other half
9
Figure 1.1. The place conditioning paradigm.
Place conditioning is an example of a classical conditioning procedure where animals experience
two similar but distinct neutral environments, usually differing in the wall colour and floor
texture. During the conditioning phase, the animal is passively administered a drug by the
investigator and placed in one of the environments. During the alternate conditioning treatment,
the animal is administered a vehicle (usually saline) and placed in the other distinct environment.
These drug- and vehicle-pairings are performed for a fixed amount of time and number of cycles,
during which the animal associates the motivational effects of the drug in question with the
environments it was repeatedly paired with. During preference testing, the animals are given a
choice with equal opportunity to enter and explore either environment, and the amount of time
spent in the previously drug-paired environment versus the vehicle-paired environment is taken
as a measure of the motivational value of the drug. If the choice is made to spend more time in
the drug-paired environment relative to the vehicle-paired environment, the drug is considered to
be rewarding, and the motivational effect is a conditioned place preference. Conversely, if the
animal chooses to spend less time in the drug-paired environment relative to the vehicle-paired
environment, the drug is considered to have an aversive motivational effect and a conditioned
place aversion is observed.
10
11
receiving vehicle first, and half of each of those groups receiving one of the two different
environments first. In this sense, the place conditioning procedure uses an unbiased design. A
biased design is less appealing and more time-consuming, requiring a pre-conditioning phase to
assess pretest preferences, after which the drug is usually paired to the least-preferred
environment for each individual animal. Using this design, increases in the amount of time spent
in the drug-paired environment in comparison to pretest time are taken as a measure of the drug’s
rewarding properties. The behavioural work presented in this thesis was completed using an
unbiased, fully counterbalanced place conditioning paradigm, which will be described in further
detail in chapter 2.
The most important benefit of the place conditioning procedure is that it permits
measurement of both rewarding and aversive motivational effects of abused drugs. Furthermore,
the motivational response can often be observed after a single conditioning session with the drug
for a variety of abused drugs (Bardo et al., 1986; Grieder et al., 2010; Mucha et al., 1982),
allowing for the observation of drug reward or aversion without any induction of tolerance or
sensitization (Bardo and Bevins, 2000). Place conditioning procedures are relatively simple to
perform in comparison to self-administration procedures, requiring no pre-training and no
surgical implantation of a catheter, therefore the time required to set up and perform a place
conditioning experiment is considerably less, with some place conditioning experiments being
performed in just one week. Also, since the drug is usually not administered during testing, place
conditioning is independent of motor responses. In this sense, drugs that reduce motor activities
can be readily used in the place conditioning paradigm. However, it is also possible in some
studies to examine the effects of administration of the drug during testing on the expression of
motivational responses, which serves as a control for the presence of state-dependent learning
(learning that is exhibited only during a specific physiological and/or mental state).
Place conditioning procedures also allow the investigator to maintain precise control over
the amount of drug administered throughout the conditioning phase. However, this benefit also
can be considered a drawback, as the drug-seeking and -taking aspect that is modeled by self-
administration is lost when experimenters passively administer a drug during place conditioning
12
procedures. Another benefit of the place conditioning paradigm in terms of drug administration
is that testing can be and usually is performed in a drug-free state, thus there is no satiety or
motor effects on the results obtained.
Furthermore, most conditioning apparatuses can measure locomotion simultaneously
while recording motivational effects, providing an extra experimental measure for investigation.
The place conditioning paradigm is also adaptable to a wide variety of animal models, and could
even be modeled in human subjects due to the ease of experimental set up (Bardo and Bevins,
2000).
A common criticism of place conditioning procedures is that the animal may have an
innate motivational response to a novel environment. To address this concern, control
experiments can be performed at the same time with separate groups of animals that are tested
for a novelty preference or aversion. Similarly, handling the animals prior to conditioning to
familiarize them with injection and handling procedures that will be carried out during the
conditioning phase may control for any criticisms about stress effects on conditioning. Finally,
drugs often produce motivational effects with narrower dose ranges in place conditioning versus
self-administration procedures, leading to dose-response curves that are considered to be less
informative.
The study of drug motivation by use of operant or classical conditioning procedures
represents a similar phenomenon to Olds and Milner’s electrical self-stimulation in that drugs of
abuse represent stimuli that are not physiologically important. Although abused drugs such as
nicotine, opiates, alcohol or cocaine actually act in the exact opposite way as natural rewards
such as food and water, producing detrimental effects after continued use, they are nevertheless
capable of producing robust appetitive responding in a comparable way to natural rewards.
Research examining the neurobiological substrates important for the reinforcing effects of drugs
of abuse has shown that they are the same as those that are important for brain stimulation
reward (Koob and Le Moal, 2006). This research refined the theories put forth by Olds and
Milner and identified specific neurochemical and neuroanatomical pathways that are important
13
for the occurrence of motivational responses to drugs of abuse, leading to the development of a
variety of theoretical models describing drug motivation.
1.3 Models of Drug Motivation
Over the course of decades of drug addiction research, a variety of neurobiological
theories have been developed that attempt to identify and explain the neurocircuitry behind the
motivation to consume drugs of abuse. The following are brief summaries of the major
hypotheses that have been proposed since the discovery of electrical brain self-stimulation.
These theories are both complementary and contradictory, without one single theory having the
ability to fully explain drug motivation.
The Mesolimbic Dopamine Reward Hypothesis
One of the original theories of drug actions on the brain was the DA hypothesis, which
suggested that all drugs of abuse acted on dopaminergic neurotransmission in the brain reward
system (Wise, 1980). This hypothesis was formulated based on the considerable body of
evidence suggesting that the brain reward system, consisting of the fast-conducting myelinated
fibers of the medial forebrain bundle and the VTA dopaminergic neurons projecting to the
nucleus accumbens (NAc) of the ventral striatum, served as a final common pathway for the
transmission of rewarding motivational information (Wise, 1980). This theory originally focused
on the ventral tegmental-striatal projection, but later came to focus more broadly on the
mesolimbic DA system and its various inputs (Wise, 2002). Since the development of this
theory, a variety of experiments using a wide range of techniques have reported that activity of
the mesolimbic DA system is necessary for the motivational response to both natural rewards
and drugs of abuse.
Compelling evidence for this theory comes from studies showing that abused drugs act
directly on DA synapses (amphetamine and cocaine) or cell bodies (opiates) in the mesolimbic
DA system (Wise, 1980). Furthermore, studies that utilized pharmacologic manipulation of DA
14
signaling through DA receptor (DAR) antagonist drugs reported that blockade of DA signaling
strongly reduces or completely attenuates the reinforcing properties of natural reinforcers, such
as food reward (Wise et al., 1978), as well as the rewarding effects of various drugs of abuse,
such as morphine, nicotine, cocaine, amphetamine and ethanol (Acquas et al., 1999; Corrigall
and Coen, 1991; Price and Middaugh, 2004; Yokel and Wise, 1975). Additional evidence for this
theory came from studies showing that destruction of the mesolimbic DA system using 6-
hydroxydopamine lesions induces an anhedonic motivational state, wherein a general loss of
interest in both natural and other rewarding stimuli occurs (Wise, 1982). This suggested that
inactivation of the DA system rendered the subject incapable of feeling pleasure and without
motivation to seek reinforcing events or stimuli. Taken together, these studies implied that DA
function and the activation of the DA system is both necessary and sufficient for the mediation of
reward-related motivational signals, leading to the formulation of the mesolimbic DA
hypothesis.
The main idea behind this DA hypothesis was that the common feature of all drugs of
abuse is their ability to activate the mesolimbic DA system to produce reward. It was thus
postulated that a single neural system is the final common pathway for the transmission of all
rewarding motivational information. This theory is elegantly simple, but therein lays one of the
criticisms of this hypothesis: Drugs of abuse can, and do, act through other non-DA
neurobiological substrates. For example, studies in previously drug naive animals given acute
nicotine or cocaine demonstrated that the rewarding effects of these drugs are DA-independent
(Lanca et al., 2000; Laviolette et al., 2002; Mackey and van der Kooy, 1985). Furthermore, the
rewarding response to acute morphine is present in both wild-type (WT) and DAR subtype-2
(D2R) knockout (KO) mice (Dockstader et al., 2001). Rewarding responses after chronic drug
use can also be DA-independent, as the rewarding effects of chronic ethanol are not blocked by
DAR antagonism (Ting-A-Kee et al., 2009), and DA is not even necessary for the pursuit of
natural reinforcers such as food reward (Bechara and van der Kooy, 1992). Finally, mice that are
DA-deficient will still develop a conditioned place preference for cocaine (Hnasko et al., 2007).
Furthermore, DA has been shown in some situations to not signal reward at all, but rather
that changes in DA activity signal arousing or novel sensory events, or even aversive stimuli
15
(Schultz et al., 1992; Shultz, 2001). This predictive quality of DA was shown to be independent
of hedonic processing in monkeys that were conditioned over a very large number of trials to
receive sweetened juice after a neutral stimulus: Although DA neurons initially responded to the
juice reward, after conditioning the DA neurons no longer responded to the reward, but would
then respond to the presence (or absence) of the neutral stimulus alone (Schultz et al., 1992).
Further, some neurons showed activation following aversive, non-noxious stimuli (Schultz,
2001). Other studies have also shown that aversive footshocks increase DA release (Joseph et al.,
2003) and populations of DA neurons have been found in the VTA that are strongly excited by
footshocks (Brischoux et al., 2009). These results stand in direct contrast to the mesolimbic DA
hypothesis, as activation of the DA system did not occur during the experience of reward, but
rather during aversive experiences.
Perhaps the most compelling argument against the hypothesis that the DA system is the
exclusive mediator of reward signals, as well as the most relevant to this thesis, is that the
mesolimbic DA system can also be activated during aversive events. For example, this
hypothesis would assume that nicotine, a widely abused drug, would produce reward through a
DA-mediated system. However, acute nicotine administration in nondependent subjects can
produce an aversive motivational response in a place conditioning paradigm that is DA-
mediated, being blocked by DAR antagonists (Laviolette and van der Kooy, 2004; Tan et al.,
2009; also see chapters 2 and 3) or lesions of dopaminergic afferents to the NAc (Sellings et al.,
2008). DA is also released in the NAc after aversive footshocks in rats (Young et al., 1993), and
extracellular DA concentrations in the forebrain are increased after footshock or anxiogenic drug
administration in rats (Dazzi et al., 2001). Human imaging studies have also shown increased
DA signaling in the ventral tegmentum during aversive thermal stimulation (Becerra et al.,
2001). Taken together, these results suggest that increased DA does not necessarily signal reward
and largely disprove the hypothesis that the mesolimbic DA pathway represents a common and
sufficient system to explain reward.
Another problem with the DA hypothesis that drug-taking and -seeking is performed due
to its DA-increasing euphoric effects is the evidence that tolerance develops after repeated drug
administration. When tolerance develops, the pleasurable effects of drugs are decreased and may
16
be completely absent, but the subject continues to seek out and take drugs (Koob and Le Moal,
2006). Although the DA hypothesis remains as a prominent and influential theory, it is clear that
the role of DA and the mesolimbic system in motivation is far more complex than a simple
reward signal. Indeed, the studies reported in this thesis, as well as other data, suggest that
specific patterns of DA signaling actually mediate aversive motivational responses.
Consequently, other theories that have proven more relevant and encompassing in the
explanation of drug motivation have persisted.
The DA reward prediction error model
Both complementary and in contrast to the idea that DA signals the receipt of a reward or
reinforcing motivational stimuli, Schultz and colleagues have proposed the DA reward prediction
error model, whereby the slower activity of DA neurons is hypothesized to code the uncertainty
associated with rewards (Schultz, 2001; Schultz, 2007; Schultz et al., 2000; Schultz et al., 2002).
A series of experiments performed by Schultz and colleagues suggested that mesolimbic DA
projections from the midbrain to the striatum and frontal cortex show increases in activity
following primary food and liquid rewards (consistent with the DA hypothesis) as well as after
the presentation of conditioned, reward-predicting stimuli or novel stimuli (Schultz, 2001). They
also show depression by the attention-generating omission of reward or during aversive events
(Schultz et al., 2003; Schultz, 2007). This data led to the hypothesis that DA neurons are not in
general activated by salient stimuli, but are reporting rewards as far as they occur differently than
predicted, producing a ‘prediction error’ message that serves as a powerful teaching signal for
behaviour and learning (Schultz, 2001). This short-acting, subsecond DA message was
hypothesized to be different from the more long-term (ie. minutes) DA function in behavioural
responses and the much more long-term DA function (ie. hours to days) that is deficient in
Parkinson’s disease, suggesting that DA neurons serve different functions at different time
scales.
The prediction error hypothesis suggests that all responses to rewards, aversive events,
and reward-predicting stimuli depend on the predictability of an event. After extensive training,
17
Schultz’s monkeys were fully trained and the DA neurons no longer responded to receipt of a
liquid reward because it was fully predicted. However, the DA neurons were activated or
depressed if the predicted reward occurred sooner or failed to occur, respectively, at its habitual
presentation time (Schultz et al., 1993). These changes in DA activity reflect an expectation
process that is based on an internal clock that measures the precise timing of a predicted reward.
The DA response is hypothesized to be equal to the reward occurrence minus the reward
prediction (Schultz, 2001). This theory posits that the response to a reward does not occur
unconditionally, but rather codes the prediction error such that an unpredicted reward elicits
activation, a fully predicted reward elicits no response, and the omission of a predicted reward
(an aversive event) induces a depression (Schultz, 2007). In this sense, DA neurons do not
discriminate between or indicate different rewards, rather emitting an alerting message about the
surprising presence or absence of rewards. The prediction error process continues until the
behavioural outcome matches the prediction and the prediction error becomes nil.
Since the proposal of this theory, reward-responsive signals have been demonstrated in
the tegmental pedunculopontine nucleus (TPP) (Okada et al., 2009), the lateral habenula
(Bromberg-Martin et al., 2010) and the frontal cortex (Roesch and Olson, 2004). DA prediction
error signals have also been reported that respond to aversive stimuli in the rostromedial
tegmental nucleus (Jhou et al., 2009) and the lateral habenula (Matsumoto and Hikosaka, 2009).
Furthermore, DA firing has been suggested to code not only for the predicted timing of a reward,
but also for the size of a prospective reward (Bromberg-Martin and Hikosaka, 2009), leading to
the hypothesis that midbrain DA neurons are involved in information processing as well as
reward and aversive signaling.
However, in addition to having the same major criticism of the DA hypothesis, that
reward has been demonstrated in the absence of DA (see for example Bechara et al., 1992;
Hnasko et al., 2007; Vargas-Perez et al., 2009), the prediction error model has its own set of
drawbacks. Some have suggested that DA firing is more likely to play a central role in
identifying which aspects of context and behavioural output are crucial in causing unpredicted
events rather than precisely signaling the error in timing of the predicted event (Redgrave and
Gurney, 2006). In support of this idea, the results mentioned above showing that DA reward- and
18
aversion-responsive signaling occurs in a variety of areas other than the midbrain (Bromberg-
Martin et al., 2010; Jhou et al., 2009; Matsumoto and Hikosaka, 2009; Okada et al., 2009;
Roesch and Olsen, 2004) and demonstrate that DA neurons use a variety of activity patterns to
signal different properties of rewarding and aversive stimuli, suggesting that DA activity may
indeed be signaling more than a reward prediction error.
Another major drawback of this theory is that it does not address drug dependence and
withdrawal, or make any suggestions or hypotheses about DA neurons signaling these important
areas of the addictive process. Thus, although a variety of experiments have supported the
hypothesis that DA neurons signal a prediction error and research continues in this area, other
groups have pursued alternative avenues of research in an attempt to explain the complexities of
drug addiction and motivation.
The incentive-sensitization theory
This hypothesis speaks against the fundamental importance of both pleasure (the DA
hypothesis) and withdrawal (the opponent process theory, described below) in the establishment
of addiction to drugs of abuse, rather suggesting that an addict’s neural system wrongfully
attributes salience to drugs and drug cues, leading to pathological wanting of the abused drug.
There is a conceptual and neurobiological distinction made between the hedonic aspects of drugs
of abuse, or liking, and the motivational factors mediating their use, or wanting (Robinson and
Berridge, 2003). This distinction was hypothesized based on a variety of experiments examining
facial expressions and movements in a taste reactivity paradigm designed to measure hedonic
impact, where the researchers assessed rhythmic mouth movements, tongue movements and
protrusions, and gapes and various accompanying facial movements in a frame-by-frame camera
analysis. It was observed that lesions of the mesolimbic DA system had no effect on the hedonic
impact or liking of taste stimuli, leading to the idea that the DA system has no role in the liking
of a drug (Robinson and Berridge, 1993). However, the DA system remains important for the
incentive salience, or perceived value, which leads to wanting a drug of abuse after repeated
exposure (Robinson and Berridge, 2003). The postulates of the incentive-sensitization theory
19
suggest that repeated drug exposure increases the incentive salience of the drug and leads to a
hypersensitivity, or sensitization, of the DA system, which results in compulsive motivation, or
pathological wanting, of the drug. However, the implication is not that DA neurons themselves
mediate incentive salience, rather that the attribution of incentive salience coincides with the
activation of DA neurons, and that incentive salience is the reward component most directly
altered by manipulations of DA systems (Berridge and Robinson, 1998). DA manipulations can
reveal dissociations between liking and wanting of drug rewards, but do not reveal the full nature
of the psychological process or its neurobiological substrates, thus the involvement of other
systems in the rewarding effects of drugs of abuse are not ruled out by this theory (Berridge and
Robinson, 1998).
The incentive-sensitization theory posits that reward is a multiplex process, comprising
hedonic activation (liking), associative learning of the relationship between neutral events and
their hedonic consequences, and subsequent attribution of incentive salience to those events
(Berridge and Robinson, 1998). DA is not needed for the hedonic or the associative prediction
components, but is required for the incentive salience component of reward. Dysregulation of the
DA system responsible for wanting increases the motivation to seek the stimulus, such as a drug
of abuse, and causes an increased pursuit of it, leading to drug-seeking behaviour and later, the
negative consequences of addiction.
This theory provides a more complete picture of drug addiction than its preceding
neurobiological theories of motivation, however there are still many valid criticisms of its
suggestions. First, in common with the DA hypothesis and prediction error hypothesis, there is
no explanation of how drug motivation and ‘wanting’ occur in the absence of DA (see for
example Bechara et al., 2002; Hnasko et al., 2007; Laviolette and van der Kooy, 2003; Vargas-
Perez et al., 2009). The evidence that motivated behaviour for abused drugs occurs in the
absence of dopaminergic activity, therefore preventing the DA-mediated attribution of incentive
salience, stands in contrast to this theory. Although, as mentioned above, the involvement of
other systems in drug motivation is not completely ruled out by this theory, no direct evidence
for any alternative systems in the attribution of incentive salience has been reported to date by
this group or others. Furthermore, some DAR antagonism studies have reported an increase in
20
drug-seeking behaviour after DAR activity is blocked (Ettenberg et al., 1982) or even a switch in
the motivational valence of a drug, whereby administration of a DAR antagonist switches a
conditioned place aversion to a conditioned place preference (Laviolette and van der Kooy,
2003; Sturgess et al., 2010).
Another criticism of this theory comes from the lack of evidence of a true distinction
between the two components of liking and wanting. Indeed, infusions of µ-opioid-receptor,
glutamate receptor, or gamma-aminobutyric acid (GABA) receptor drugs into various brain
regions are all capable of modulating the hedonic reactions to taste stimuli, or the liking
component, and increasing the wanting component as well (Pecina et al., 2006; Reynolds and
Berridge, 2002; Reynolds and Berridge, 2003) implying that the two components are at least
partially linked, being dependent on the same neurobiological substrates. This lack of a double
dissociation – whereby a manipulation affects only one process and not the other – indicates that
further investigation of this hypothesis and its postulates is required.
The non-deprived/deprived hypothesis
The main idea behind this model, proposed by van der Kooy and colleagues, is that the
rewarding properties of both natural and drug rewards are mediated by either a dopaminergic or
a non-dopaminergic motivational system depending on the deprivation state of the animal
(Bechara et al., 1992). In contrast to the theories discussed above, this hypothesis proposes that
the mechanisms underlying both natural and drug rewards are not rigid, but rather are transient,
and are changing depending on the current motivational state of the organism (Bechara et al.,
1992). This theory is not a specific alternative to the hypotheses involving DA, but rather
imposes a constraint on when DA mediates reward. In a satiated or non-deprived motivational
state, reward is mediated through a non-dopaminergic system, involving the VTA GABA
neurons projecting to the TPP nucleus of the brainstem (Bechara et al., 1998). In the non-
satiated, drug withdrawn or deprived motivational state, the TPP no longer is thought to mediate
the rewarding effects of food or drugs, but rather the mesolimbic DA system is responsible for
21
motivational drive (Bechara et al., 1998). Therefore, DA can and does mediate reward, but only
when the subject is in a deprived motivational state.
The two separate systems postulated to mediate reward in the brain are mutually
exclusive: they contribute similarly to behaviour, but are operative at different times, depending
on the deprivation state. This hypothesis was developed based on a series of experiments that
showed that administration of the broad-spectrum DAR antagonist α-flupenthixol (α-flu), but not
lesions of the TPP, diminished food and opiate reward when rats were hungry or opiate-
withdrawn, respectively, but not when they were sated or opiate-naive (Bechara et al., 1992;
Bechara et al., 1995; Nader et al., 1997). Conversely, excitotoxic lesions of the TPP but not α-flu
administration disrupted food and morphine reward only if rats were tested while sated or drug-
naive, respectively, but not if they were hungry or in withdrawal from chronic opiate exposure
(Bechara and van der Kooy, 1989; Bechara et al., 1992; Olmstead et al., 1998). More recent
studies support this hypothesis, finding that brain-derived neurotrophic factor in the VTA
promotes a shift from a DA-independent to a DA-dependent opiate reward system, involving a
switch from inhibitory to excitatory signaling in the GABA receptors on VTA neurons (Vargas-
Perez et al., 2009). Unlike the incentive salience model, this work demonstrated a double
dissociation between the motivational state of the animal (non-deprived or deprived) and the
mechanism responsible for mediating reward (TPP- or DA-dependent).
The switch from a non-deprived, TPP-dependent, to a deprived, DA-dependent
motivational state is not permanent. One study showed that opiate-dependent and withdrawn rats
(whose conditioned place preferences are DA-dependent) given opiates to relieve their
withdrawal are in a TPP-dependent, non-deprived motivational state, having their conditioned
place preferences blocked by TPP lesions but not DAR antagonism (Bechara and van der Kooy,
1992). These results suggest that the presence or absence of withdrawal, or being in a deprived or
non-deprived motivational state, respectively, determined which neurobiological substrate
mediated reward. Similarly, if deprived animals are given enough time to recover fully from the
effects of withdrawal, the mechanisms underlying opiate reward are again TPP-dependent
(Nader et al., 1994). These results are in contrast to the incentive-sensitization theory in that the
22
non-deprived/deprived theory does not require that a permanent switch in brain neurochemistry
occur (Robinson and Berridge, 1993).
Criticisms of this hypothesis come from studies showing that the DA system is activated
(DA overflow is observed in the NAc) by sex, food and drug reward in non-deprived or drug-
naive rats (Di Chiara and Imperato, 1998; Fiorino et al, 1997; Martel and Fantino, 1996). This
hypothesis cannot account for cocaine or amphetamine reward, and cannot completely account
for nicotine motivation, as nicotine reward is DA-mediated in the deprived motivational state and
many studies have shown that nicotine reward in nondeprived subjects is also DA-mediated
(Acquas et al., 1989; Lecca et al., 2006; Merlo Pich et al., 1999; Pak et al., 2006; Sellings et al.,
2008; Spina et al., 2006; Tanabe et al., 2008). However, the van der Kooy group and others have
demonstrated DA-independent (TPP-dependent) nicotine reward (Corrigall et al., 2001; Lanca et
al., 2000; Laviolette et al., 2003), suggesting that nicotine and opiates act in a similar way in
terms of TPP- and DA-mediation. Much debate remains about the precise neurobiological
substrates mediating nicotine reward in the non-deprived motivational state, therefore this thesis
will focus on another theory of motivation, the opponent process theory, which attempts to
explain the dual motivational effects of acute and chronic nicotine in both the non-deprived and
deprived motivational states.
The opponent process theory
The opponent process theory of motivation was first described by Solomon and Corbit
(1974) and later expanded and refined by Koob and colleagues. It is a two-sided hedonic
hypothesis that has gone by many different names, such as positive-negative reinforcement,
opponent processes, hedonic dysregulation, and reward allostasis (Koob and Le Moal, 2006;
Solomon and Corbit, 1973). This theory all but abandons the idea that DA and the pleasant
feelings of drug administration drive compulsive drug use, rather focusing on the unpleasantness
of withdrawal as the driving force behind continued drug-taking and relapse to compulsive drug
seeking. It was originally hypothesized that many hedonic, affective, or emotional states, both
pleasant and aversive, are automatically opposed by neurobiological mechanisms that reduce the
23
intensity of the state (Solomon and Corbit, 1974). Koob and colleagues elaborated on this theory,
suggesting that drugs are taken at first because they are pleasant, but with repeated use certain
neuroadaptations lead to tolerance and dependence, such that drug taking is no longer pleasant
and in fact, unpleasant withdrawal symptoms ensue and eventually dominate upon cessation of
drug use (Koob and Le Moal, 2006). Compulsive drug taking is thus maintained simply in order
to escape the negative experience of withdrawal that occurs upon cessation of chronic drug use.
The opponent process theory posits that initial pleasant stimuli activate a dose-dependent,
relatively short-acting a-process, which in turn triggers the activation of a longer-lasting
opponent b-process. The b-process is thought to restore homeostasis in the brain, bringing the
activity states back to normal, being strengthened by use and weakened by disuse (Solomon and
Corbit, 1974). An initially aversive stimulus similarly activates a dose-dependent, aversive a-
process, that triggers and is followed by a longer lasting, slower to decay, rewarding b-process
(Figure 1.2). With repeated activation, the opponent b-process is strengthened, growing in
magnitude and duration, and causes a tolerance effect to the a-process. In the example of an
initially rewarding drug of abuse, the a-process is not as pleasant during subsequent drug
exposures because of the adaptation of the b-process. The a-process is not affected by use, being
a relatively stable, unconditioned reaction, but with repeated use, the b-process shows a shorter
latency in response to the a-process, a quicker rise, and a longer decay time (Solomon and
Corbit, 1974). Furthermore, unpleasant effects of withdrawal are caused when the rewarding
effects of a drug wear off, as the b-process is slow to decay and opposite in direction to the a-
process, thus the aversive effects of the b-process remain after the pleasantness of the a-process
has worn off. Similar to the DA hypothesis, the opponent process theory postulates that the a-
process is caused by mesolimbic DA activity (Koob and Le Moal, 2008). The b-process also
involves DA neurotransmission and a loss of function of the brain reward system (a within-
system neuroadaptation), but it has been hypothesized that the key player in the anxiogenic and
aversive effects of withdrawal experienced during the b-process to drugs of abuse are mediated
by recruitment of the corticotropin-releasing factor (CRF) brain stress anti-reward system (a
between-system neuroadaptation) in the amygdala and other brain areas (Koob and Le Moal,
1997; Koob and Le Moal, 2006; Koob and Le Moal, 2008). This combination of decreased
24
Figure 1.2. The opponent process theory of motivation.
Solomon and Corbit (1974) postulated that any stimulus would trigger an initial a-process that
will closely follow the stimulus and will be fast to occur and fast to end. The initial a-process can
be rewarding or aversive and will be followed by a later occurring opponent b-process that is
longer lasting, slower to end and is opposite in direction to the a-process. For drugs of abuse, a
rewarding dose will produce an initial rewarding a-process followed by an aversive opponent b-
process (top), while an aversive dose will produce an initial aversive a-process followed by a
rewarding opponent b-process (bottom).
Grieder et al., 2010
Appetitive Stimulus (Reward)
Aversive Stimulus
a processb process
Stimulus Response
a processb process
25
reward function and recruitment of an anti-reward system is hypothesized to lead to an
“allostatic” state, or chronic deviation from the normal homeostatic state, where the a-process is
less rewarding and the b-process is much more intense (Koob and Le Moal, 2001). This process
provides a strong source of negative reinforcement, whereby the drug is taken to relieve the
negative effects of the b-process. This progression contributes to and is hypothesized to drive
relapse and addiction.
Koob and colleagues hypothesized that an individual who does not frequently use a drug,
allowing sufficient time between re-administering the drug, will not experience allostasis and
will retain the a-process, experiencing a positive hedonic motivational state after the cessation of
drug use (Koob and Le Moal, 2001). In other words, an appropriate counteradaptive opponent b-
process that balances the a-process does not lead to an allostatic state. However, the changes in
an individual with repeated frequent drug use represent a transition to an allostatic state in the
brain reward systems and therefore a transition to drug addiction and withdrawal (Figure 1.3). In
an allostatic state, the b-process never returns to the original homeostatic level before drug-
taking begins again, thus creating a greater and greater allostatic state in the brain reward system
(Koob and Le Moal, 2001; Koob and Le Moal, 2006). In the allostatic motivational state, the
counteradaptive opponent b-process no longer balances the a-process.
The opponent process theory of motivation is very sound in that it incorporates both
pleasure and withdrawal in its descriptions of opponent motivational a- and b-processes. It can
also account for both rewarding and aversive stimuli, leading to rewarding and aversive a-
processes, respectively. The opponent process theory is unique in that the original theory did not
view addiction as an abnormality, but rather as an inevitable consequence of a normally
functioning system that opposes affective or hedonic states (Solomon and Corbit, 1974).
However, criticisms of this theory come from data showing that previously addicted subjects
often experience intense cravings and subsequent relapse to drug-taking months or years after
complete abstinence, and long after the negative effects of withdrawal have subsided, the b-
process having decayed (Lu et al., 2004). It appears from these studies that elimination of
withdrawal symptoms does not protect against relapse. Furthermore, withdrawal is not as
26
Figure 1.3. The allostatic state of drug addiction.
The changes in an individual after repeated drug use lead to an allostatic state, where the normal
homeostatic state is chronically deviated from, and a new homeostatic point is reached. At top,
the initial experience of a drug with no prior drug history is depicted, modeling the original
opponent process theory of motivation put forth by Solomon and Corbit (1974). The a-process is
positive and the opponent b-process is negative. The motivational state is the sum of the a-
process and b-process. An individual whom experiences a positive hedonic mood state from a
drug of abuse with sufficient time between re-administering the drug is hypothesized to retain the
a-process and does not experience an allostatic state. At bottom, the changes in the affective state
in an individual with repeated frequent drug use leading to an allostatic motivational state. In an
allostatic state, the b-process does not balance the a-process because the system never returns to
the original homeostatic level before drug-taking begins again.
Koob and Le Moal, 2001.
27
powerful a motivator for relapse to drug-taking as was originally implied in this theory, as
studies have shown that activating the a-process is far more effective at reinstating drug-seeking
and -taking in recovered drug addicted rats than activating the b-process (Stewart and Wise,
1992).
Additional evidence against this theory, which predicts that withdrawal motivates drug-
seeking and -taking, comes from studies in opiate dependent animals. In opiate withdrawn rats,
no predictive relationship between the demonstration of a somatic withdrawal syndrome and the
aversiveness of withdrawal measured by conditioned place aversion was observed (Mucha,
1987). Furthermore, opiates will be self-administered in the absence of withdrawal symptoms
(Ternes et al., 1985). These studies suggest that withdrawal is actually not as strong a predictor
of drug-seeking than postulated in this theory.
As with the other theories of motivation, the opponent process theory cannot fully explain
the process of drug addiction and the motivation to seek drugs of abuse. However, it is the most
relevant theory to this thesis, and will be explained further in relation to acute nicotine intake and
nicotine withdrawal in Chapter 2 and in the discussion.
1.4 The Anatomy of the Ventral Tegmental Area
The VTA is home to one of the major populations of DA cells in the brain (Kalivas,
1993), thus it is not surprising that this area has received much attention in the study of drug
motivation. The VTA lies close to the midline on the floor of the midbrain, bordered laterally by
the substantia nigra, rostrally by the mammillary bodies and posterior hypothalamus, and
caudally by the pons and hindbrain (Oades and Halladay, 1987).
28
Neurons and Projections
The VTA is mainly composed of DA and GABA neurons (Kalivas, 1993). Although the
exact proportions of these two major cell types have not been precisely determined, it has been
suggested using a variety of methods that the VTA is approximately 55-60% DA and 5-33%
GABA cell types (Kalivas, 1993; Margolis et al., 2006). There are numerous methods that may
be utilized to identify DA and GABA cells, including histology, pharmacology, and
electrophysiology. DA cells are very large in size, stain positively for tyrosine hydroxylase (TH,
the rate limiting enzyme responsible for DA synthesis), and when measured electro-
physiologically will demonstrate well established features: (I) a relatively longer action potential
width (>2.5 ms); (II) a triphasic (+/-/+) waveform consisting of a notch on the rising phase
followed by a delayed after-potential; (III) a characteristic low tone by audio monitoring; (IV) a
slow, irregular or bursting firing pattern, and (V) a spontaneous firing rate of 2 - 5 Hz or less
(Grace and Bunney, 1983; Tan et al., 2009;). GABA cells are smaller in size, stain positively for
GABA and glutamic acid decarboxylase (GAD, an enzyme responsible for GABA synthesis),
and when measured electrophysiologically will demonstrate spontaneous activity, and higher
firing frequencies and shorter action potentials than DA neurons (Cameron et al., 1997;
Korotkova et al., 2004). There are also glutamate neurons present in the VTA, comprising
approximately 1-15% of VTA cells, that are suggested to provide local excitatory modulation of
the DA and GABA neurons (Dobi et al., 2010; Yamaguchi et al., 2007). These neurons can be
identified through the detection of mRNA encoding vesicular glutamate transporters, which
transport glutamate into synaptic vesicles at presynaptic terminals, and are known to be
independent from DA and GABA cells because they do not co-stain for TH or GAD,
respectively (Dobi et al., 2010; Yamaguchi et al., 2007). Dopaminergic, GABAergic and
glutamatergic receptors are located throughout the VTA.
Axon terminals derived from both GABAergic and glutamatergic neurons establish local
intrinsic synapses as well as extrinsic inputs on dendrites of both dopaminergic and non-
dopaminergic neurons in the VTA (Morales and Pickel, 2012). The principal excitatory extrinsic
inputs to the VTA are glutamatergic projections from prefrontal cortex, bed nucleus of the stria
terminalis, amygdala, and the pontomesencephalic tegmental nuclei (TPP and lateral dorsal
29
tegmental nucleus) (Mao and McGehee, 2010). The pontomesencephalic tegmental projections
are also cholinergic and GABAergic, and have been shown to be important for the rewarding
effects of nicotine (Corrigall et al., 2002; Lanca et al., 2000) probably because of their
contribution to the phasic burst firing of VTA DA neurons (Floresco et al., 2003; Lodge and
Grace, 2005). The principal inhibitory inputs to the VTA are GABAergic and include local
interneurons (mentioned above) and projections from the NAc, ventral pallidum, and
pontomesencephalic tegmental nuclei (Mao and McGehee, 2010). However, because of the
presence of inhibitory GABAergic interneurons, excitatory inputs from extrinsic sites can also
cause inhibition of VTA DA firing, as in the case of lateral habenular suppression of DA
neuronal activity (Matsumoto and Hikosaka, 2007).
The VTA receives CRF inputs from the extended amygdala and paraventricular nucleus
of the hypothalamus (PVN), from which CRF is co-released with GABA and glutamate
(Tagliaferro and Morales, 2008). VTA dopaminergic and nondopaminergic neurons express both
CRF1Rs and CRF2Rs (George et al., 2012; Ungless et al., 2003).
The dopaminergic projections from VTA to NAc are very well established, however,
some studies have demonstrated that in addition to VTA dopaminergic innervations, the NAc
receives inputs from both GABAergic (Van Bockstaele et al., 1995) and glutamatergic VTA
neurons (Yamaguchi et al., 2011). Mesocortical DA neurons also project to the prefrontal cortex,
however, retrograde tracing studies indicate that approximately half of the mesocortical
projection neurons from the VTA are nondopaminergic (Morales and Pickel, 2012). The VTA
also sends dopaminergic projections to the extended amygdala and PVN (Eliava et al., 2003).
A summary figure of the VTA neurons, projections, and inputs is shown in Figure 1.4.
Nicotinic Receptors
Ultimately, nicotine influences neuronal activity in the VTA and causes its motivational
effects by binding to nicotinic acetylcholine receptors (nAChRs), which are pentameric, ligand-
gated ion channels (Koob, 2001; Mansvelder and McGehee, 2002). There are 12 neuronal
30
Figure 1.4. The VTA: Neurons, receptors, inputs and projections.
The VTA (blue dotted box) is composed of DA, GABA, and glutamate (Glu) neurons. Both
inhibitory GABAergic and excitatory glutamatergic neurons establish local intrinsic synapses
(white arrows) on both dopaminergic and non-dopaminergic neurons. Extrinsic inputs to the
VTA (yellow arrows) come from the prefrontal cortex (PFC), amygdala, TPP, NAc, ventral
pallidum (VP), habenula, and other regions not shown in this figure. The VTA neurons send
reciprocal projections (yellow arrows) back to the areas pictured here, as well as other areas not
shown in this figure. VTA dopaminergic and nondopaminergic neurons express CRF receptors
(both CRF1Rs and CRF2Rs) and a variety of nAChRs, as well as DA and GABA receptors,
which are expressed throughout and not labeled on this diagram.
31
nAChR subunits identified to date, the α2-α10 and β2-β4, of which only α8-α10 have not been
reported as being expressed in the VTA. The nAChR receptor profiles that are associated with DA
and GABA neurons differ considerably, with dopaminergic VTA neurons mainly expressing α7,
α4β2* and α6β2* nAChRs, with the asterisk denoting the possibility of other subunits, such as
α5 and β3, being incorporated into these receptors (Mao and McGehee, 2010). On
nondopaminergic neurons, less than 25% have been reported to express the α3, α5, α6, and β4
subunits, thus α4β2 combinations predominate, with some α7 homomeric nAChRs being found
as well (Laviolette and van der Kooy, 2004; Mao and McGehee, 2010). nAChR activation leads
to increased cation flow through the central channel, which induces depolarization and increased
excitability in the neuron (Mao and McGehee, 2010). Nicotine produces its motivational effects
by first acting on its receptors, and modulating the activity of VTA neurons.
1.5 The Neurobiology of Nicotine Motivation: DA and CRF
The Use and Abuse of Nicotine
Nicotine is a highly toxic alkaloid, one of over 4000 chemicals that may be obtained from
smoking dried tobacco leaves of the cultivated plant Nicotiana tabacum (Koob and Le Moal,
2006). Nicotine is the major reinforcing component of tobacco smoke that leads to dependence
in humans (Stolerman and Jarvis, 1995). Its use by indigenous peoples of the Americas for both
medicinal and ceremonial purposes has been traced back 8000 years, but the first documented
practice of smoking the dried leaves of the tobacco plant has been attributed to European
explorers in 1492 (Koob and Le Moal, 2006). The first cigarette-making machine was produced
in 1880, and since then, cigarette production boomed due to the ease of production and
distribution, the ability of the mass media to market their product, and increased demand
(Akehurst, 1968).
Today, tobacco smoking and the addiction to nicotine that comes from it, is a worldwide
health problem. Tobacco addiction is the leading avoidable cause of disease and premature death
32
in North America (Fellows et al., 2002). The cost to society associated with nicotine addiction
leading to health care problems that often result in death, medical costs, and human suffering is
significant (CDC, 2008). The most common reason for relapse reported by quitting smokers is
the desire to relieve the discomforts that come with nicotine abstinence and withdrawal (Allen et
al., 2008). Similar to nicotine-withdrawn humans, nicotine-dependent experimental animals that
undergo nicotine withdrawal demonstrate an observable somatic nicotine abstinence syndrome
(Epping-Jordan et al., 1998; Malin et al., 1992; Stoker et al., 2008). Most animal studies involve
antagonist-precipitated nicotine withdrawal (George et al., 2007; Kenny and Markou, 2001;
Laviolette et al., 2008; Watkins et al., 2000), although a spontaneous withdrawal procedure
would more closely model the human response to withdrawal from chronic nicotine. Rodents
experiencing spontaneous withdrawal from chronic nicotine will show a conditioned aversive
response to a withdrawal-paired environment in place conditioning paradigms (Merritt et al.,
2008; also see Chapter 2). This withdrawal response is modeled by the opponent process theory
of motivation and represents the opponent b-process to the a-process of chronic nicotine reward
in dependent animals. The neurobiological substrates mediating these opponent motivational
processes in nicotine-dependent animals are essentially unknown. In nondependent animals,
acute nicotine administered directly into the VTA will produce both rewarding and aversive
effects (Laviolette et al., 2002; Sellings et al., 2008), thus two different a-processes, one
rewarding and another aversive, would be stimulated after acute nicotine administration. These
acute nicotine a-processes are mediated by different neural substrates, with reward being TPP-
mediated and aversion being DA-mediated (Laviolette et al., 2002). In many studies over many
years of research on the neurobiology of nicotine motivation and withdrawal, focus has been on a
variety of neurobiological substrates, including VTA DA, the TPP, CRF, epinephrine, serotonin,
brain-derived neurotrophic factor, GABA, and others. However, this thesis will focus on the DA
and CRF systems involvement in nicotine’s acute and chronic motivational effects.
33
VTA Dopamine
Like most drugs of abuse, nicotine acutely produces both aversive and positive
motivational effects (Grunberg, 1994; Laviolette and van der Kooy, 2004; Perkins et al., 2008)
by increasing the extracellular concentration of DA in the mesolimbic system (Di Chiara and
Bassareo, 2007; Grace, 2000; Nestler, 2005; Picciotto and Corrigall, 2002) as well as non-
DAergic neural substrates (Fowler et al., 2011; Lanca et al., 2000; Laviolette et al., 2002; Levin
et al., 1996; Picciotto and Corrigall, 2002). Nicotine given peripherally has centrally mediated
effects on DA release (Seppa et al., 2000) and selectively activates DA neurons in the pVTA, but
not aVTA (Zhao-Shea et al., 2011). DA signaling has been implicated in the aversive
motivational response to acute nicotine (Laviolette and van der Kooy, 2003; Tan et al., 2009);
however, DA-dependent acute nicotine reward has also been demonstrated (Acquas et al., 1989;
Lecca et al., 2006; Merlo Pich et al., 1999; Pak et al., 2006; Sellings et al., 2008; Spina et al.,
2006; Tanabe et al., 2008).
DA acts at five different receptor subtypes, D1-D5, of which only D1Rs and D2Rs are
found on VTA neurons (Le Foll et al., 2009). DA neurons in the VTA exhibit burst firing that
produces a fast and large DA release that mainly activates postsynaptic D1Rs, as well as
population firing that produces a slower tonic DA release that mainly activates higher affinity,
mostly presynaptic D2Rs (Floresco et al., 2003; Goto and Grace, 2005; Grace, 2000). A single
systemic injection of acute nicotine in nondependent animals increases the firing rate and phasic
burst activity of VTA DA neurons, elevating DA in the projection sites of VTA DA neurons
(Mameli-Engvall et al., 2006; Zhang et al., 2009). Consistent with the idea that phasic activation
leads to D1R activation, D1R antagonism blocks acute nicotine motivation in nondependent
mice (David et al., 2006). Conversely, chronic exposure to nicotine decreases tonic but not
phasic DA activity in the VTA (Tan et al., 2009) and spontaneous and mecamylamine-
precipitated nicotine withdrawal in dependent animals is associated with a decrease in
extracellular DA levels in the NAc (Carboni et al., 2000; Rahman et al., 2004.) These effects
reflect changes in both DA release and reuptake (Duchemin et al., 2009) that lead to a decrease
in DA signaling in the brain (Kalivas and Volkow, 2005), results that are similar to those
obtained in drug-dependent human subjects, who show marked decreases in D2R availability
34
(Fehr et al., 2008) and thus presumably in DA release (Volkow et al., 2009). These results and
many others demonstrate that VTA DA signaling through D1Rs and D2Rs is involved in both
the rewarding and aversive motivational effects of acute and chronic nicotine, suggesting that
different DA neurons in the same brain area may signal both reward and aversion, possibly due
to the different patterns of DA activity, different timing of DA release, or different activation of
DA receptors in VTA projection sites.
In the striatum, the main VTA projection site, adenosine receptors subtype-2A (A2ARs)
and D2Rs are colocalized (Tozzi et al., 2011) and form A2AR-D2R heteromers (Fuxe et al.,
2010). The A2AR and D2R interact antagonistically, such that agonism of A2ARs decreases
signaling at D2Rs (Tanganelli et al., 2004) and antagonism of A2ARs increases signaling at
D2Rs (Fuxe et al., 2010). In terms of nicotine motivation, very few studies have examined the
role of A2ARs, however one study found that genetic deletion of the A2AR would prevent the
rewarding motivational effects of acute nicotine in nondependent mice without affecting the
conditioned taste aversion for acute nicotine (Castañé et al., 2006). These results suggest that the
A2AR plays a role in nicotine motivation, possibly through a DAR-mediated mechanism.
Although there have been a variety of studies on DA activity, DA receptors, and less so
A2ARs in nicotine motivation, the role of tonic and phasic DA activity and activation of the
various DARs (and possibly of A2ARs because of their antagonistic activities) in signaling the
motivational effects of both acute and chronic nicotine is essentially unknown. The studies
described in Chapters 2 and 3 of this thesis will address this gap in the nicotine motivation
research.
Corticotropin-Releasing Factor (CRF)
CRF is a 41-amino acid polypeptide isolated initially in 1981 from ovine hypothalamus
(Vale et al., 1981) that controls hormonal, sympathetic and behavioural responses to stressors
(Koob, 2008). CRF is present in the prefrontal cortex, extended amygdala, medial septum,
hypothalamus, thalamus, cerebellum, locus coeruleus, and midbrain and hindbrain nuclei
35
(Swanson et al., 1983). The anatomical distribution of CRF indicates a role for the neuropeptide
in responses to stress, food intake and cognition (Koob, 2008). There are two known CRF
receptors, CRF1R and CRF2R, both of which can be found on VTA neurons (Sauvage and
Steckler, 2001; Ungless et al., 2003).
CRF release occurs during ethanol (Funk et al., 2006; Merlo-Pich et al., 1995) and opiate
(Weiss et al., 2001) withdrawal in dependent rats, and CRF receptor antagonists can reduce
ethanol (Rassnick et al., 1993), cocaine (Basso et al., 1999), and opiate (Stinus et al., 1995)
withdrawal-induced anxiety-like behaviour. Fewer studies examining CRF and nicotine
motivation have been performed, but it has been shown that CRF release is increased in the
amygdala during nicotine withdrawal (George et al., 2007) and that CRF1R antagonists can
block the anxiogenic effects of withdrawal from chronic nicotine (George et al., 2007; Tucci et
al., 2003). The very few studies on the involvement of CRF in nicotine motivation have
demonstrated that CRF plays a role in the anxiogenic effects of nicotine withdrawal, suggesting a
possible role for CRF in the opponent motivational effects of chronic nicotine.
Dopamine and CRF in drug motivation
The mesolimbic DA system and the CRF brain stress system have been extensively
studied independently and are usually considered to be mutually exclusive in terms of drug
motivation. However, recent results have demonstrated that these two systems do indeed interact
with each other (reviewed in George et al., 2012), suggesting that dysregulation of this newly
discovered DA-CRF interaction may produce motivational effects that contribute to the
development of drug addiction.
There are reciprocal connections between CRF-producing neurons in the extended
amygdala and PVN and the DA-producing neurons of the VTA. The VTA sends heavy
dopaminergic projections to the extended amygdala and PVN (Liposits and Paull, 1989), directly
innervating CRF-containing neurons in the central nucleus of the amygdala (CeA) (Eliava et al.,
2003). In return, CRF neurons from the extended amygdala and PVN project to the VTA
36
(Rodaros et al., 2007). DARs are expressed throughout CRF-producing areas of the brain, and
the VTA expresses both CRF1Rs and CRF2Rs (Sauvage and Steckler, 2001; Ungless et al.,
2003). Neuroanatomical studies show that CRF is colocalized in glutamatergic and GABAergic
afferents to the VTA, and that these afferents synapse with dopaminergic as well as non-
dopaminergic VTA neurons (Tagliaferro and Morales, 2008). Furthermore, corticotropin
releasing factor-binding protein (CRF-BP), a protein that participates in the regulation and
potentiation of CRF signaling at the synapse (Ungless et al., 2003), has been identified in a
subset of DA and GABA neurons within the VTA (Wang and Morales, 2008). This CRF/DA
mechanism in the VTA has been implicated in the process of relapse to cocaine seeking (Wang
et al., 2007), suggesting that a CRF/DA mechanism operating in the VTA may be involved in
drug addiction, withdrawal, and relapse.
Consistent with anatomical data, CRF dose-dependently increased VTA DA neuronal
firing (Hahn et al., 2009; Wanat et al., 2008) and locomotor activity (Kalivas et al., 1987), which
was prevented by antagonism of CRF1Rs but not CRF2Rs, and was mimicked by CRF1R
agonists (Wanat et al., 2008). CRF1R activation can also facilitate slow, D2R-mediated
neurotransmission (Beckstead et al., 2009) as well as DA release (Bagosi et al., 2006;
Muramatsu et al., 2006), while CRF1R knock down in the VTA reduces DA release in the
prefrontal cortex (Refojo et al., 2011). CRF1R antagonism was also found to significantly
increase DA neuron tonic population activity without affecting phasic burst firing, average firing
rate, or NAc DA levels (Lodge and Grace, 2005). These results suggest that modulating CRF
activity may have effects on DA neuronal firing activity and DA release in various brain areas.
Although a variety of recent studies have demonstrated a definite interaction between the
mesolimbic DA system and the CRF brain stress system, there are no known studies that have
directly investigated nicotine addiction and motivation in regards to this DA-CRF interaction.
Chapter 4 of this thesis will address this gap in the research.
37
1.6 Research Aims and Hypotheses
Identification of the involvement of VTA DA and CRF activity in mediating the opponent
motivational responses to acute and chronic nicotine
In the following dissertation, I have investigated the role of the neurotransmitters DA and
CRF in the VTA in mediating the initial aversive response and opponent rewarding response to
acute nicotine, as well as the initial rewarding response to chronic nicotine and opponent
aversive motivational response of withdrawal from chronic nicotine. I have explored in depth the
changes in VTA phasic and tonic dopaminergic neuronal activity that occur during the
administration of acute aversive nicotine versus chronic nicotine and withdrawal, and
investigated which type of DA receptor activation is required for these aversive motivational
responses to acute nicotine and chronic nicotine withdrawal. I have also examined the activation
of a VTA CRF system during the transition from a nicotine nondependent to a dependent
motivational state. Furthermore, I have studied the connection between the DA and CRF systems
in the VTA of nicotine dependent and withdrawn animals. Previous studies showing that CRF-
BP is present in VTA neurons (Wang and Morales, 2008) and that chronic experience with
cocaine, another drug of abuse, enhances CRF-dependent potentiation of VTA DA neurons
(Hahn et al., 2009) suggest that such a DA/CRF link might not be unexpected. My overall
hypothesis is that nicotine dependence upregulates CRF and modifies DA activity in the VTA,
and that both the increase in CRF and the specific pattern of DA activity in the VTA are
necessary for the experience of the aversive motivational response to nicotine withdrawal.
In the first series of experiments described in chapter 2, I tested whether the mesolimbic
DA system is involved in the motivational response to nicotine withdrawal. Previous work
suggests that the DA system is involved in the processing of the aversive motivational effects of
opiate withdrawal (Bechara and van der Kooy, 1992). I thus hypothesized that blocking DA
signaling at dopaminergic receptors would prevent the aversive response to nicotine withdrawal.
I began by examining whether rodents would experience withdrawal after cessation of
chronic nicotine administration. I assessed the effect of spontaneous withdrawal of chronic
38
nicotine administration on the expression of a somatic nicotine abstinence syndrome in rats and
mice. Previous work suggests that antagonist-precipitated nicotine withdrawal induces an
abstinence syndrome in rodents (Isola et al., 1999; Malin et al., 1992). I thus hypothesized that
the spontaneous removal of chronic nicotine in a dependent animal would produce an observable
and significant nicotine abstinence syndrome in both mice and rats.
I next tested whether rodents would show a motivational response to nicotine withdrawal
by examining the effect of spontaneous withdrawal from chronic nicotine in a place conditioning
paradigm in rats and mice. Previous work suggests that withdrawal from chronic opiate
administration in rats leads to conditioned place aversions to a withdrawal-paired environment
(Bechara and van der Kooy, 1992). The opponent process theory of motivation suggests that this
aversive response to withdrawal is the opponent process to chronic drug reward. I hypothesized
that withdrawal from chronic nicotine would lead to conditioned place aversions in both rats and
mice, and tested whether this motivational response would coincide with the demonstration of a
nicotine abstinence syndrome.
I also investigated whether acute aversive nicotine in nondependent mice would produce
opponent motivational processes that could be measured in the place conditioning paradigm. The
opponent process theory of motivation suggests that an initial aversive stimulus will lead to a
later occurring and longer lasting rewarding stimulus (Solomon and Corbit, 1974), which implies
that acute aversive nicotine would stimulate an initial aversive motivational response and a later
occurring rewarding motivational response.
The most important and novel finding in the set of experiments detailed in Chapter 2
tested the effect of administration of a DA antagonist, which disrupts dopaminergic signaling by
blocking DA receptors, on the opponent motivational processes occurring after both acute and
chronic nicotine. We showed using the place conditioning paradigm that acute aversive nicotine
in nondependent mice produces an initial aversive response followed by a rewarding opponent
motivational response, chronic nicotine in dependent mice produces a rewarding motivational
response, and withdrawal from chronic nicotine in dependent mice will produce an opponent
aversive motivational response. Previous results have demonstrated that DA antagonism blocks
39
the conditioned aversive response to acute nicotine in rats (Laviolette and van der Kooy, 2004)
and that DA activity is involved in nicotine motivation in dependent animals (Bruijnzeel and
Markou, 2005; Kenny and Markou, 2001; Laviolette et al., 2008; Smolka et al., 2004). I
hypothesized that the mesolimbic DA system mediates the motivational response underlying the
opponent process to chronic but not acute nicotine. Similarly, I also examined the effect of D2R
KO on the opponent motivational effects of acute and chronic nicotine by utilizing D2R KO
mice in the place conditioning paradigm. I hypothesized that if both genetic deletion of a DA
receptor and antagonism of DA receptors could block the aversive motivational effects of
nicotine withdrawal, a process that changes the activity of DA neurons in the VTA and the
release of DA in the NAc (Hildebrand et al., 1998; Liu and Jin, 2004; Rada et al., 2001), then
this suggests that the modification of DA signaling prevents a specific pattern of activity that
may signal nicotine withdrawal.
In chapter 3, I thoroughly investigated the specific pattern of DA signaling that mediates
nicotine motivation by testing the effect of withdrawal from chronic nicotine on tonic and phasic
VTA DA activity and whether the specific pattern of signaling through D1Rs and D2Rs mediates
the conditioned motivational responses to nicotine withdrawal and acute nicotine. I began by
examining whether increasing or decreasing dopaminergic signaling at receptors could prevent
nicotine withdrawal aversions. Opiate withdrawal aversions can be blocked with DA agonist or
antagonist pretreatment (Laviolette et al., 2002), leading to the hypothesis that a specific pattern
of signaling at DA receptors mediates the expression of opiate withdrawal aversions. I thus
tested whether increasing or decreasing DA signaling at receptors by using a DA agonist or
antagonist, respectively, would prevent the expression of nicotine withdrawal aversions.
I next examined the pattern of VTA DA signaling electrophysiologically. Previous
research has shown that DA neurons exhibit burst- and population-firing activity that leads to
phasic and tonic DA release, respectively (Floresco et al., 2003; Goto and Grace, 2005; Grace,
2000), and that acute nicotine affects phasic VTA DA activity (Mameli-Engvall et al., 2006)
while chronic nicotine affects tonic VTA DA activity (Tan et al., 2009). I thus investigated both
phasic and tonic VTA DA activity using in vivo electrophysiology after administration of a dose
of acute nicotine that produces conditioned place aversions, and after nicotine dependence and
40
spontaneous withdrawal from chronic nicotine. I hypothesized that acute nicotine would modify
phasic DA activity, while withdrawal from chronic nicotine would modify tonic DA activity.
Furthermore, I examined whether blockade of phasic DA activity using antagonist drugs that
were known to selectively modify phasic but not tonic DA activity would prevent the expression
of acute nicotine aversions or nicotine withdrawal aversions, hypothesizing that only the phasic
DA-mediated acute nicotine aversions would be blocked by the selective antagonists.
I also examined whether modification of D1Rs and D2Rs would differentially affect
acute nicotine and nicotine withdrawal aversions. Since previous research has shown that phasic
DA release mainly activates D1Rs and tonic DA release mainly activates D2Rs (Floresco et al.,
2003; Goto and Grace, 2005), I hypothesized that modifying D1R activity by using D1R agonists
or antagonists, or genetic deletion of the D1R, would prevent the expression of acute nicotine
aversions in nondependent mice but not aversions to withdrawal from chronic nicotine in
dependent mice. I also hypothesized that modifying D2R activity using D2R agonists and
antagonists, or genetic deletion of the D2R, would prevent aversions to withdrawal from chronic
nicotine in dependent mice but not to acute nicotine in nondependent mice. Similarly, the
adenosine A2AR is colocalized with D2Rs on neurons in the mesolimbic system (Tozzi et al.,
2011) and acts antagonistically to the D2R (Tanganelli et al., 2004). I thus hypothesized that
modifying A2AR activity would prevent aversions to chronic nicotine withdrawal, but not acute
nicotine, and tested this idea using A2AR agonists and antagonists as well as A2AR KO mice.
In chapter 4 I examined the involvement of the CRF system in the VTA in nicotine
motivation. It is well known that the mesolimbic DA system originating in the VTA is important
in mediating nicotine’s motivational effects, and that CRF is involved in the negative effects of
withdrawal from drugs of abuse. Previous work has demonstrated that CRF1 receptors are
present in the VTA (Sauvage and Steckler, 2001), and centrally administered CRF1 receptor
antagonists can mediate the motivational effects of nicotine (Bruijnzeel et al., 2009; Tucci et al.,
2003). Furthermore, chronic cocaine administration leads to the recruitment of CRF1 receptors in
the VTA that may control VTA activity (Hahn et al., 2009; Lodge and Grace, 2005), and CRF-
BP has been identified in VTA neurons (Wang and Morales, 2008), suggesting that a CRF/DA
mechanism operating in the VTA may be involved in drug addiction, withdrawal, and relapse. I
41
thus hypothesized that chronic nicotine and withdrawal would recruit and activate the CRF-
CRF1 system in the VTA, and that this newly activated CRF would mediate the aversive
response to withdrawal from chronic nicotine but not to acute nicotine.
To test whether nicotine dependence and withdrawal upregulates brain CRF levels, I
measured CRF mRNA in the PVN, CeA and VTA using quantitative real-time polymerase chain
reaction (rtPCR) and in situ hybridization, as well as CRF protein levels using
immunohistochemistry. I then observed the effect on the motivational responses to acute nicotine
and chronic nicotine withdrawal after blocking CRF mRNA and CRF1R activation using siRNA
knockdown and antagonist drugs, respectively. If these modifications of CRF activity prevented
the expression of nicotine withdrawal aversions, the hypothesis that CRF activity mediates
nicotine withdrawal motivation would be supported.
42
Chapter 2
Dopaminergic Signaling Mediates the Motivational Response Underlying the Opponent Process to
Chronic but Not Acute Nicotine
Taryn E. Grieder, Laurie H. Sellings, Hector Vargas-Perez, Ryan Ting-A-Kee, Eric C. Siu,
Rachel F. Tyndale and Derek van der Kooy
This chapter is adapted from the paper published in Neuropsychopharmacology, vol. 35, p 943-954, 2010. Reprinted with permission.
43
Abstract
The mesolimbic DA system is implicated in the processing of the positive reinforcing
effect of all drugs of abuse, including nicotine. It has been suggested that the dopaminergic
system is also involved in the aversive motivational response to drug withdrawal, particularly for
opiates, however the role for dopaminergic signaling in the processing of the negative
motivational properties of nicotine withdrawal is largely unknown. We hypothesized that
signaling at dopaminergic receptors mediates chronic nicotine withdrawal aversions and that
dopaminergic signaling would differentially mediate acute versus dependent nicotine motivation.
We report that nicotine dependent rats and mice demonstrated conditioned place aversions to an
environment paired with withdrawal from chronic nicotine that were blocked by the DA receptor
antagonist α-flu and in D2R KO mice. Conversely, α-flu pretreatment had no effect on
preferences for an environment paired with abstinence from acute nicotine. Taken together, these
results suggest that dopaminergic signaling is necessary for the opponent motivational response
to nicotine in dependent, but not non-dependent, rodents. Further, signaling at the D2R is critical
in mediating withdrawal aversions in nicotine dependent animals. We propose that the
alleviation of nicotine withdrawal primarily may be driving nicotine motivation in dependent
animals.
44
Introduction
Nicotine is the major reinforcing constituent of tobacco smoke that is responsible for
smoking dependence in humans (Stolerman and Jarvis, 1995). Nicotine causes its motivational
effects by acting on nicotinic receptors localized in the mesocorticolimbic DA system (Koob,
2001; Mansvelder and McGehee, 2002). Like most drugs of abuse, nicotine increases the
extracellular concentration of DA in the mesolimbic system (Di Chiara and Bassareo, 2007;
Picciotto and Corrigall, 2002). Nicotine also produces motivational effects through non-
dopaminergic neural systems such as the cholinergic TPP (Lanca et al., 2000; Laviolette et al.,
2002; Levin et al., 1996; Picciotto and Corrigall, 2002). The mesolimbic DA system has been
implicated in the processing of the acute motivational properties of nicotine (Laviolette et al.,
2003, 2008; Spina et al., 2006; Tanabe et al., 2008); however, the involvement of dopaminergic
signaling in the aversive response to chronic nicotine withdrawal is largely unknown.
The most common reason for relapse reported by quitting smokers is the desire to relieve
the discomforts of withdrawal (Allen et al., 2008). The aversive abstinence syndrome
experienced by quitters as well as the ability of renewed nicotine use to relieve this syndrome
likely contributes to relapse. Similar to nicotine-withdrawn humans, rodents that undergo
spontaneous withdrawal show a somatic nicotine abstinence syndrome (Epping-Jordan et al.,
1998; Malin et al., 1992; Stoker et al., 2008). The negative affective state of withdrawal and its
alleviation by nicotine is one of the primary factors driving nicotine craving in nicotine
dependent subjects. Most studies on nicotine motivation in dependent animals involve
antagonist-precipitated withdrawal (George et al., 2007; Kenny and Markou, 2001; Laviolette et
al., 2008; Watkins et al., 2000). However, a spontaneous withdrawal procedure more closely
models human withdrawal.
When a psychoactive drug triggers a motivational response, animals will experience a
rebound motivational state (Koob et al., 1989; Koob and Le Moal, 2001; Robinson and Berridge,
2003; Wise, 1996) that is predicted by the opponent process theory of motivation (Solomon and
Corbit, 1974). This theory postulates that any motivational stimulus activates two opposing
motivational processes: The a-process has a fast onset and offset and the b-process is opposite in
45
direction, lasts longer and is slower to start and end (Figure 2.1a). Similar to other drugs of
abuse, chronic nicotine produces a negative withdrawal syndrome that can be viewed as the
opponent process to the rewarding effects of nicotine in dependent subjects (Gutkin et al., 2006;
Koob and Le Moal, 1997). Acute nicotine produces both rewarding and aversive stimulus
properties (Laviolette and van der Kooy, 2004; Sellings et al., 2008; Wilkinson and Bevins,
2008). Dopaminergic signaling is involved in acute nicotine aversion (Laviolette and van der
Kooy, 2003; Tan et al., 2009) and chronic nicotine motivation (Bruijnzeel and Markou, 2005;
Kenny and Markou, 2001; Laviolette et al., 2008; Smolka et al., 2004); however, little is known
about the role of dopaminergic signaling in the opponent motivational processes of acute and
chronic nicotine.
We thus investigated the role of dopaminergic signaling in the acute and chronic nicotine
a- and b-processes. We first studied the correlation between somatic and affective nicotine
withdrawal by examining the timing of the maximal somatic withdrawal syndrome and
motivational withdrawal in a place conditioning paradigm. Next, we subjected previously drug
naive and nicotine dependent rodents to place conditioning after acute and chronic nicotine,
respectively, and examined the opponent motivational processes after nicotine exposure. The
involvement of dopaminergic signaling in the aversive a-process and rewarding b-process of
acute nicotine and the rewarding a-process and aversive b-process of chronic nicotine was
investigated by treatment with the DA receptor antagonist α-flu prior to conditioning. We also
examined D2 receptor involvement in chronic nicotine withdrawal aversions. The D2R has been
implicated in nicotine dependence (Fehr et al, 2008) and withdrawal (Laviolette et al, 2008). Our
results demonstrate that acute aversive and chronic rewarding nicotine lead to opponent a- and b-
processes and that dopaminergic signaling, specifically at the D2R, mediates the opponent
motivational process of chronic aversive but not acute rewarding nicotine.
Materials and Methods
Animals
46
Figure 2.1. The opponent process theory of motivation and its modeling by use of the place
conditioning paradigm.
(a) The opponent process theory of motivation. Solomon and Corbit (1974) postulated that any
stimulus would trigger an initial a-process that will closely follow the stimulus and will be fast to
occur and fast to end. The a-process can be rewarding or aversive and will be followed by a later
occurring b-process that is longer lasting, slower to end and is opposite in direction to the a-
process. At the dose used in the present experiments, acute nicotine is aversive and the a-process
is therefore negative. The acute nicotine b-process will be later occurring and positive or
rewarding. We postulate that chronic nicotine elicits a rewarding a-process in dependent animals
and the aversion to nicotine withdrawal is the conditioned opponent b-process. (b) The B, N, and
W procedures. In procedure B, each animal experienced the effects of nicotine in one
environment (Nic) and the lack of nicotine (or the effects of withdrawal) in the other
environment (WD). Procedure B measures both the rewarding value of the drug itself and the
aversiveness associated with drug withdrawal, and models both the a- and b-process of the
opponent process theory. In procedure N, each animal was conditioned only while experiencing
the effects of chronic nicotine. On the alternate day, the animals experienced withdrawal in their
home cage. Procedure N measures the rewarding value of the drug itself, modeling only the a-
process of the opponent process theory. In procedure W, conditioning took place only while the
animals experienced withdrawal from nicotine. On the alternate day, the animal was confined to
its home cage during chronic nicotine exposure. Procedure W measures only the aversive
motivational effects of drug withdrawal, separate from the rewarding value of the drug itself, and
is used as a model to measure the b-process of the opponent process theory of motivation.
47
Appetitive Stimulus (Reward)
Aversive Stimulus
a processb process
Stimulus Response
a processb process
B procedure
N procedure
W procedure
B Nic
Nic
WD
WD
A
48
Male WT mice were C57BL/6 (n = 329; Charles River, Montreal, Canada) weighing 25-
35 g. Heterozygous 5th generation D2 breeder mice were received as a gift (Kelly et al., 1997)
and crosses were bred at the University of Toronto to obtain homozygous male D2R KO mice (n
= 26) and their controls (n = 20). Mice were housed individually in plastic cages in a sound-
attenuated room at a temperature of 22°C with lights on from 7:00 AM to 7:00 PM. Male Wistar
rats (Charles River) weighing 330-380 g (n = 128) were individually housed in Plexiglas cages in
a room kept at a temperature of 22°C with lights on from 7:00 AM to 7:00 PM. All animals had
ad libitum access to food and water except during behavioral testing. All procedures were
approved by the University of Toronto Animal Care Committee in accordance with the Canadian
Council on Animal Care guidelines.
Chronic nicotine treatment
(-)-nicotine hydrogen tartrate salt (Sigma-Aldrich, Ontario) titrated to a pH of 7.0±0.3 or
saline was administered to mice (n = 263) and rats (n = 128) using osmotic minipumps (models
1002 and 2001, respectively; Alzet, Cupertino, CA). Animals were anesthetized by inhalation of
5% isofluorane in oxygen (1-2% maintenance) and the minipump placed subcutaneously
between the scapulae parallel to the spine. Nicotine was administered at doses of 1.4 and
7 mg/kg/day (free base) for 13 days in mice and 1 and 3.15 mg/kg/day (free base) for 7 days in
rats based on previous studies showing that these doses induce nicotine dependence with the
expression of spontaneous somatic withdrawal signs (Damaj et al., 2003; Malin et al., 1992,
2006; Watkins et al., 2000). After minipump implantation, the surgical wound was sutured and
treated with Polysporin antibiotic cream. Due to the faster metabolism of mice in comparison to
rats (Matta et al., 2007), mice were exposed to chronic nicotine for 6 additional days.
Blood Analysis
Blood was collected by cardiac perfusion from nicotine dependent mice (n = 3) after 12
days of exposure at the 7 mg/kg/day dose. Samples were then analyzed by high performance
49
liquid chromatography as described previously (Siu and Tyndale, 2007).
Somatic withdrawal assessment
Wild-type mice (n = 24) were observed for somatic signs of nicotine withdrawal at 30
minutes, 4, 8, 12, 24 and 48 hours after minipump removal. A group of D2R KO mice (n = 6)
was also observed for somatic signs of withdrawal at 8 hours following minipump removal. Rats
(n = 24) were observed at 30 minutes, 4, 8, 12, 16, 24, 36 and 48 hours after minipump removal.
A group of rats pretreated with α-flu (n = 8) were observed for somatic signs 16 hours following
minipump removal. Experimenters were blind to the drug treatment of each subject. Typical
abstinence signs in mice included head shakes, paw tremors, writhing, scratching, backing and
jumping (Isola et al., 1999; Stoker et al., 2008). Rats were observed for body and head shakes,
cheek tremors, eye blinks, ptosis, foot and genital licks, scratches, writhes and gasps (Malin et
al., 1992).
Place Conditioning Procedure
Mice and rats were conditioned in an apparatus as described previously (Dockstader et
al., 2001; Vargas-Perez et al., 2009). Briefly, mice were conditioned in an apparatus consisting
of two different environments measuring 15 x 15 x 15 cm. One environment was black with a
smooth Plexiglas floor that was wiped with 5% acetic acid and the other environment was white
with a wire mesh floor. The boxes were separated by a removable wall that was painted with the
corresponding color on each side. During preference testing, the dividing wall was removed and
mice were given free access to both environments. Rats were conditioned in boxes measuring 41
x 41 x 38 cm. One environment was black with a Plexiglas bottom wiped with a 3% acetic acid
solution prior to conditioning. The other environment was white with a smooth aluminum
bottom covered by a mesh grid. The test cage consisted of a black and a white conditioning cage
separated by a middle grey area. For preference testing, the rats were placed in the neutral grey
zone and given free choice between the different environments.
50
Each cage was cleaned between animals and each group was fully counterbalanced. A
single 10 min preference testing session was performed 3-5 days after the last conditioning day,
when subjects were drug- and withdrawal-free. Behavioral testing for rats consisted of three
phases: pre-exposure, conditioning and testing. The pre-exposure phase comprised a single 20
minute session in separate boxes painted grey with a grey floor. The conditioning phase
comprised one to two sessions of 40 min each for rats and 1 hour for mice, depending on the
procedure (B, N, or W - see below for details). All place conditioning and testing was performed
between 8:30 AM and 7 PM.
Procedure B (both drug and withdrawal pairing) was adapted from the method described
by Bechara et al. (1992). Procedure B involved two pairings, the first having one environment
paired to the administration of nicotine that was continuously delivered through a minipump. For
the second pairing, the minipump was removed and the animal was paired to the other
environment while experiencing withdrawal. The drug-paired environment was counterbalanced
within groups. Before the withdrawal-paired conditioning, each mouse (n = 100) and rat (n = 16)
underwent 8 hours and 16 hours of abstinence from nicotine, respectively. Thus, in procedure B,
each animal experienced the effects of chronic nicotine in one environment and the lack of
nicotine (or the effects of withdrawal) in the other environment (Figure 2.1b). The difference
score for each animal was calculated by subtracting the time spent in the withdrawal-paired
environment from the time spent in the nicotine-paired environment. This method of place
conditioning (procedure B) measures both the rewarding value of the drug itself and the
aversiveness associated with drug withdrawal (Bechara et al., 1992).
Procedure N (nicotine only) was a modified place conditioning procedure for which
conditioning took place in only the nicotine-paired environment of the place conditioning
apparatus. As in procedure B, each animal was chronically nicotine treated and confined to one
of the environments. On the alternate day, the minipump was removed and the animal
experienced withdrawal in its home cage. Thus, in contrast to procedure B, the mice (n = 23) and
rats (n = 10) were never allowed to experience withdrawal in the other compartment of the place
conditioning apparatus. The difference score for each animal was calculated by subtracting the
time spent in the non-paired environment from the time spent in the nicotine-paired environment.
51
This place conditioning method (procedure N) measures the rewarding value of the drug itself
(Bechara and van der Kooy, 1992).
In procedure W (withdrawal only), conditioning took place in only the withdrawal-paired
environment of the place conditioning apparatus. The key difference between this procedure and
procedure N was that withdrawal only (but not the direct effects of chronic nicotine) was paired
with one compartment of the place conditioning apparatus. On the first day, each mouse (n = 60)
and rat (n = 14) received a sham surgery where the minipump was removed and replaced
immediately, controlling for any effects of surgery on conditioning. For the remainder of the day,
the animal was confined to its home cage. On the conditioning day, the minipump was removed
and when the animal was experiencing withdrawal they were confined to one of the conditioning
environments. Mice were conditioned at 4 hours (n = 10), 8 hours (n = 38) and 12 hours (n = 12)
following pump removal. Rats were conditioned at 16 hours following pump removal. The
difference score for each animal was calculated by subtracting the time spent in the withdrawal-
paired environment from the time spent in the non-paired environment. This method of place
conditioning (procedure W) measures only the aversive motivational effects of drug withdrawal,
separate from the rewarding value of the drug itself (Bechara and van der Kooy, 1992).
Effects of α-flupenthixol
Mice (n = 51) and rats (n = 12) were made nicotine dependent and conditioned according
to procedure B or W as described above except that subjects were pretreated (i.p.) with either
saline or α-flu (Sigma-Aldrich). This DAR antagonist has no motivational effects of its own at
the doses and times used in this study (Laviolette and van der Kooy, 2003) and is known to
antagonize both D1Rs and D2Rs (Creese et al., 1976). Mice (n = 12) were also conditioned
according to procedure N. Mice were pretreated with 0.8 mg/kg (i.p.) at 60 minutes and rats with
0.1 mg/kg (i.p.) α-flu at 120 minutes prior to conditioning.
Acute nicotine conditioning
52
Previously drug naive WT mice (n = 62) were given a single dose of nicotine (1.75
mg/kg free base, s.c.) in one environment and saline in the other environment. This dose of
nicotine was expected to produce an acute aversive motivational response (Rauhut et al, 2008).
The mice were conditioned in the B procedure in the same way as dependent and withdrawn
mice (described above).
To examine the acute nicotine a-process, previously drug naive mice (n = 20) were
conditioned immediately following nicotine administration. To examine the effect of DA system
blockade on the acute nicotine a-process, an injection of α-flu or saline was administered one
hour prior to nicotine (n = 18).
To examine the acute nicotine b-process, mice (n = 42) were conditioned 8 hours after
nicotine administration for one hour. To examine DA system involvement in the acute nicotine
b-process, α-flu or saline was administered one hour prior to conditioning (n = 24; blocking the
b-process) or one hour prior to nicotine administration (n = 18; blocking the a-process) in
separate groups of mice.
Statistical analysis
Somatic withdrawal results were analyzed with SYSTAT software using a two-way
repeated measures ANOVA at each somatic withdrawal point after dependence assessment. For
conditioned place preference experiments, statistical analysis was performed with a one- or two-
way ANOVA. Posthoc Student-Newman-Keuls tests or Student’s t-tests were performed where
appropriate. P values of less than 0.05 were considered to be significant.
Results
Both rats and mice exhibit somatic signs upon withdrawal from chronic nicotine
Discontinuing the administration of chronic nicotine after 7 days in rats and 12 days in
53
mice produced a spontaneous somatic nicotine abstinence syndrome. The severity of this
syndrome at various time points following chronic pump removal is depicted in Figure 2.2a for
mice and 2.2b for rats, shown as mean abstinence scores taking saline as 100%. A two way
repeated measures ANOVA comparing mouse abstinence scores revealed a significant dose x
time interaction (F10,75 = 2.761, p < 0.05). Nicotine withdrawn mice displayed significantly
increased somatic withdrawal signs compared to saline-treated mice in both the 1.4 and 7
mg/kg/d group at 8 hours following pump removal (p < 0.05), but not at 4 hours (p > 0.05) or 12
hours (p > 0.05) following pump removal. The 7 mg/kg/day nicotine dose was selected for use in
subsequent mouse experiments due to the largest abstinence syndrome being observed with this
dose at the 8 hour time point.
To test whether DA signaling and the D2R specifically is involved in the emergence of
spontaneous nicotine withdrawal after chronic nicotine exposure, a group of D2R (-/-) KO mice
(n = 6) was observed for abstinence signs at 8 hours after removal of pumps containing the 7
mg/kg/day nicotine dose. D2R KO mice exhibited spontaneous somatic signs of withdrawal at a
similar level as WT mice given the 7 mg/kg/day dose of nicotine (t10 = 1.041, p > 0.05; Figure
2.2a), demonstrating that the D2R is not involved in somatic withdrawal.
Nicotine withdrawn rats after 7 days of exposure displayed the largest abstinence
syndrome at 16 hours following pump removal. A two way repeated measures ANOVA
comparing abstinence scores in rats revealed a significant dose x time interaction (F18,189 =
5.740, p <0.05; Figure 2.2b). Somatic withdrawal signs were significantly increased compared
to saline-treated rats in both the 3.15 mg/kg/d group (p < 0.05) and 1 mg/kg/d group (p < 0.05) at
16 hours following pump removal, but not at baseline prior to pump removal (F2,21 = 0.190, p >
0.05) nor after 48 hours (F2,21 = 2.900, p > 0.05) following pump removal.
An additional group of rats (n = 8) pretreated with α-flu was observed for somatic signs
of nicotine withdrawal 16 hours after pump removal. Nicotine withdrawn rats treated with α-flu
showed significant somatic withdrawal signs in comparison to saline treated animals (t14 = 6.20,
p < 0.05; Figure 2.2b), therefore it appears that somatic withdrawal is not mediated by signaling
at DA receptors.
54
Figure 2.2. The time course of spontaneous nicotine somatic and motivational withdrawal.
(A) Mice were given chronic nicotine in osmotic minipumps (7 and 1.4 mg/kg/day) for 13 days.
Somatic withdrawal signs were recorded at 30 minutes, 4, 8, 12, 24 and 48 hours following
minipump removal. An abstinence syndrome compared to saline-treated mice was observed that
peaked at 8 hours following pump removal, suggesting that chronic nicotine and spontaneous
withdrawal will induce a somatic withdrawal syndrome in mice that peaks 8 hours after the
removal of chronic nicotine. A group of D2R KO mice given 7 mg/kg/day nicotine for 13 days
was also observed for abstinence signs 8 hours after pump removal. The D2R KO mice showed a
somatic withdrawal syndrome that did not differ from WT mice, suggesting that the D2R does
not mediate the expression of a somatic nicotine withdrawal syndrome. (B) Somatic withdrawal
signs were recorded in rats at 30 minutes, 4, 8, 12, 24 and 48 hours following minipump
removal. After 7 days of nicotine minipumps (1 and 3.16 mg/kg/day), an abstinence syndrome
compared to saline-treated rats was observed that peaked at 16 hours following pump removal. A
group of rats treated with α-flu were observed for abstinence signs 16 hours after pump removal.
These rats showed a somatic withdrawal syndrome similar to nicotine-dependent and -withdrawn
rats, suggesting that DA receptor antagonism does not affect the expression of a somatic
withdrawal syndrome. (C) Mice were trained in the W procedure at 4, 8, and 12 hours following
pump removal at the 7 mg/kg/day chronic nicotine dose. A significant aversive motivational
response to the withdrawal-paired environment was observed in nicotine-dependent and -
withdrawn mice only at the 8-hour time point. These results suggest that the motivational
response to withdrawal corresponds to the time point of somatic withdrawal. Data represent
means +/- SEM (*p < 0.05).
55
0 100 200 300 400 500 600
30 m 4 h 8 h 12 h 24 h 48 h % A
bstin
ence
sco
re
Time since pump removal
Saline 1.4 mg/kg/d 7 mg/kg/d D2 KO
A Mice
*
0 100 200 300 400 500 600
30 m 4 h 8 h 12 h 16 h 24 h 36 h 48 h % A
bstin
ence
sco
re
Time since pump removal
Saline 1 mg/kg/d 3.16 mg/kg/d α-flu pretreatment
B
*
-250 -200 -150 -100
-50 0
50 100
4 h 8 h 12 h Saline
Diff
eren
ce s
core
(s)
Time since pump removal
C
*
Rats
56
Chronic nicotine exposure did not noticeably affect the subjects during the exposure
period (based on observations of locomotor activity, feeding patterns and general behaviour). To
compare our dose of nicotine in mice to the human condition, we analyzed plasma levels of
nicotine in mice treated with the 7 mg/kg/day dose and found an average of 29.9 ng/mL ± 15.5, a
result that is similar to the average maximum arterial blood concentration of human chronic
smokers (range ~20-40 ng/mL) (Armitage et al., 1975; Matta et al., 2007; O’Dell et al., 2006).
Somatic and affective nicotine withdrawal occur coincidentally
To determine if the time when maximal somatic withdrawal signs were observed
corresponded to the time of motivational response in a place conditioning paradigm, chronic
nicotine-treated mice were conditioned in the W procedure at 4 and 12 hours following pump
removal (when few somatic withdrawal signs were observed in mice) and compared to 8 hours
following pump removal (when most somatic withdrawal signs were observed in mice). Saline-
treated mice conditioned at 4, 8, and 12 hours following pump removal showed no significant
difference for time of conditioning (F2,25 = 0.279, p > 0.05) and were therefore analyzed as one
group. A two-way ANOVA showed a significant treatment x withdrawal time interaction (F2,44 =
3.414, p < 0.05; Figure 2.2c). Nicotine-treated mice demonstrated a significant motivational
effect only at the 8 hour time point (p < 0.05), a result which validates the use of the 8 hours
following pump removal time point as our maximal withdrawal conditioning time and suggests
that somatic withdrawal coincides with motivational withdrawal over time.
Mice and rats exhibit conditioned place aversions to an environment paired with nicotine
withdrawal
To dissociate the rewarding from the aversive motivational effects of nicotine and
nicotine withdrawal in dependent subjects, we performed place conditioning using the B, N and
W procedures. A one-way ANOVA comparing the B, N, W procedures and saline in mice
showed a significant effect of chronic nicotine treatment (F3,47 = 15.372, p < 0.05; Figure 2.3a).
Saline-treated mice in each of the B, N, and W procedures were not significantly different (F2,13
57
Figure 2.3. The opponent processes of chronic and acute nicotine and the effect of DA
receptor antagonism.
(a) Separation of the rewarding effects of chronic nicotine from the aversive effects of
withdrawal in nicotine dependent mice, as revealed by a modified place conditioning paradigm
for assessing the rewarding effects of nicotine only (Nicotine procedure), the aversive effects of
nicotine withdrawal (Withdrawal procedure), or both the rewarding effects of nicotine and the
aversive effects of withdrawal (Both procedure). Nicotine dependent mice conditioned in all
three procedures showed a significant motivational response as compared to saline-treated
animals, with negative and positive difference scores representing aversive and rewarding
motivational responses, respectively. (b) α-flu pretreatment attenuated the motivational response
in each of the B, N, and W procedures, and had no motivational effect on its own in saline-
treated mice, suggesting that both the rewarding response to chronic nicotine and the aversive
response to withdrawal are DA-mediated. (c) Separation of the rewarding effects of chronic
nicotine from the aversive effects of withdrawal in nicotine dependent rats. Significant effects as
measured by place conditioning were observed in the Both and Withdrawal procedures, but not
in the Nicotine procedure as compared to saline. (d) α-flu pretreatment attenuated the B and W
effects. (e) Previously drug naive mice administered a single dose of nicotine and conditioned
immediately (0 hours) in the B procedure demonstrated a significant conditioned place aversion
for the nicotine-paired environment. Mice conditioned 8 hours after acute nicotine administration
demonstrated a significant preference for the nicotine-paired environment. Mice conditioned 4 or
12 hours after nicotine administration showed no significant motivational effect. These results
suggest that after acute nicotine administration, an aversive a-process is followed by a rewarding
b-process 8 hours later. Data represent means +/- SEM (*p < 0.05).
58
times to those used above in nicotine dependent andwithdrawn mice. We administered a single dose of nicotine(1.75mg/kg, s.c.) and conditioned separate groups of miceimmediately (0 h) and 4, 8, and 12 h after nicotine admin-istration. A one-way ANOVA showed a significant effectof conditioning time (F3,36¼ 7.827, po0.05; Figure 3e).When drug naive mice were given an acute dose of nicotineand conditioned immediately after nicotine administra-tion, they showed a significant conditioned place aversion(po0.05). The opponent process theory of motivationsuggests that an acute aversive effect of nicotine (a-process)will set up a longer lasting opponent b-process that shouldmanifest as a rewarding response to the environment pairedwith 8 h of abstinence from acute aversive nicotine. Weobserved a significant preference for the nicotine-pairedenvironment at 8 h after nicotine administration (po0.05).When mice were conditioned 4 and 12 h after acute nicotineadministration, they showed no significant motivationaleffect (p40.05). These results show that an acute aversivedose of nicotine stimulates two separate opposing motiva-tional effects: an aversive response when mice are condi-tioned immediately after nicotine administration and a
rewarding response when mice are conditioned 8 h afternicotine administration.
DA Receptor Blockade Attenuates Chronic NicotineWithdrawal Aversions
We examined whether the DA system mediates themotivational responses to nicotine and withdrawal in thenicotine-dependent state. Mice were given a-flu 1 h beforeconditioning in the B, N, and W procedures. A two-wayANOVA using procedure (B, N, W, or saline) and pre-treatment (a-flu or saline) as independent factors showeda procedure" pretreatment interaction (F3,106¼ 11.593,po0.05; Figure 3b). Saline-treated mice in each of the B,N, and W procedures were not significantly different(F2,19¼ 0.043, p40.05) and were therefore analyzed as onegroup. DA receptor blockade prevented the conditionedmotivational effect in the B, N, and W procedures (p40.05).Mice conditioned in the B procedure with a-flu in oneenvironment and saline in the other environment showedno motivational preference (p40.05), showing that the dose
-300
-200
-100
0
100
200
300
Both Nicotine Withdrawal Saline
Dif
fere
nce s
co
re (
s)
Procedure
Mice
-150
-100
-50
0
50
100
150
Both Nicotine Withdrawal Saline
Dif
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re (
s)
Procedure
α-flu
-300
-200
-100
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100
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300
Both Nicotine Withdrawal Saline
Dif
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co
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s)
Procedure
Rats
-150
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Both Withdrawal Saline D
iffe
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s)
Procedure
α-flu
-300
-200
-100
0
100
200
300
0 4 8 12
Dif
fere
nce S
co
re (
s)
Time since acute nicotine administration (h)
Figure 3 The opponent processes of chronic and acute nicotine and the effect of dopamine (DA) antagonism. (a) Separation of the rewarding effects ofchronic nicotine from the aversive effects of withdrawal in nicotine-dependent mice, as revealed by a modified place conditioning paradigm for assessing therewarding effects of nicotine only (Nicotine procedure; n¼ 11), the aversive effects of nicotine withdrawal (Withdrawal procedure; n¼ 14), or both therewarding effects of nicotine and the aversive effects of withdrawal (Both procedure; n¼ 15). Nicotine-dependent mice conditioned in all three proceduresshowed a significant motivational response as compared with saline-treated animals (*po0.05). (b) a-flu (0.08mg/kg, i.p.) pretreatment attenuated themotivational response in each of the B (n¼ 16), N (n¼ 12), and W (n¼ 14) procedures, and had no motivational effect in saline-treated mice. (c)Separation of the rewarding effects of chronic nicotine from the aversive effects of withdrawal in nicotine-dependent rats. Significant effects were observed inthe Both (n¼ 8) and Withdrawal (n¼ 10) procedures, but not in the Nicotine (n¼ 10) procedure (*po0.05) as compared with saline. (d) a-flu (0.1mg/kg)pretreatment attenuated the Both (n¼ 8) and Withdrawal (n¼ 4) effects. (e) Previously drug naive mice (n¼ 13) administered a single dose of nicotine(1.75mg/kg, s.c.) and conditioned immediately (0 h) in the B procedure showed a significant conditioned place aversion for the nicotine-paired environment(*po0.05). Mice (n¼ 14) conditioned 8 h after acute nicotine administration showed a significant preference for the nicotine-paired environment(*po0.05). Mice conditioned 4 or 12 h after nicotine administration showed no significant effect. Data represent means±SEM.
Dopamine and nicotine motivationTE Grieder et al
948
Neuropsychopharmacology
59
= 0.447, p > 0.05) and were therefore analyzed as one group. Mice showed a significant aversion
to the withdrawal-paired side as compared to the nicotine-paired side in the B procedure (p <
0.05), demonstrating that either nicotine is rewarding to dependent animals, or withdrawal is
aversive, or both. To determine which of these motivational effects was responsible for the B
procedure results, we used the N and W procedures, which separate the rewarding effects of a
drug from the aversiveness of withdrawal (Bechara and van der Kooy, 1992). Nicotine-
dependent mice demonstrated a significant preference for the nicotine-paired side versus the non-
paired side in the N procedure (p < 0.05), suggesting that the presence of nicotine in a nicotine-
dependent animal is rewarding. In the W procedure, mice demonstrated a significant aversion to
the withdrawal-paired side compared to the non-paired side (p < 0.05), suggesting that
withdrawal from chronic nicotine is indeed aversive. To control for a bias in novelty seeking in
the N and W procedures, we tested for a place preference for a novel, previously unpaired
environment in saline-treated animals. Saline-treated mice did not show a preference for the
novel side over a previously paired side (t5 = 0.932, p > 0.05; data not shown), demonstrating
that a novelty effect cannot account for the N and W procedure results.
To evaluate whether the rewarding and aversive properties of chronic nicotine would
generalize to another species we conditioned rats using the same protocols as mice. A one-way
ANOVA comparing the B, N, W procedures and saline in rats showed a significant effect of
procedure (F3,44 = 18.896, p < 0.05; Figure 2.3c). Saline-treated rats in each of the B, N, and W
procedures were not significantly different (F2,21 = 0.016, p > 0.05) and were therefore analyzed
as one group. Rats demonstrated a significant aversion to the withdrawal-paired side as
compared to the nicotine-paired side in the B procedure (p < 0.05). In the N procedure, nicotine-
dependent rats did not prefer the nicotine-paired or the non-paired environment (p > 0.05). Rats
tested in the W procedure demonstrated an aversion to the withdrawal-paired side compared to
the non-paired side (p < 0.05), suggesting that withdrawal from chronic nicotine is indeed
motivationally aversive. Results from the N and W procedures in rats suggest that the
motivational effect in the B procedure may be attributable primarily to an aversive response to
nicotine withdrawal.
60
Acute nicotine stimulates opposing motivational processes
We next examined whether acute nicotine would elicit opposing motivational processes
in previously drug naive mice, using the B procedure and identical conditioning times to those
used above in nicotine dependent and withdrawn mice. We administered a single dose of
nicotine (1.75 mg/kg, s.c.) and conditioned separate groups of mice immediately (0 hours) and 4,
8 and 12 hours after nicotine administration. A one-way ANOVA showed a significant effect of
conditioning time (F3,36 = 7.827, p < 0.05; Figure 2.3e). When drug naive mice were given an
acute aversive dose of nicotine and conditioned immediately following nicotine administration,
they demonstrated a significant conditioned place aversion (p < 0.05). The opponent process
theory of motivation suggests that an acute aversive effect of nicotine (a-process) will set up a
longer lasting opponent b-process that should manifest as a rewarding response to the
environment paired with 8 hours of abstinence from acute aversive nicotine. We observed a
significant preference for the nicotine-paired environment at 8 hours following nicotine
administration (p < 0.05; Figure 2.3e). When mice were conditioned 4 and 12 hours after acute
nicotine administration, they showed no significant motivational effect (p > 0.05). These results
demonstrate that an acute aversive dose of nicotine stimulates two separate opposing
motivational effects: an aversive a-process response when mice are conditioned immediately
after nicotine administration and a rewarding b-process response when mice are conditioned 8
hours after nicotine administration.
DA receptor blockade attenuates chronic nicotine withdrawal aversions
We examined if the DA system mediates the motivational responses to nicotine and
withdrawal in the nicotine dependent state. Mice were given α-flu 1 hour prior to conditioning in
the B, N, and W procedures. A two way ANOVA using procedure (B, N, W, or saline) and
pretreatment (α-flu or saline) as independent factors showed a procedure x pretreatment
interaction (F3,106 = 11.593, p < 0.05; Figure 2.3b). Saline-treated mice in each of the B, N, and
W procedures were not significantly different (F2,19 = 0.043, p > 0.05) and were therefore
analyzed as one group. DA receptor blockade prevented the conditioned motivational effect in
the B, N, and W procedures (p > 0.05). Mice conditioned in the B procedure with α-flu in one
61
environment and saline in the other environment showed no motivational preference (t6 = 0.861,
p > 0.05; data not shown), demonstrating that the dose of α-flu used in the present experiments
does not have any motivational effects on its own.
Consistent results were obtained in rats when they were pretreated with α-flu 1 hour prior
to conditioning in the B and W procedures. A two way ANOVA using procedure (B, W, or
saline) and pretreatment (α-flu or saline) as independent factors showed a procedure x
pretreatment interaction (F2,61 = 9.174, p < 0.05; Figure 2.3d). Saline-treated rats in the B and W
procedures were not significantly different (F1,14 = 0.032, p > 0.05) and were therefore analyzed
as one group. We did not include a group of rats pretreated with α-flu in the N procedure, as no
motivational effect was observed in the dependent group (Figure 2.3c). DA receptor blockade
prevented the conditioned motivational effect in the B and W procedures (p > 0.05). The results
from both rats and mice receiving α-flu pretreatment in the W procedure demonstrate that the
DA system is mediating the aversive response to withdrawal after chronic nicotine.
Dopaminergic signaling specifically mediates chronic nicotine withdrawal aversions
In order to further investigate dopaminergic mediation of the rewarding effects of
nicotine versus the aversiveness of withdrawal in nicotine-dependent animals, we gave α-flu to
mice in either the nicotine-paired or withdrawal-paired environment in the B procedure and
observed the effect on the aversive motivational response after 8 hours of abstinence from
chronic nicotine. A one-way ANOVA showed a significant effect of treatment (F2,33 = 10.941, p
< 0.05; Figure 2.4a). Mice given saline on both conditioning days demonstrated an aversive
motivational response to chronic nicotine withdrawal, as observed previously (saline; p < 0.05).
When mice were pretreated with α-flu on the nicotine-paired side and given saline on the
withdrawal-paired side, a preference for the nicotine-paired environment over the withdrawal-
paired environment was observed (no delay; p < 0.05). Mice that received the opposite treatment
(α-flu on the withdrawal-paired side and saline on the nicotine-paired side) did not show an
aversive response to withdrawal from chronic nicotine (delay; p > 0.05). These results suggest
that the dopaminergic system mediates the aversive response to withdrawal from chronic
nicotine.
62
Figure 2.4. Dopaminergic signaling differentially mediates the opponent motivational
process after acute and chronic nicotine.
(a) Mice chronically treated with nicotine and pretreated with saline demonstrate a conditioned
place aversion to the withdrawal-paired environment in the B procedure. α-flu given 1 hour prior
to conditioning during chronic nicotine administration (no delay) did not prevent the aversive
response from occurring in dependent mice. However, α-flu given prior to conditioning after 8
hours of withdrawal from chronic nicotine (delay) blocked the aversive response from occurring.
These results suggest that DA activity is necessary for the b-process occurring after chronic
nicotine. (b) Previously drug naive mice given acute nicotine, pretreated with saline and
conditioned after 8 hours of abstinence from nicotine demonstrate a preference for the
environment paired with the acute nicotine b-process. α-flu administered prior to the acute
nicotine a-process (no delay) prevented this rewarding response from occurring. However, α-flu
given prior to b-process conditioning after 8 hours of abstinence from acute nicotine (delay) did
not prevent the occurrence of the rewarding response, suggesting that DA activity is not required
for the acute nicotine b-process. Data represent means +/- SEM (*p < 0.05).
63
-250
-200
-150
-100
-50
0
50
100
Diff
eren
ce s
core
(s)
Chronic nicotine A
*
-50
0
50
100
150
200
Saline α-flu, no delay α-flu, delay
Diff
eren
ce s
core
(s)
Acute nicotine B
*
64
DA antagonism does not block the rewarding motivational response 8 hours after acute nicotine
We next investigated if the DA system mediates the positive b-process response observed
8 hours after acute nicotine in the same way as it does the aversive b-process response observed
8 hours after chronic nicotine. The immediate aversive effects of acute nicotine are blocked by
pretreatment with α-flu in rats (Laviolette and van der Kooy, 2003). Similarly, we observed that
the immediate aversive response to acute nicotine (the a-process) in mice was blocked by α-flu
pretreatment (t6 = 1.785, p>0.05; data not shown). To investigate the effect of DAR antagonism
on the rewarding motivational b-process 8 hours after acute aversive nicotine, we administered
α-flu 1 hour prior to conditioning for either the immediate aversive or the delayed rewarding
effect, similar to the procedure followed with nicotine dependent mice. A one-way ANOVA
showed a significant effect of treatment (F2,39 = 4.068, p < 0.05; Figure 2.4b). Mice pretreated
with saline showed a preference for an environment paired with 8 hours of abstinence from acute
nicotine (the b-process), as observed previously (Saline; p < 0.05). However, α-flu pretreatment
prior to acute nicotine prevented the later occurring rewarding response (no delay; p > 0.05).
This result suggests that the immediate aversive a-process response to acute nicotine is required
for the delayed rewarding b-process response to occur. Mice treated with α-flu prior to
conditioning for the delayed rewarding response exhibited a similar conditioned place preference
to saline-treated mice (delay; p < 0.05). These results demonstrate that dopaminergic signaling is
required for conditioning to the immediate aversive effect of acute nicotine, but not the delayed
rewarding b-process effect occurring 8 hours after acute nicotine.
The D2R mediates the aversive response to withdrawal from chronic nicotine
The behavior of male D2 (+/+) WT and D2 (-/-) KO littermate mice was examined using
the N and W place conditioning procedures to determine the role of the D2R in mediating the
motivational response to chronic nicotine and withdrawal. A two-way ANOVA using genotype
and procedure (N or W) showed a genotype x procedure interaction (F2,42 = 20.260, p < 0.05;
Figure 2.5). WT mice conditioned in the N procedure showed a significant preference for an
environment paired with chronic nicotine (p<0.05) that was not demonstrated by D2R KO mice
(p > 0.05). WT mice conditioned in the W procedure demonstrated a significant aversion to
65
Figure 2.5. The D2R mediates the aversive response to chronic nicotine withdrawal.
WT mice conditioned in procedure N showed a preference for chronic nicotine that was not
present in D2R KO mice. D2R KO mice conditioned in procedure W did not exhibit nicotine
aversions while WT mice showed normal aversions to chronic nicotine withdrawal. These results
suggest that the D2R is important for signaling both the rewarding response to chronic nicotine
and the aversive response to nicotine withdrawal in dependent mice. Data represent means +/-
SEM (*p < 0.05).
-200 -150 -100
-50 0
50 100 150 200 250
Nicotine Withdrawal
Diff
eren
ce S
core
(s)
Procedure
WT D2 KO *
*
66
an environment paired with withdrawal from chronic nicotine (p < 0.05), similar to the WT mice
in previous experiments. However, D2R KO mice did not show an aversion to withdrawal (p >
0.05), indicating that the D2R mediates the aversive response to withdrawal from chronic
nicotine.
Discussion
Understanding the neurobiological substrates mediating the motivational response
experienced by smokers during nicotine withdrawal has important implications for improving
smoking cessation. We show here that dopaminergic signaling at the D2R mediates affective but
not somatic nicotine withdrawal. Moreover, we have dissociated the role of dopaminergic
signaling in the opponent motivational processes of acute and chronic nicotine. Indeed,
dopaminergic signaling, specifically at the D2R, is required for the delayed motivational
response to chronic nicotine in dependent subjects. In contrast, dopaminergic signaling is
required for the immediate but not the delayed motivational response to acute nicotine in non-
dependent subjects.
Somatic signs observed in the present experiments resemble those described previously
in mice (Isola et al., 1999; Stoker et al., 2008) and rats (Epping-Jordan et al., 1998; Malin et al.,
1992; Watkins et al., 2000); however the time course of peak nicotine somatic withdrawal
differs. The use of a variety of time points in our measurements of somatic withdrawal symptoms
allowed us to find the most appropriate time for withdrawal motivation studies in both mice and
rats. Although the dose used in mice (7 mg/kg/day) and rats (3.15 mg/kg/day) exceeds the
amount of nicotine smoked by the heaviest smokers of high-yield cigarettes (Armitage et al.,
1975; Epping-Jordan et al., 1998), it is important to consider that rats and mice have much
higher metabolic and drug clearance rates (Matta et al., 2007) than humans. Furthermore, plasma
levels of nicotine measured presently were similar to those observed in humans and measured in
previous rodent studies (Guillem et al., 2005; O’Dell et al., 2006). We showed that nicotine
somatic withdrawal coincides with affective withdrawal, such that the largest abstinence
syndrome in both mice and rats occurs when the aversive response to withdrawal can be
67
conditioned in the place preference paradigm. Dopaminergic signal disruption by α-flu
pretreatment or genetic deletion of the D2R blocked the aversive motivational response to
affective nicotine withdrawal but not the somatic signs of withdrawal. These results suggest that
somatic and affective motivational withdrawal occur coincidentally, but are not causally related.
Similarly, motivational withdrawal from opiates can be blocked without attenuating somatic
withdrawal signs (Bechara et al., 1995), lending support to the idea that somatic withdrawal
signs do not necessarily reflect the motivational impact of withdrawal (Watkins et al., 2000).
Although self-administration more closely models human nicotine intake (Rose and
Corrigall, 1997), separating drug motivation due to the rewarding effects of nicotine or the
alleviation of withdrawal is more easily performed using a place conditioning procedure (Mucha
et al., 1982). Our place conditioning experiments showed that nicotine withdrawal is aversive in
both dependent mice and rats. Furthermore, results from the N and W procedures in rats suggest
that the motivational effect in the B procedure may be attributable primarily to an aversive
response to nicotine withdrawal, and that the motivational effects observed in the N procedure in
mice might reflect the ability of nicotine to overcome the aversiveness of withdrawal. Nicotine
reward in dependent animals has been previously demonstrated in conditioned place preference
(Acquas et al., 1989; Sellings et al., 2008; Wilkinson et al., 2008), self administration
(Tammimaki et al., 2008) and ICSS (Kenny and Markou, 2001) paradigms, however the N place
conditioning procedure used in the present experiments was not sensitive enough to measure the
rewarding effect of nicotine in dependent rats. An alternative hypothesis would be that the dose
of nicotine (compared to mice) used in the present experiments was not sufficient to produce a
motivational response, and that a higher dose of nicotine in rats would elicit a preference for the
nicotine-paired environment in the N procedure.
We demonstrated that dopaminergic signaling through activation of D2Rs is critical for
the expression of chronic nicotine withdrawal aversions. It is unlikely that the present results are
due to a learning deficit in D2R KO mice as these mice can learn morphine (Dockstader et al.,
2001) and ethanol place preferences (Ting-A-Kee et al., 2009). This result confirms previous
work showing that D1R and D2R antagonists block conditioned aversions to nicotine withdrawal
using pharmacologically precipitated withdrawal (Laviolette et al., 2008) and further extends
68
these findings to spontaneous withdrawal, which more closely models the human condition.
The opponent process theory of motivation (Solomon and Corbit, 1974) postulates that
any motivational stimulus activates two opposing motivational processes, the a-process having a
fast onset and offset and the b-process being slower to start, longer lasting and occurring in an
opposite direction to the a-process (Ettenberg, 2004; Koob and Le Moal, 2008; Koob et al.,
1989). We have shown that an aversive dose of nicotine will act as a negative motivational
stimulus in previously drug naive mice that will manifest as a negative a-process, in turn causing
the activation of a delayed positive b-process. In nicotine dependent animals, chronic nicotine
will act as a rewarding motivational stimulus that will manifest as a positive a-process, in turn
causing the activation of a delayed aversive b-process during withdrawal. Each of these opponent
motivational effects was modeled using the place conditioning paradigm. We demonstrated that
signaling at dopaminergic receptors is required for the immediate aversive response (a-process)
but not for the delayed rewarding effect after acute nicotine (b-process). In nicotine dependent
animals, we showed that signaling at dopaminergic receptors is required for both the immediate
rewarding response to chronic nicotine (a-process) and for the delayed aversive response (b-
process) to withdrawal from chronic nicotine.
The immediate aversive response to nicotine in previously drug naive mice could be due
to central and/or peripherally mediated effects. Indeed, conditioned place aversions to acute
nicotine could be due to peripheral effects since nicotine is known to induce nausea (Perkins et
al., 2008). However, conditioned place aversions to acute nicotine have been demonstrated after
intra-cerebral administration (Laviolette et al., 2003). Furthermore, nicotine given peripherally
has centrally mediated effects on DA release (Seppa et al., 2000), therefore it is unlikely that the
acute aversive motivational response is simply due to nausea or another peripheral effect.
The role of the dopaminergic and TPP systems in the opponent motivational effects
produced by nicotine bears striking resemblance to those produced by opiates. The aversive a-
process is DA-mediated in both acute opiate (Zito et al., 1988) and acute nicotine motivation, as
shown presently and previously (Laviolette et al., 2003). The rewarding a-process for acute
opiates (Bechara et al., 1992) and acute nicotine are TPP mediated (Laviolette et al., 2002). We
69
have shown that the rewarding b-process after acute aversive nicotine is not DA-mediated, which
resembles the DA-independent acute b-process for opiates (Bechara et al., 1992). Although the
TPP appears to be a good candidate to mediate the acute b-process in both nicotine and opiate
motivation, it was recently suggested that the TPP does not mediate the acute opiate b-process
(Vargas-Perez et al., 2009). However, TPP involvement in the acute nicotine b-process cannot be
completely ruled out as the b-process after acute aversive nicotine is rewarding, and TPP
involvement in acute nicotine reward has been previously demonstrated (Laviolette et al., 2002).
Results from this study showed that nicotine given acutely in a previously drug naive
animal elicited a DA-mediated aversion in a place conditioning paradigm, while other groups
have demonstrated DA-dependent acute nicotine reward (Acquas et al., 1989; Lecca et al., 2006;
Merlo-Pich et al., 1999; Pak et al., 2006; Sellings et al., 2008; Spina et al., 2006). Acute nicotine
administered directly into the brain produces both rewarding and aversive motivational effects
(Laviolette et al., 2002; Sellings et al., 2008) that can be segregated within the nucleus
accumbens (Sellings et al., 2008). Therefore it is not surprising that different groups have
reported that different paradigms (place conditioning vs. self-administration) or routes of nicotine
administration (intracerebral, subcutaneous, intravenous) produce differences in the direction of
the observed motivational response.
It is possible that nicotine intake may occur during withdrawal to restore previous levels
of dopaminergic signaling in the dependent user’s brain and therefore to blunt the negative
experience of withdrawal. The N procedure results in mice may be due to the ability of nicotine
to alleviate withdrawal. In support of this idea are the present data showing that the motivational
response observed in the N and W procedures do not add to give the B (both) motivational effect.
The argument then follows that nicotine is not actually rewarding in dependent animals, and the
rewarding effect observed in mice conditioned in the N procedure is simply an alleviation of the
aversiveness of withdrawal. Furthermore, when dopaminergic signaling was blocked during
withdrawal-paired conditioning but left intact during nicotine-paired conditioning in the B
procedure, no motivational response was observed. When the opposite experiment was
performed, where the DA system was blocked during nicotine-paired conditioning but left intact
during withdrawal-paired conditioning, a conditioned place aversion to the withdrawal-paired
70
environment was observed. These results suggest that nicotine motivation in nicotine-dependent
and -withdrawn animals is driven by a DA-dependent aversion to nicotine withdrawal.
Dopaminergic signaling after nicotine administration is a complex phenomenon
involving tonic and phasic DA activity (Rice and Cragg, 2004; Zhang and Sulzer, 2004; Zhang et
al., 2009). We hypothesize that the motivational response to withdrawal from chronic nicotine is
mediated by a dysregulated pattern of DA signaling at the receptor resulting from a decreased
level of DA in the NAc during withdrawal (Hildebrand et al., 1998; Rada et al., 2001; Rahman et
al., 2004). We pharmacologically and genetically modified the specific dopaminergic signal that
occurs during withdrawal and therefore blocked the negative affective component of withdrawal.
These results suggest that the specific pattern of DA signaling mediates the aversive motivational
response to nicotine withdrawal.
The present study suggests that dopaminergic signaling is necessary for the opponent
motivational response to nicotine in dependent, but not non-dependent, animals. Further,
signaling at the D2R is critical in mediating withdrawal aversions in nicotine-dependent animals.
These results suggest that different neurobiological substrates mediate the opponent motivational
process for nicotine in drug dependent and non-dependent animals and that the alleviation of
nicotine withdrawal primarily may be driving nicotine motivation in dependent animals. These
findings may have implications in understanding motivational processes in dependent smokers
and may therefore inform targeted drug development in this population.
71
Chapter 3
Phasic D1 and Tonic D2 Dopamine Receptor Signaling Double Dissociate the Motivational Effects of
Acute Nicotine and Chronic Nicotine Withdrawal
Taryn E. Grieder, Olivier George, Huibing Tan, Susan R. George, Bernard Le Foll, Steven R.
Laviolette, Derek van der Kooy
This chapter is adapted from the paper published in Proceedings of the National Academy of Sciences,
vol. 109, issue 8, p 3101-3106, 2012. Reprinted with permission.
72
Abstract
Nicotine, the main psychoactive ingredient of tobacco smoke, induces negative motivational
symptoms during withdrawal that contribute to relapse in dependent individuals. The
neurobiological mechanisms underlying how the brain signals nicotine withdrawal remain poorly
understood. Using electrophysiological, genetic, pharmacological and behavioral methods, we
demonstrate that tonic but not phasic activity is reduced during chronic nicotine withdrawal in
VTA dopaminergic neurons, and that this pattern of signaling acts through D2Rs and adenosine
A2ARs, but not D1Rs. Selective blockade of phasic DA activity prevents the expression of
conditioned place aversions to a single injection of nicotine in nondependent mice, but not to
withdrawal from chronic nicotine in dependent mice, suggesting a shift from phasic to tonic
dopaminergic mediation of the conditioned motivational response in nicotine dependent and
withdrawn animals. Either increasing or decreasing activity at D2Rs or A2ARs prevents the
aversive motivational response to chronic nicotine withdrawal, but not to acute nicotine.
Modification of D1R activity prevents the aversive response to acute nicotine, but not to nicotine
withdrawal. This double dissociation demonstrates that the specific pattern of tonic dopaminergic
activity at D2Rs is a key mechanism in signaling the motivational effects experienced during
nicotine withdrawal, and may represent a novel target for therapeutic treatments for nicotine
addiction.
73
Introduction
Tobacco addiction is the leading avoidable cause of disease and premature death in North
America (Fellows et al., 2002). Of more than 3000 chemicals present in tobacco smoke, nicotine
is the main psychoactive ingredient responsible for tobacco addiction (Koob and Le Moal, 2006).
Withdrawal from nicotine is hypothesized to represent a powerful source of negative
reinforcement that drives relapse and compulsive tobacco use (George et al., 2007), therefore
understanding the neurobiological substrates mediating the motivational properties of nicotine
withdrawal is an important step in the development of new treatments for nicotine addiction.
Current hypotheses suggest that nicotine withdrawal leads to a decrease in DA signalling in the
brain (Kalivas and Volkow, 2005) and that DA neurons exhibit two activity states, phasic and
tonic, that mediate separate aspects of goal-directed behaviour (Floresco et al., 2003; Goto and
Grace, 2005; Grace, 2000). However, the role of these two activity states in the motivational
effects of nicotine is unknown.
Nicotine acutely produces both aversive and positive motivational effects (Grunberg,
1994; Laviolette and van der Kooy, 2004; Sellings et al., 2008) by activating the mesolimbic DA
system (Grace, 2000; Nestler, 2005) as well as non-dopaminergic neural substrates (Fowler et
al., 2011; Laviolette et al., 2003). DA neurons exhibit burst and population firing activity that
leads to phasic and tonic DA release, respectively (Floresco et al., 2003; Goto and Grace, 2005;
Grace, 2000). Burst firing produces a fast and large DA release that mainly activates
postsynaptic D1Rs, while population firing produces a slower tonic DA release that mainly
activates the higher affinity (Hikida et al., 2010) D2Rs (Floresco et al., 2003; Goto and Grace,
2005). The phasic and tonic activities of DA neurons are thought to mediate different aspects of
goal-directed behavior; phasic activity facilitates cue-reward association and acquisition of
incentive salience, whereas tonic activity is involved in response inhibition and behavioral
flexibility (Floresco et al., 2003; Zweifel et al., 2009; Goto and Grace, 2005). Consistent with its
motivational properties, a single systemic nicotine injection increases phasic activity in the VTA
(Mameli-Engvall et al., 2006) and the release of DA in the ventral striatum (Zhang et al., 2009;
Tan et al., 2009), while chronic exposure to nicotine decreases tonic but not phasic DA activity
in the VTA (Tan et al., 2009). However, the role of tonic activity and the D2R vs. phasic activity
74
and the D1R in signaling the motivational effects of both acute nicotine and chronic nicotine
withdrawal is unknown. Here we tested whether withdrawal from chronic nicotine differentially
affects tonic and phasic dopaminergic activity in the VTA and whether the specific pattern of
signaling through D1Rs and D2Rs mediates the expression of the aversive motivational
responses to acute nicotine and chronic nicotine withdrawal.
Materials and methods
Animals
All animal use procedures were approved by the University of Toronto Animal Care
Committee, in accordance with the Canadian Council on Animal Care guidelines. Adult male
Wistar rats and C57BL/6 mice (Charles River, Montreal, Canada) and D1R, D2R and A2AR
knockout mice were housed in a temperature-controlled room with lights on from 6 AM to 8 PM.
Heterozygous twelfth-generation D1R and fifth-generation D2R breeder mice were received as a
gift from DK Grandy and MJ Low and heterozygous A2AR mice from M Schwarszchild and J
Chen. Crosses were bred at the University of Toronto to obtain homozygous male D1R, D2R and
A2AR knockout mice and their wild-type controls.
Drugs
Nicotine hydrogen tartrate salt (Sigma-Aldrich, Ontario) was dissolved in saline at
pH 7.0±0.3 and administered via osmotic minipumps (chronic nicotine, 7 mg/kg/day) or s.c.
injection (acute nicotine, 1.75 mg/kg). The DAR agonist apomorphine (2.5 mg/kg), DAR
antagonist α-flu (0.8 mg/kg), D1R antagonist SCH23390 (0.01 mg/kg), D1R agonist A-77636
(0.1 mg/kg), D2R agonist quinpirole (0.05 mg/kg), D2R antagonist eticlopride (1.0 mg/kg),
A2AR antagonist SCH58261 (0.5 mg/kg) and A2AR agonist CGS21680 (0.1 mg/kg) were
purchased from Sigma-Aldrich, Ontario, dissolved in PBS and administered i.p. at 0, 60, 10, 0,
15, 20, 30 and 20 minutes prior to conditioning, respectively. The NMDA receptor antagonist
CGP39551 (2.5 mg/kg) was purchased from Tocris, Missouri, dissolved in PBS and
administered i.p. immediately prior to conditioning. Rimonabant (3.0 mg/kg; National Institute
75
on Drug Abuse) was suspended in 0.3% Tween80 in saline and administered i.p. 45 minutes
prior to conditioning. Additional groups of D1R KO mice and their controls received the D1R
agonist A-77636 at a dose of 1.0 mg/kg. All doses of drugs are expressed as mg of free base/kg
of body weight. Doses and time of injections were selected based on previous studies (Chausmer
and Katz, 2002; Fontinha et al., 2009; Grieder et al., 2010; Le Foll and Goldberg, 2004;
Natividad et al., 2010; Ralph and Caine, 2005; Sotak et al., 2005).
Electrophysiology
Rats were subcutaneously implanted with osmotic minipumps (model 2001, Alzet,
Cupertino, CA) delivering either saline (non-dependent) or nicotine (nicotine dependent; 3.14
mg/kg/day) for 7 - 10 days. Nicotine dependent and withdrawn rats had their minipump removed
16-24 hours prior to electrophysiological recordings, a time that corresponds to peak
motivational withdrawal (George et al., 2010; Grieder et al., 2010). Rats were anaesthetized with
urethane (1.5 mg/kg, i.p.) and placed in a stereotaxic apparatus. A scalp incision was made and a
hole was drilled in the skull overlaying the VTA. Electrodes were pulled from borosilicate glass
with average impedance between 6 and 8 MΩ. Microelectrodes were filled with a 2% Pontamine
Sky Blue solution and lowered in to the VTA (AP: -5.3 mm caudal to bregma; ML: ±0.5 – 0.8
mm; DV: 7 – 8.5 mm ventral to the brain surface) (Tan et al., 2009). Extracellular signals were
amplified using a MultiClamp 700B amplifier (Molecular Devices) and recorded through a
Digidata 1440A acquisition system (Molecular Devices) using Clamp10 software. Extracellular
recordings were typically filtered at 1 kHz and sampled at 5 kHz. Body temperature of the rats
was monitored and maintained at 37 ± 1℃ by a thermostatically regulated heating pad. DA
neurons were identified according to well established electrophysiological features: (I) a
relatively longer action potential width (>2.5 ms); (II) a triphasic (+ /-/ +) waveform consisting
of a notch on the rising phase followed by a delayed after-potential; (III) a characteristic low tone
by audio monitoring; (IV) a slow, irregular or bursting firing pattern, and (V) a spontaneous
firing rate of 2 - 5 Hz or less (Grace and Bunney, 1983; Tan et al., 2009). Phasic bursting activity
of DA neurons was defined as the occurrence of two or more consecutive spikes with an
interspike interval lower than 80 ms and terminating with an interspike interval greater than 160
76
ms. Tonic activity was defined as the baseline firing rate (2 - 5 Hz) of the DA neuron (Tan et al.,
2009) and did not include a measure of the number of neurons firing.
Place Conditioning
The place conditioning apparatus was obtained from Med Associates (SOF-700RA-25
Two Chamber Place Preference Apparatus). One environment was black with a metal rod floor
and the other was white with a wire mesh floor. An intermediate gray area housed a removable
partition. Each cage was cleaned between animals and each group was fully counterbalanced.
Mice were implanted with osmotic minipumps (model 1002; Alzet) or given acute nicotine and
pretreated i.p. with saline, apomorphine, α-flu, rimonabant, SCH23390, A-77636, quinpirole,
eticlopride, SCH58261, or CGS21680 and conditioned according to modified place-conditioning
procedures, as described previously (Grieder et al., 2010) and hereafter.
Each cage was cleaned between animals and each group was fully counterbalanced.
During preference testing, the dividing partition was removed and mice were given free access to
both environments. A single 10-min preference testing session was performed 3–5 days after the
last conditioning day, when subjects were drug- and withdrawal-free. All place conditioning and
testing was performed between 10:00 AM and 6:00 PM.
Nicotine-dependent and -withdrawn mice were conditioned according to modified place
conditioning procedure W. Conditioning took place in only the withdrawal-paired environment
of the place conditioning apparatus, so that the motivational effects of withdrawal (but not the
direct effects of chronic nicotine) were paired with that compartment. Mice were implanted with
osmotic minipumps (model 1002; Alzet) that were removed 13 days later. Eight hours after
pump removal, when the mouse was experiencing motivational withdrawal from chronic nicotine
(Grieder et al., 2010), it was pretreated i.p. with saline, apomorphine (2.5 mg/kg), α-flu (0.8
mg/kg), rimonabant (3 mg/kg), SCH23390 (0.01 mg/kg), A-77636 (0.1 or 1.0 mg/kg), quinpirole
(0.05 mg/kg), eticlopride (1.0 mg/kg), SCH58261 (0.5 mg/kg), or CGS21680 (0.1 mg/kg) and
confined to one of the conditioning environments for 1 h. The difference score for each animal
was calculated by subtracting the time spent in the non-paired environment from the time spent
in the withdrawal-paired environment.
77
For acute nicotine experiments, mice were pretreated i.p. with saline, rimonabant (3
mg/kg), SCH23390 (0.01 mg/kg), A-77636 (0.1 or 1.0 mg/kg), quinpirole (0.05 mg/kg),
eticlopride (1.0 mg/kg), SCH58261 (0.5 mg/kg), or CGS21680 (0.1 mg/kg) and given a s.c.
injection of nicotine (1.75 mg/kg) or saline, and confined immediately to one of the conditioning
environments for 1 hour. The difference score for each animal was calculated by subtracting the
time spent in the saline-paired environment from the time spent in the nicotine-paired
environment.
Statistical Analysis
Results were analyzed using a one- or two-way ANOVA or Student’s t-test with alpha
level of 0.05 (two-tailed). In all cases a normality test and equal variance test were performed
before the ANOVA to ensure its validity. Post hoc Bonferroni or Duncan’s tests were used where
appropriate. Data are shown as mean ± SEM.
Results
Activation or blockade of DA receptors prevents the expression of chronic nicotine withdrawal
aversions
Pharmacological blockade of DA activity at receptors attenuates the expression of food
(Sotak et al., 2005) and drug motivation (Grieder et al., 2010; Laviolette et al., 2002) in place
conditioning paradigms. Interestingly, pharmacological activation of DA receptors also prevents
food motivation (Sotak et al., 2005) and the expression of conditioned morphine withdrawal
aversions (Laviolette et al., 2002). We hypothesized that a specific pattern of signaling at DA
receptors could mediate nicotine withdrawal, and thus that either pharmacologically increasing
or decreasing activity at DA receptors would prevent the expression of conditioned place
aversions to nicotine withdrawal. Mice were given chronic nicotine (7 mg/kg/d) via osmotic
minipumps for 13 days and subjected to place conditioning after 8 hours of spontaneous
withdrawal (Grieder et al., 2010). The motivational response to a withdrawal-paired environment
78
was assessed after pretreatment with saline vehicle, the DA receptor agonist apomorphine (2.5
mg/kg) or the DA receptor antagonist α-flu (0.8 mg/kg). A one-way ANOVA showed a
significant effect of pharmacological pretreatment (F2,42 = 17.1, P < 0.05) (Figure 3.1a), where
nicotine dependent and withdrawn mice pretreated with vehicle (n=15) showed a significant
aversion to a withdrawal-paired environment (P < 0.05) that was blocked with apomorphine
(n=15; P < 0.05) or α-flu (n=15; P < 0.05) pretreatment. Each drug pretreatment had no
motivational effects on its own (F2.26 = 0.26, P > 0.05) (Figure 3.2). Similar to previous results in
chronic opiate withdrawn rats (Laviolette et al., 2002), these results suggest that disruption of the
specific pattern of dopaminergic signaling by either increasing or decreasing activity at DA
receptors prevents the expression of nicotine withdrawal aversions in dependent mice.
Tonic but not phasic dopaminergic activity in the VTA is altered in nicotine dependent and
withdrawn rats
We next directly investigated the specific patterns of DA neuron firing that mediate the
motivational response to nicotine withdrawal. Using defined criteria to measure phasic bursting
activity and tonic population activity of DA neurons (Grace and Bunney, 1983; Tan et al., 2009),
we measured tonic and phasic VTA DA activity with in vivo extracellular single-unit recordings
in saline control, previously drug-naive given acute nicotine (1.5 mg/kg), nicotine dependent
(3.14 mg/kg/day), and nicotine-dependent and spontaneously withdrawn rats (Grieder et al.,
2010; George et al., 2010). Analysis of tonic DA neuron activity with one-way ANOVA
revealed a significant effect of drug treatment (F3,35 = 9.7, P < 0.05) (Figure 3.1b). Saline- treated
(n = 11) and acute nicotine-treated (n = 11) rats showed no difference in tonic DA neuron
activity (P > 0.05). However, similar to previous studies (Rahman et al., 2004; Tan et al., 2009),
nicotine-dependent rats receiving chronic nicotine (n = 11) showed a significant decrease in tonic
DA activity in comparison with both saline controls and acute nicotine-treated groups (P < 0.05).
Most interesting, rats experiencing withdrawal from chronic nicotine (n = 6) showed a further
decrease in tonic DA activity compared with nicotine-dependent rats that were not in withdrawal
(P < 0.05). This result is consistent with the hypothesis that dependent human smokers have
decreased DA activity during withdrawal (Kalivas and Volkow, 2005), and suggests that the
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Figure 3.1. Phasic DA activity mediates aversions to acute nicotine while the specific
pattern of tonic DA activity mediates aversions to withdrawal from chronic nicotine.
(A) Both increasing or decreasing DA receptor activity prevents the expression of motivational
withdrawal aversions. Nicotine-dependent and -withdrawn mice subjected to place conditioning
and pretreated with vehicle showed a conditioned place aversion to the withdrawal-paired
environment. This aversive motivational response was blocked in separate groups of mice that
were made nicotine-dependent and -withdrawn and pretreated with the DAR agonist
apomorphine or DA receptor antagonist α-flu. (B) Electrophysiological recordings of tonic and
phasic VTA DA activity during acute and chronic nicotine and withdrawal show that phasic
activity is modified after acute nicotine administration while tonic DA activity is modified after
chronic nicotine and withdrawal. Top: Representative electrophysiological recordings from VTA
DA neurons in rats treated with saline vehicle, acute nicotine, chronic nicotine (nicotine
dependent) and chronic nicotine with spontaneous withdrawal. Middle: Acute nicotine did not
affect tonic DA firing. Nicotine dependent rats exhibited a decrease in tonic VTA DA activity
compared to saline control rats that was further significantly decreased in rats undergoing
withdrawal from chronic nicotine (*p < 0.05 in comparison to all other groups). Bottom: Only
acute nicotine increased phasic activity in VTA DA neurons. Chronic nicotine exposure and
withdrawal from chronic nicotine did not alter phasic activity. (C) Selective blockade of phasic
DA activity using antagonist pretreatment prevents conditioned place aversions to acute nicotine,
but not to withdrawal from chronic nicotine. Nondependent mice that were given acute nicotine
and pretreated with saline showed a conditioned place aversion to the nicotine-paired
environment. Selectively blocking phasic dopaminergic activity at receptors with the
cannabinoid-1 inverse agonist rimonabant or the NMDA receptor antagonist CGP39551
prevented the expression of the conditioned aversive response to acute nicotine. Nicotine-
dependent and -withdrawn mice pretreated with saline showed conditioned place aversions to the
withdrawal-paired environment that were not blocked with rimonabant or CGP39551
pretreatment. Separate groups of mice given acute or chronic saline and pretreated with saline,
rimonabant or CGP39551 showed no motivational response to any pretreatment, suggesting that
80
at the doses used in the present study, these drugs have no motivational effect on their own. Data
represent mean ± SEM (*p < 0.05).
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Figure 3.2. The DA receptor agonist and antagonist have no motivational effects on their
own.
Separate groups of mice given chronic saline in minipumps were place conditioned after
pretreatment with saline (n=8), apomorphine (n=14) or α-flu (n=7). The saline-treated group
showed no motivational response to the novel environment. The apomorphine or α-flu pretreated
groups showed no motivational response to the drug-paired environment. These results suggest
that novelty plays no role in the motivational responses demonstrated by mice in this study, and
that apomorphine and α-flu have no motivational effects on their own at the concentrations used
in the present study. Data represent mean ± SEM.
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aversive motivational state of spontaneous nicotine withdrawal is signaled by a further patterned
decrease in tonic DA activity than that observed during the nicotine-dependent state. Analysis of
phasic VTA DA activity with one-way ANOVA revealed a significant effect of drug treatment
(F3,35 = 5.0, P < 0.05) (Figure 3.1b). Acute nicotine increased phasic DA firing rates (P < 0.05),
in comparison with nondependent, dependent, and nicotine-dependent and -withdrawn groups
(all P > 0.05), suggesting that the specific pattern of phasic activity may mediate the
motivational response to acute nicotine.
Blockade of phasic DA activity prevents aversions to acute nicotine, but not to withdrawal from
chronic nicotine
To test if phasic DA activity directly mediates the aversive response to acute nicotine, but
not to chronic nicotine withdrawal, we examined the effect of blocking phasic DA activity on
conditioned place aversions for acute nicotine and withdrawal from chronic nicotine using the
cannabinoid receptor-1 inverse agonist rimonabant (3.0 mg/kg) and the NMDA receptor
antagonist CGP39551 (2.5 mg/kg). Previous studies suggested that rimonabant blocks phasic DA
release without affecting baseline DA transients (Cheer et al., 2000; Cheer et al., 2007; Cohen et
al., 2002); however, these were performed in vitro (Cheer et al., 2000) or measured the absolute
amount of DA release using voltammetry (Cheer et al., 2007) or microdialysis (Cohen et al.,
2002). We thus performed in vivo electrophysiological recordings of VTA DA neurons in drug-
naive rats given rimonabant (n = 10) to test the hypothesis that the drug selectively decreases
phasic but not tonic baseline DA firing. Rimonabant significantly decreased phasic DA activity
(t9 = 2.715, P < 0.05) but not tonic DA activity (t9 = 0.2018, P > 0.05) (Figure 3.3). CGP39551 is
another pharmacological tool that selectively disrupts phasic DA activity without affecting tonic
activity and blocks nicotine-induced VTA DA bursting (Schilström et al., 2004). A two-way
ANOVA showed a significant interaction of pharmacological pretreatment and nicotine history
(F6,110 = 4.291, P < 0.05) (Figure 3.1c). Nondependent mice given acute nicotine after saline
pretreatment (n = 9) showed a conditioned place aversion to a nicotine-paired environment (P <
0.05) that was blocked with rimonabant (n = 9; P > 0.05) or CGP39551 (n = 13; P > 0.05)
83
Figure 3.3. The cannabinoid-1 receptor inverse agonist rimonabant significantly decreases
phasic VTA DA activity but does not affect tonic DA activity.
Rats were given rimonabant and their VTA DA neuronal activity was recorded
electrophysiologically. A representative trace from a rat VTA DA neuron is depicted.
Rimonabant significantly decreased the amount of bursts per minute (28.0±5.06 to 22.9±4.96)
but did not decrease tonic DA activity (4.34±0.61 to 4.50±1.22).
Supporting InformationGrieder et al. 10.1073/pnas.1114422109SI Materials and MethodsPlace Conditioning. Each cage was cleaned between animals andeach group was fully counterbalanced. During preference testing,the dividing partition was removed and mice were given freeaccess to both environments. A single 10-min preference testingsession was performed 3–5 d after the last conditioning day, whensubjects were drug- and withdrawal-free. All place-conditioningand testing was performed between 10:00 AM and 6:00 PM.Nicotine-dependent and -withdrawn mice were conditioned
according to modified place-conditioning procedure W. Con-ditioning took place in only the withdrawal-paired environmentof the place conditioning apparatus, so that the motivationaleffects of withdrawal (but not the direct effects of chronicnicotine) were paired with that compartment. Mice wereimplanted with osmotic minipumps (model 1002; Alzet) thatwere removed 13 d later. Eight hours after pump removal, whenthe mouse was experiencing motivational withdrawal fromchronic nicotine (1), it was pretreated intraperitoneally with
saline, apomorphine (2.5 mg/kg), α-flupenthixol (0.08 mg/kg),rimonabant (3 mg/kg), SCH23390 (0.01 mg/kg), A-77636 (0.1 or1.0 mg/kg), quinpirole (0.05 mg/kg), eticlopride (1.0 mg/kg),SCH58261 (0.5 mg/kg), or CGS21680 (0.1 mg/kg) and confinedto one of the conditioning environments for 1 h. The differencescore for each animal was calculated by subtracting the timespent in the nonpaired environment from the time spent in thewithdrawal-paired environment.For acute nicotine experiments, mice were pretreated in-
traperitoneally with saline, rimonabant (3 mg/kg) SCH23390(0.01mg/kg), A-77636 (0.1 or 1.0 mg/kg), quinpirole (0.05mg/kg),eticlopride (1.0 mg/kg), SCH58261 (0.5 mg/kg), or CGS21680(0.1 mg/kg) and given a subcutaneous injection of nicotine (1.75mg/kg) or saline, and confined immediately to one of the con-ditioning environments for 1 h. The difference score for eachanimal was calculated by subtracting the time spent in the saline-paired environment from the time spent in the nicotine-pairedenvironment.
1. Grieder TE, et al. (2010) Dopaminergic signaling mediates the motivationalresponse underlying the opponent process to chronic but not acute nicotine.Neuropsychopharmacology 35:943–954.
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Fig. S1. The dopamine receptor (DAR) agonist and antagonist have no motivational effects on their own. Mice given chronic saline in minipumps andpretreated with saline (n = 8), apomorphine (n = 14), or α-flupenthixol (n = 7) showed no motivational response to the novel environment or the drug-pairedenvironment (F2.26 = 0.26, P > 0.05), suggesting that apomorphine and α-flupenthixol have no motivational effects on their own at the concentrations used inthe present study. Data represent mean ± SEM.
Fig. S2. Rimonabant decreases phasic but not tonic dopamine (DA) activity. In this representative trace from a rat VTA DA neuron, rimonabant (3.0 mg/kg,i.p.) significantly decreased the amount of bursts per minute (28.0 ± 5.06–22.9 ± 4.96; t9 = 2.715, P < 0.05) but did not decrease tonic DA activity (4.34 ± 0.61–4.50 ±1.22; t9 = 0.2018, P > 0.05).
Grieder et al. www.pnas.org/cgi/content/short/1114422109 1 of 2
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pretreatment. In contrast, nicotine-dependent and withdrawn mice given saline (n = 11) showed a
conditioned place aversion to the withdrawal-paired environment (P < 0.05) that was not blocked
by rimonabant (n = 13; P < 0.05) or CGP39551 (n = 12, P < 0.05). Mice given chronic or acute
saline and saline (n = 14), rimonabant (n = 7), or CGP39551 (n = 6) showed no motivational
response to the drugs (P > 0.05). These results suggest that phasic DA activity is required for the
motivational response to acute nicotine, but not to withdrawal from chronic nicotine.
Genetic deletion of D2Rs versus D1Rs double dissociate chronic versus acute nicotine
motivation
Phasic and tonic DA signalling act through D1Rs and D2Rs, respectively (Floresco et al.,
2003; Goto and Grace, 2005), and we demonstrated here that acute nicotine modifies phasic DA
activity, but withdrawal from chronic nicotine modifies tonic DA activity. We thus hypothesized
that genetic deletion of the D2R would prevent aversions to nicotine withdrawal in dependent
mice, but D1R deletion would prevent acute nicotine aversions in non-dependent mice. D1R and
D2R KO mice and their WT littermates were place-conditioned after receiving acute nicotine
(1.75 mg/kg) or during spontaneous withdrawal from chronic nicotine (7 mg/kg/d). One-way
ANOVA revealed a significant group effect (F2,24 = 3.43, P < 0.05) (Figure 3.4a) in dependent
and withdrawn mice. WT mice in withdrawal from chronic nicotine (n = 11) showed a
conditioned place aversion to a withdrawal-paired environment (P < 0.05); aversions that were
shown as well by D1R KO mice (n = 9) but not by D2R KO mice (n = 7; P > 0.05). For acute
nicotine-treated mice, one-way ANOVA revealed a significant group effect (F2,27 = 8.27, P <
0.05) (Figure 3.4b). D2R KO (n = 7) and previously drug-naive WT mice (n = 14) given acute
nicotine showed a significant conditioned place aversion to a nicotine-paired environment (P <
0.05) that was blocked in D1R KO mice (n = 9; P > 0.05). Taken together, these results doubly
dissociate the role of D1Rs and D2Rs in nicotine motivation; D2Rs (but not D1Rs) are required
for the aversive motivational response to withdrawal in nicotine-dependent mice, and D1Rs (but
not D2Rs) are necessary for acute nicotine aversions in non-dependent mice.
85
Figure 3.4. A specific pattern of signaling at D1Rs is required for aversions to acute
nicotine in nondependent mice, while a specific pattern of D2R activity is required for
aversions to nicotine withdrawal in dependent mice.
(A) WT mice that are made nicotine-dependent and -withdrawn and place conditioned in the W
procedure will show a conditioned place aversion to the withdrawal-paired environment. This
aversive motivational response to chronic nicotine withdrawal is observed in a group D1R KO
mice, but is not shown by D2R KO mice. These results suggest that D2Rs but not D1Rs are
required for the expression of the motivational response to nicotine withdrawal. (B) WT
nondependent mice given acute nicotine prior to place conditioning will demonstrate an aversive
motivational response to the nicotine-paired environment. These conditioned place aversions to
acute nicotine are blocked in D1R KO mice but are shown by D2R KO mice, suggesting that
D1Rs but not D2Rs are required for the expression of acute nicotine aversions. (C) Nicotine-
dependent and -withdrawn mice pretreated with vehicle show a conditioned place aversion to the
withdrawal-paired environment that is blocked with D2R antagonist eticlopride and D2R agonist
quinpirole pretreatment, but not with D1R antagonist SCH23390 or D1R agonist A-77636
pretreatment. These results suggest that either increasing or decreasing D2R but not D1R activity
will prevent the expression of nicotine withdrawal aversions in dependent mice. (D)
Nondependent mice given an injection of nicotine and pretreated with vehicle showed an
aversion to the nicotine-paired environment that was blocked with A-77636 and SCH23390
pretreatment, but not with quinpirole or eticlopride pretreatment, suggesting that either
increasing or decreasing D1R but not D2R activity prevents acute nicotine aversions. Data
represent mean ± SEM (*p<0.05).
86
87
Pharmacological manipulations of D2Rs but not D1Rs block withdrawal aversions in nicotine-
dependent mice
Modification of the specific pattern of activity at DA receptors, and D2R but not D1R
deletion, blocked the aversive response to withdrawal from chronic nicotine. We thus
hypothesized that either increasing or decreasing activity at D2Rs but not D1Rs would block
conditioned withdrawal aversions in dependent mice. We examined the effect of the D1R agonist
A-77636 (1.0 mg/kg), the D1R antagonist SCH23390 (0.01 mg/kg), the D2R agonist quinpirole
(0.05 mg/ kg), and the D2R antagonist eticlopride (1.0 mg/kg) on conditioned place aversions to
withdrawal. A one-way ANOVA revealed a significant effect of pharmacological pretreatment
(F4,60 = 4.11, P < 0.05) (Figure 3.4c). Nicotine-dependent and -withdrawn mice that received
vehicle (n = 31) before conditioning showed an aversion to a withdrawal- paired environment (P
< 0.05) that was blocked in mice that received quinpirole (n = 7; P > 0.05) and eticlopride (n = 7;
P > 0.05), but not in mice that received A-77636 (n = 12; P < 0.05) or SCH23390 (n = 8; P <
0.05). No motivational response to any of the drugs on their own was observed (F4,44 = 0.08,
p>0.05) (Figure 3.5a). However, groups of mice tested with a higher dose of the D1R agonist A-
77636 (10.0 mg/kg) showed a nonspecific block of learning (F1,25 = 15.08, P < 0.05) (Figure
3.5b,c). These results suggest that either increasing or decreasing D2R activity blocks the
specific pattern of signaling that mediates the aversive motivational response to chronic nicotine
withdrawal, and that activity at D1Rs is not required for the experience of nicotine withdrawal
aversions.
Pharmacological manipulations of D1Rs but not D2Rs block aversions to acute nicotine in
nondependent mice
We next tested the hypothesis that either increasing or decreasing activity at D1Rs but not
D2Rs would prevent the aversive motivational response to acute nicotine by examining the effect
of A-77636, SCH23390, quinpirole, and eticlopride on conditioned place aversions to a nicotine-
paired environment in nondependent mice. There was a significant effect of pharmacological
pretreatment on acute nicotine aversions (F4,44 = 4.99 P < 0.05) (Figure 3.4d) that occurred
exactly opposite to dependent and withdrawn mice. Nondependent mice given acute aversive
88
Figure 3.5. D1R and D2R agonists and antagonists have no motivational effects on their
own at the doses used in this study, however a high dose of the D1R agonist prevents
learning.
(A) Separate groups of mice given chronic saline in minipumps and place conditioned after
pretreatment with saline vehicle (n=14), A-77636 (n=7), SCH23390 (n=10), quinpirole (n=8) or
eticlopride (n=10) showed no motivational response to any of the drugs. These results suggest
that there was no motivational response to novelty (in the saline-pretreated group) and that the
D1R and D2R agonists and antagonists have no motivational effects on their own at the
concentrations used in the present study. However, a higher dose of the D1R agonist A-77636
prevents learning in nicotine-dependent and -withdrawn WT or D1R KO mice (B) and in
nondependent mice given acute nicotine (C). Nicotine-dependent and -withdrawn mice
pretreated with saline showed an aversive motivational response to the withdrawal-paired
environment that was not observed in WT or D1R KO mice pretreated with A-77636 at 10.0
mg/kg. Previously drug-naive mice given acute nicotine and pretreated with saline show an
aversion to an acute nicotine-paired environment that is blocked in mice pretreated with A-77636
at 10.0 mg/kg. These results suggest that A-77636 at a dose of 10.0 mg/kg blocks learning in our
paradigm. Data represent mean ± SEM (*p < 0.05).
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nicotine and pretreated with vehicle (n = 17) showed a conditioned place aversion to a nicotine-
paired environment (P < 0.05) that was blocked in mice pretreated with the D1R agonist A-
77636 (n = 8; P > 0.05) and the D1R antagonist SCH23390 (n = 10; P > 0.05), but not with the
D2R agonist quinpirole (n = 7; P < 0.05) or the D2R antagonist eticlopride (n = 7; P < 0.05).
These results demonstrate that either increasing or decreasing activity at D1Rs blocks aversions
to acute nicotine, and suggest that D2R activation is not necessary for the aversive response to
acute nicotine.
Pharmacological and genetic modifications of A2ARs specifically block chronic nicotine
withdrawal aversions
In the striatum, A2ARs and D2Rs are colocalized (Tozzi et al., 2011) and form A2AR-
D2R heteromers (Fuxe et al., 2010). The A2AR and D2R interact antagonistically, such that
agonism of A2ARs decreases signaling at D2Rs (Tanganelli et al., 2004) and antagonism of
A2ARs increases signaling at D2Rs (Fuxe et al., 2010). If the specific pattern of activity at D2Rs
is a key factor in mediating aversions to nicotine withdrawal, and colocalized A2ARs and D2Rs
act antagonistically in the striatum (Fuxe et al., 2010; Tozzi et al., 2011), then genetic and
pharmacological manipulation of A2ARs should also affect nicotine withdrawal aversions in
dependent animals. We examined the effect of A2AR manipulation on the conditioned aversive
responses to acute nicotine and withdrawal from chronic nicotine in A2AR KO mice and WT
mice pretreated with the A2AR agonist CGS21680 (0.1 mg/kg) or the A2AR antagonist
SCH58261 (0.5 mg/kg). One-way ANOVA revealed a significant effect of A2AR manipulation
in nicotine-dependent and -withdrawn mice (F3,51 = 6.2, P < 0.05) (Figure 3.6a) but not in
nondependent mice given acute nicotine (F2,24 = 0.06, P > 0.05) (Figure 3.6b). Dependent and
withdrawn WT mice that received vehicle (n = 23) showed a conditioned place aversion to a
withdrawal-paired environment (P < 0.05) that was blocked in A2AR KO mice (n = 14; P >
0.05) and in WT mice that received CGS21680 (n = 7; P > 0.05) or SCH58261 (n = 11; P >
0.05). Previously drug-naive mice given acute nicotine and pretreated with vehicle (n = 13)
showed a conditioned place aversion to a nicotine-paired environment that was not blocked in
mice pretreated with CGS21680 (n = 7; P > 0.05) or SCH58261 (n = 7; P > 0.05). No
91
Figure 3.6. Manipulations of the A2AR block the aversive response to withdrawal from
chronic nicotine but not acute nicotine.
(A) Nicotine-dependent and -withdrawn WT mice pretreated with vehicle and place conditioned
showed an aversive motivational response to the withdrawal-paired environment. These
conditioned place aversions were blocked in groups of A2AR KO mice, in mice pretreated with
the A2AR agonist CGS21680 and in mice pretreated with the A2AR antagonist SCH58261.
These results suggest that any modification of the A2AR will prevent the expression of the
aversive motivational response to nicotine withdrawal in dependent mice. (B) Nondependent
mice given an acute injection of nicotine and pretreated with vehicle prior to place conditioning
showed conditioned place aversions to the nicotine-paired environment that were not blocked
with CGS21680 or SCH58261 pretreatment. These results suggest that the A2AR does not play a
role in mediating the aversive motivational response to acute nicotine in nondependent mice. (C)
Mice given chronic or acute saline in minipumps and pretreated with vehicle (n=6), CGS21680
(n=7) or SCH58261 (n=7) showed no motivational response to the drugs, suggesting that the
A2AR agonist and antagonist have no motivational effects on their own at the doses used in the
present study. Data represent mean ± SEM (*p<0.05).
92
C
Figure 3.
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93
motivational response to the drugs on their own was observed (F2,17 = 0.15, P > 0.05) (Figure
3.6c). These results suggest that either increasing or decreasing activity at A2ARs blocks
aversions to withdrawal from chronic nicotine but not the aversive response to acute nicotine,
possibly via modification of D2R activity.
Discussion
Withdrawal from nicotine has been hypothesized to represent a powerful source of
negative reinforcement (Grieder et al., 2010; George et al., 2010) that drives relapse and
compulsive tobacco use (George et al., 2007; Koob and Le Moal, 2006). Therefore,
understanding the neurobiological substrates mediating the motivational properties of withdrawal
from chronic nicotine is an important step in the development of new treatments for nicotine
addiction. Previous reports have suggested that a neurobiological switch occurs during the
transition from a drug-naive to a drug-dependent motivational state (Vargas-Perez et al., 2009).
The transition from acute nicotine use to nicotine dependence has been hypothesized to result
from neuroadaptive changes that produce the powerful withdrawal syndrome and negative
emotional state observed upon cessation of nicotine use (George et al., 2007). The present results
demonstrate that a shift in VTA DA signaling from phasic to tonic, and of receptor mediation
from D1 to D2, occurs upon dependence and withdrawal from nicotine, and doubly dissociates
the role of D1Rs vs. D2Rs in nicotine motivation. We suggest that phasic DA activity at D1Rs
mediates acute nicotine aversions, whereas tonic DA activity at D2Rs (and indirectly, A2ARs)
mediates aversions to withdrawal from chronic nicotine.
Rodents experiencing spontaneous withdrawal from chronic nicotine show a conditioned
place aversion to a withdrawal-paired environment in place conditioning paradigms (Grieder et
al., 2010; Merritt et al., 2008). Similarly, previously drug-naive mice given a single aversive
dose of nicotine will show a conditioned place aversion to the nicotine-paired environment
(Grieder et al., 2010). The important difference in these two effects is the exposure to nicotine:
acute exposure in nondependent animals versus chronic exposure and withdrawal in dependent
94
animals. The present results demonstrate that modifying activity at D2Rs prevented the
expression of nicotine-withdrawal aversions in dependent animals. A previous study suggested
that both increasing or decreasing DA signaling at DA receptors blocked the expression of
conditioned place aversions to withdrawal from chronic morphine (Laviolette et al., 2002). Our
present work suggests that a similar phenomenon occurs during nicotine withdrawal in
dependent animals, such that treatment with a broad-spectrum DA receptor agonist or antagonist,
or a specific D2R agonist or antagonist, prevented the expression of nicotine withdrawal
aversions in dependent mice. Furthermore, genetic deletion of the D2R (but not the D1R)
prevented nicotine withdrawal aversions. In nondependent animals exposed to acute nicotine,
exactly the opposite phenomenon occurred: Both D1R-specific agonism or antagonism, as well
as D1R deletion, selectively prevented aversions to acute nicotine in nondependent mice, without
affecting aversions to withdrawal in nicotine-dependent mice. These results doubly dissociate the
role of the D1R versus the D2R in nicotine motivation, such that the motivational response to
withdrawal in dependent mice is D2R-mediated and acute nicotine motivation is D1R-mediated.
These results are in line with previous studies showing that drug-dependent human subjects have
marked decreases in D2R availability (Fehr et al., 2008) and presumably in DA release (Volkow
et al., 2009), which is consistent with the hypothesis that a pattern of DA activity signals nicotine
motivation and the present results showing that nicotine-dependent and -withdrawn mice have a
decrease in tonic activity of VTA DA neurons. Furthermore, animal studies have shown that
D1R antagonism blocks nicotine motivation in nondependent mice (David et al., 2006). A recent
study showed that blockade of D1R but not D2R transmission prevented acquisition of opiate-
reward memory in nondependent rats, and D2R but not D1R blockade prevented opiate-reward
encoding in dependent and withdrawn rats (Lintas et al., 2011). However, previous studies
suggest that both acute nicotine and opiate reward are mediated by the non-dopaminergic
brainstem TPP nucleus (Laviolette and van der Kooy, 2004; Laviolette et al., 2002), and thus
must involve separate cells in the TPP that are thought to mediate burst-firing of VTA DA
neurons (Floresco et al., 2003); burst-firing that we show here is involved in the response to
acute nicotine. The present data show that only the acute aversive motivational effects of
nicotine are mediated by D1Rs, leading to the suggestion that the induction of nicotine
dependence switches the neurobiological substrate mediating the aversive motivational effects of
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nicotine from D1R to D2R-mediated.
We have suggested that a specific pattern of tonic DA activity through D2Rs signals
withdrawal from chronic nicotine. The D2R system is important for learning to shift behavior in
response to change in motivation (Goto and Grace, 2005). It is thus possible that animals have a
tonic pattern of DA activity that does not shift with nicotine dependence and withdrawal. D2R
KO mice did not demonstrate a conditioned aversive response to withdrawal, possibly because
these mice never experience a change in tonic DA activity that signals withdrawal. However, this
block of the motivational response is not simply because of an effect on learning, as both D1R
and D2R KO mice can learn a motivational response to nicotine in our paradigm. Indeed, both
hyperdopaminergic and hypodopaminergic mice can learn various tasks although their
motivation is altered (Zweifel et al., 2009), suggesting that DA mediates motivation rather than
learning. Our block of nicotine withdrawal aversions with both D2R agonist and antagonist drugs
provides further support for the hypothesis that the specific pattern of DA release at D2Rs
signals withdrawal, and that any deviation from this pattern, whether an increase or a decrease of
DA activity and release, at receptors will prevent the aversive motivational response to nicotine
withdrawal.
The present results doubly dissociate the role of phasic and tonic dopaminergic activity in
the motivational response to acute nicotine in nondependent mice and to withdrawal in nicotine
dependent mice. Previous studies have shown that tonic DA activity is decreased in nicotine
dependent animals (Tan et al., 2009) and that precipitated withdrawal from chronic nicotine
leads to decreased DA levels (Natividad et al., 2010). Using defined electrophysiological
methods to measure VTA DA activity (Grace and Bunney, 1983; Tan et al., 2009), we confirmed
and extended these results to dependent animals experiencing spontaneous withdrawal, showing
that tonic DA activity is further decreased during withdrawal from chronic nicotine. Acute
nicotine increased phasic DA activity in nondependent animals; pharmacologically blocking
phasic activity via cannabinoid-1 (Cheer et al., 2000; Cheer et al., 2007; Cohen et al., 2002) or
NMDA (Schilström et al., 2004) receptor modulation prevented the aversive motivational
response to acute nicotine, but not to withdrawal from chronic nicotine. Taken together, these
results suggest that nicotine withdrawal is signaled by a pattern of tonic but not phasic DA
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activity, and that there is a decrease in tonic DA release during withdrawal in dependent animals.
Although the amount of DA released via tonic neuronal activity is small in comparison with that
via phasic activity, a previous study showed that tonic DA activity is independent of burst firing
and provides sufficient DA to engage behavior (Zweifel et al., 2009), thus it is plausible that a
tonic DA signal mediates the behavioral response to nicotine withdrawal. A single injection of
nicotine leads to the large-scale phasic release of DA (Rice and Cragg, 2004; Zhang et al., 2009);
therefore, it is possible that nicotine-dependent subjects who are experiencing withdrawal may
take nicotine to temporarily modulate DA levels in the brain by increasing release through phasic
activation of VTA DA neurons. This hypothesis is similar to Grace’s tonic/phasic model of DA
system regulation (Grace, 2000). Another possibility suggested by the present results is that
acute nicotine floods the DA system in a similar fashion as administration of a broad-spectrum
DA receptor agonist. These manipulations of DA activity would modify the specific pattern of
DA firing that signals withdrawal, and would thus prevent the aversive motivational effects of
withdrawal from chronic nicotine.
We suggest that modulation of D2Rs could prevent the motivational effects of nicotine
withdrawal; however, directly increasing or decreasing DA activity could potentially produce
schizophrenic or Parkinson-like symptoms, respectively. We demonstrate here that both
increasing and decreasing activity at adenosine A2ARs blocked nicotine withdrawal aversions in
dependent mice but, similar to a previous study (Castañé et al., 2006), had no effect on acute
nicotine aversions in nondependent mice. These results suggest that A2AR modulation can
prevent the aversive motivational response to nicotine withdrawal, possibly through an indirect
disruption of the specific pattern of D2R activity that mediates withdrawal. Furthermore, we
hypothesize that tonic and phasic VTA DA activity leads to effects on D1Rs, D2Rs, and A2ARs
in the ventral striatum. This idea is supported by a previous study showing that intrastriatal DA
receptor antagonism has similar effects to systemic antagonist administration (Laviolette and van
der Kooy, 2003). Activation of striatal receptors could in turn feed back to the VTA via direct
and indirect pathways (Hikida et al., 2010), and this feedback may be important in the generation
of the specific pattern of tonic DA activity that signals nicotine withdrawal aversions.
Taken together, our results suggest that a key mechanism signaling nicotine withdrawal is
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tonic activity of VTA DA neurons, which may act through D2Rs and indirectly, A2ARs, to
signal an aversive motivational state during withdrawal in dependent subjects that contributes to
relapse. Pharmacological manipulation of the tonic DA signal prevents the aversive motivational
state that is normally experienced during nicotine withdrawal, suggesting that modifying tonic
DA activity via manipulation of D2Rs or possibly A2ARs may represent a unique target for
therapeutic treatments of nicotine addiction.
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Chapter 4
Recruitment of a VTA CRF system mediates the aversive effects of nicotine withdrawal
Taryn E. Grieder, Hector Vargas-Perez, Candice Contet, Laura A. Tan, John Freiling, Laura
Clarke, Elena Crawford, Pascale Koebel, Brigitte L. Kieffer, Paul E. Sawchenko, George F.
Koob, Derek van der Kooy and Olivier George
This paper is adapted from a version that currently is submitted to Neuron.
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Abstract
The CRF system has been hypothesized to counteract the VTA DA-mediated positive rewarding
effects of drugs of abuse through upregulation of CRF and activation of CRF1Rs in the extended
amygdala. This phenomenon is known as a between-system neuroadaptation that contributes to
the transition to drug dependence and to withdrawal. Using animal models of nicotine
dependence, rtPCR, in situ hybridization, immunohistochemistry, pharmacology and viral vector
approaches, here we show that in addition to between-system neuroadaptation, CRF participates
in a within-system neuroadaptation: after chronic exposure to nicotine, CRF mRNA is expressed
in dopaminergic posterior VTA (pVTA) neurons, in the core of the brain reward system.
Moreover, upregulation of CRF mRNA, CRF release, and activation of CRF1Rs locally in the
pVTA during withdrawal directly control the motivational state of nicotine withdrawal, thus
linking the brain reward and stress systems in the same neurons in the pVTA.
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Introduction
Drug addiction has been hypothesized to be driven by two mechanisms; downregulation
of the brain reward system (Volkow et al., 2007) and upregulation of the anti-reward brain stress
system (Koob and Le Moal, 2008), concepts known as within- and between-system
neuroadaptations, respectively (Koob and Le Moal. 1997). A prominent downregulation of the
mesolimbic DA reward system originating in the VTA, and upregulation of the CRF brain stress
system originating in the extended amygdala has been observed in rodents, non-human primates
and humans during abstinence from drugs of abuse (Koob and Volkow, 2010). Several groups
have examined the mechanisms behind this between-system DA and CRF neuroadaptation
(Hahn et al., 2009; Lodge and Grace, 2005; Wanat et al., 2008; Wang et al., 2007; Wang and
Morales, 2008), but how these two systems interact in drug dependence and withdrawal is
essentially unknown. Here we tested the hypothesis that CRF participates in a within-system
neuroadaptation during nicotine dependence by examining CRF expression in the core of the
brain reward system, in dopaminergic neurons of the pVTA, and assessed whether this
neuroadaptation could directly control the motivational state of nicotine withdrawal.
Materials and methods
Animals
All animal use procedures were approved by the University of Toronto Animal Care
Committee in accordance with the Canadian Council on Animal Care guidelines and The Scripps
Research Institute Institutional Animal Care and Use Committee and were in accordance with
National Institutes of Health guidelines. Adult male C57BL/6 mice (Charles River, Montreal,
Canada, or Hollister, CA, USA) were housed in a temperature-controlled room with lights on
from 7:00 AM to 7:00 PM.
Drugs
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Nicotine hydrogen tartrate salt (Sigma-Aldrich, Ontario, Canada) was dissolved in saline,
pH 7.0 ± 0.4, and administered via osmotic minipumps (chronic nicotine, 7 mg/kg/day,
minipump model 1002, Alzet, Cupertino, CA, USA) or subcutaneous injection (acute nicotine,
1.75 mg/kg). Nicotine-dependent and -withdrawn mice had their minipumps removed 8 h prior
to experimentation at a time that corresponded to peak motivational withdrawal (Grieder et al.,
2010). The CRF1R antagonist MPZP was synthesized at The Scripps Research Institute
(Richardson et al., 2008), dissolved in HBC, and administered subcutaneously 20 min prior to
conditioning or at a concentration of 0.14 µg/0.3 µl over 10 min for intra-VTA infusions
(coordinates: AP-3.3, DV-4.4, ML±0.5).
Viral vector production
shRNA-encoding AAV2 vectors that target the CRF transcript were generated using the
same procedure as described by Darcq et al. (2011). A shRNA sequence (shCRF, sense strand
5’-GGATCTCACCTTCCACCTTCT-3’) predicted to have high silencing efficiency was
selected using Block-iT RNAi Designer (Life Technologies, Carlsbad, CA). A “universal
scramble” shRNA with no homology to any transcripts was used as a control (shSCR, sense
strand 5’-GCGCTTAGCTGTAGGATTC-3’). An AAV2 shuttle plasmid that encodes shCRF or
shSCR downstream of mU6 promoter and enhanced green fluorescent protein (EGFP) under the
control of the cytomegalovirus (CMV) promoter 3’-flanked by a β-globin intron was generated
using Invitrogen Gateway technology (see Figure 4.4b). Helper-free AAV2 particles were
produced by the triple transfection of AAV-293 cells (Agilent Technologies Inc., Santa Clara,
CA) with the AAV2 shuttle plasmid described above, a plasmid that contains AAV2 rep and cap
genes, and a plasmid that encodes the adenovirus helper functions. Two days later, cells were
collected and lysed by three freeze-thaw cycles, treated with benzonase, and clarified by
centrifugation. Viral vectors were purified by iodixanol gradient ultracentrifugation (Zolotukhin,
et al., 2002), followed by dialysis and concentration against Dulbecco phosphate-buffered saline
(PBS) using centrifugal filter units (Millipore, Billerica, CA). Genomic units were quantified by
rtPCR. The titers were 4.5-6.6 1011 GU/ml.
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Real-time PCR
RNA was isolated using a Qiagen RNeasy extraction kit (Crh, Mm01293920_s1) with
DNase to remove genomic DNA contamination, and a specified amount of cDNA was reverse-
transcribed using SuperscriptIII (Invitrogen, Foster City, CA). Quantitative PCR was performed
using Taqman Gene Expression Assays for CRF in a 7900HT Fast Real-Time PCR System (both
from Life Technologies, Carlsbad, CA). Quantification was performed using the delta Ct method
with Hprt or GAPDH as an endogenous control.
Radioactive in situ hybridization
The mice were anesthetized with chloral hydrate (350 mg/kg, i.p.) and perfused via the
ascending aorta with 0.9% saline followed by ice-cold 4% paraformaldehyde in 0.1 M borate
buffer, pH 9.5. The brains were removed, postfixed for 3 h, and cryoprotected in 20% sucrose in
0.1 M phosphate buffer overnight at 4°C. Five one-in-five series of 30 µm-thick frozen coronal
sections were cut, collected, and stored in 30% ethylene glycol and 20% glycerol in 0.1 M
phosphate buffer at -20°C until processing. In situ hybridization was performed using 35S-labeled
sense (control) and antisense cRNA probes labeled to similar specific activities using a full-
length (1.2 kb) probe for mRNA that encodes CRF (1.2 kb; Dr. K. Mayo, Northwestern
University, Evanston, IL). Sections were mounted on Superfrost plus slides and dried under
vacuum overnight. They were postfixed with 10% paraformaldehyde for 30 min at room
temperature, digested with 10 µg/ml proteinase K for 15 min at 37°C, and acetylated for 10 min.
The probes were labeled to specific activities of 1-3 × 109 dpm/µg and applied to the slides at
concentrations of ~107 cpm/ml overnight at 56°C in a solution that contained 50% formamide,
0.3 M NaCl, 10 mM Tris, 1 mM EDTA, 0.05% tRNA, 10 mM dithiothreitol, 1x Denhardt’s
solution, and 10% dextran sulfate, after which they were treated with 20 µg/ml of ribonuclease A
for 30 min at 37°C and washed in 15 mM NaCl/1.5 mM sodium citrate with 50% formamide at
70°C. The slides were then dehydrated and exposed to X-ray film (Kodak Biomax MR, Eastman
Kodak, Rochester, NY, USA) for 18 h. They were coated with Kodak NTB-2 liquid emulsion
and exposed at 4°C for 3-4 weeks as determined by the strength of the signal on film. The slides
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were developed with Kodak D-19 and fixed with Kodak rapid fixer. One series of sections that
adjoined those used for analysis was stained with thionin to facilitate the accurate localization of
hybridization signals.
Densitometry
The semiquantitative densitometric analysis of hybridization signals for CRF mRNA was
performed on emulsion-dipped slides. Photomicrographs were captured using a Leica light
microscope with a Hamamatsu Orca charge-coupled device camera through OpenLab software
(version 3.1.5) and analyzed using ImageJ software. The optical densities of hybridization
signals were determined under dark-field illumination at 400× magnification with a circular ROI
with a 20 µm diameter that was placed over individual neurons. The size of the ROI was chosen
on the basis of the average diameter of TH-immunoreactive neurons in the pVTA. The sections
were analyzed at regular 160 µm intervals across the pVTA. Optical densities were corrected for
the average background signal that was determined by sampling 20 cell-sized areas per section in
non-signal areas adjacent to the pVTA. Optical density values are expressed in gray scale values
of 1 to 256, corresponding to a gradation from low to high absorbance, respectively. Neurons on
both sides of the brain were pooled for analysis to calculate the animal mean. Animal means
were then grouped according to virus treatment, averaged, and statistically analyzed.
Immunohistochemistry
The mice were anesthetized with 3% halothane, pre-perfused transcardially with a
solution of 0.5 ml heparin/100 ml saline for 1-2 min and then perfused with a solution of 4%
paraformaldehyde in 0.1M phosphate buffer (PB), pH 7. The brains were removed from the
skull, post-fixed at 4°C in the perfusate solution for 6-18 h, rinsed in several changes of PBS that
contained 20% sucrose, and stored at 4°C in fresh 20% sucrose/PBS that contained 0.1% sodium
azide. Coronal cryostat sections (40 µm) were obtained using a Reichert Jung cryostat. The
sections were collected in strict anatomical order in a one-in-four series and stored at 4°C in PBS
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0.1% azide prior to processing. The sections were incubated free-floating with shaking in multi-
well plates, and all incubations were performed at room temperature unless otherwise specified.
The samples were incubated for 20 min in 1% hydrogen peroxide/PBS to quench endogenous
peroxidases, rinsed several times in PBS, and exposed to a blocking solution that contained
PBS/Triton-X100 (0.3%), 1 mg/ml BSA, and 5% normal donkey serum (Jackson Immuno
Research, West Grove, PA, USA) for a minimum of 60 min. The sections were incubated
overnight at 4°C in anti-CRF purified goat polyclonal antibody (Santa Cruz Biotechnology,
Santa Cruz, CA, USA) diluted 1:200 in PBS, 0.5% TWEEN 20, and 5% normal donkey serum.
Control sections were incubated in the antibody diluent. Following three rinses of 10 min each in
PBS, the sections were incubated in Vector ImmPRESS Goat (Vector Labs, Burlingame, CA,
USA) for 1 h, rinsed in PBS as above, and reacted with a DAB Substrate Kit (Vector Labs). The
sections were monitored under a microscope to determine the optimal reaction time. The reaction
was stopped in PBS. The sections were mounted on coated slides, air dried, dehydrated through a
series of ethanol and xylene, and coverslipped with Permount. All brightfield photographs for
analysis were taken with a Q Imaging Retiga 2000R color digital camera mounted on a Zeiss
Axiophot microscope.
Combined TH immunohistochemistry and CRF in situ hybridization
Brains were snap-frozen using isopentane. Twenty µm cryostat sections were mounted
onto Superfrost Plus slides. A digoxigenin (DIG)-labeled CRF riboprobe was synthesized using a
commercial kit (Roche, Indianapolis, IN) from a plasmid containing full length rat CRF cDNA
(kind donation of Dr. K. Mayo, Northwestern University, Evanston, IL). Sections were post-
fixed in 4% formaldehyde for 1 min. Following phosphate-buffered saline (PBS) washes,
proteins were acetylated in 0.1 M triethanolamine, pH 8.0, and 0.2% acetic acid. Following
washes in saline sodium citrate buffer 2x, sections were dehydrated in a graded ethanol-
chloroform series. Pre-hybridization and hybridization were then performed at 70°C in a buffer
containing 50% formamide, SSC 2x, Denhardt’s 5x, 0.5 mg/mL sheared salmon sperm DNA,
and 0.25 mg/mL yeast RNA. Probe was diluted in the hybridization buffer (800 ng/mL) and
incubated overnight on slides. Post-hybridization washes were performed in 50% formamide,
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SSC 2x, and 0.1% Tween-20. Slides were then blocked for 1 h and incubated with anti-DIG
antibody conjugated to alkaline phosphatase (Roche, 1:1000) and anti-TH antibody (Millipore,
Billerica, MA, AB152, 1:500) overnight at 4°C in TNT buffer (0.1 M Tris, pH 7.5, 0.15 M NaCl,
0.1% Tween-20) containing 1% blocking reagent (Roche). A donkey anti-rabbit secondary
antibody conjugated to Alexa Fluor 488 (Life Technologies, Carlsbad, CA, 1:200, 2 h) was used
to reveal the TH signal. Following TNT washes and incubation in 0.1 M Tris-HCl, pH 8, 0.1 M
NaCl, 0.01 M MgCl2, HNPP combined with Fast Red TR (Roche) was then used to detect
alkaline phosphatase. To enhance the signal, fresh substrate was applied three times for 30 min
and slides were rinsed in TNT in-between. Slides were washed, air dried, and coverslipped with
Vectashield HardSet-DAPI (Vector Laboratories, Burlingame, CA). Images were taken using
either epifluorescence (Zeiss Axiophot) or confocal microscopy (LaserSharp 2000, version 5.2,
emission wavelengths 488, 568, and 647 nm, Bio-Rad).
Place conditioning
The place conditioning apparatus was obtained from Med Associates (SOF-700RA-25
Two Chamber Place Preference Apparatus; St. Albans, VT, USA). One environment was black
with a metal rod floor, and the other was white with a wire mesh floor. An intermediate gray area
housed a removable partition. Each cage was cleaned between animals, and each group was fully
counterbalanced. During preference testing, the dividing partition was removed, and the mice
were given free access to both environments. A single 10 min preference test session was
performed 5 days after the last conditioning day. All place conditioning and testing were
performed between 10:00 AM and 6:00 PM.
The nicotine-dependent and -withdrawn groups of mice were conditioned according to
modified place conditioning procedures as described previously (Grieder et al., 2010; and see
Chapter 2). Conditioning occurred only during withdrawal from chronic nicotine so that the
motivational effects of withdrawal but not the direct effects of chronic nicotine were paired with
the place conditioning environment. Eight hours after minipump removal, when the mouse was
experiencing motivational withdrawal from chronic nicotine (Grieder et al., 2010; and see
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Chapters 2 and 3), it was subcutaneously pretreated with vehicle (20% HBC) or MPZP (20
mg/kg) and confined to one of the conditioning environments for 1 h. The difference score for
each animal was calculated by subtracting the time spent in the non-paired environment from the
time spent in the withdrawal-paired environment during preference testing.
For nicotine-dependent mice (not withdrawn), conditioning occurred only during
exposure to chronic nicotine so that the motivational effects of chronic nicotine in a dependent
animal were paired with the place conditioning environment. The mice were subcutaneously
pretreated with vehicle (20% HBC) or MPZP (20 mg/kg) and confined to one of the conditioning
environments for 1 h. The difference score for each animal was calculated by subtracting the
time spent in the non-paired environment from the time spent in the nicotine-paired environment
during preference testing.
For the acute nicotine experiments, previously drug-naive mice were subcutaneously
pretreated with 20% HBC or MPZP (20 mg/kg), given a subcutaneous injection of 1.75 mg/kg
nicotine, and immediately confined or confined 8 h later (Grieder et al., 2010; and see Chapter 2)
to one of the conditioning environments for 1 h. The next day, the mouse was pretreated again
with HBC or MPZP, given acute saline instead of nicotine, and confined to the other
environment. The difference score for each animal was calculated by subtracting the time spent
in the saline-paired environment from the time spent in the nicotine-paired environment.
Open field testing
Mice that were bilaterally injected with AAV2-shSCR or AAV2-shCRF (coordinates:
AP-3.3, DV-4.4, ML±0.5) were placed in the center of a grey box measuring 41 x 41 x 38 cm for
5 minutes. The room was dark with the open field testing box illuminated by a soft red light. The
locomotor activity and duration of time spent in the center square area of the box was recorded
by video camera and calculated by the monitoring program (Ethovision XT, Noldus; Leesburg,
VA, USA). The test box was cleaned with 70% alcohol between each mouse.
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Statistical analysis
The data were analyzed with Statistica software using a one- or two-way ANOVA or
Student’s t-test, where appropriate. In all cases, a normality test and an equal variance test were
performed before the ANOVA to ensure its validity. Duncan’s post hoc tests were used when
appropriate. The data are expressed as mean ± SEM.
Results
Nicotine dependence upregulates CRF mRNA in the pVTA
To test whether nicotine dependence upregulates CRF in the brain stress and reward
systems, we first measured CRF mRNA in two key regions of the CRF brain stress system, the
PVN and CeA, and in the VTA of the brain reward system using quantitative real-time
polymerase chain reaction (rtPCR). Groups of mice were made nicotine dependent by chronic
exposure to nicotine delivered by osmotic minipumps (7 mg/kg/d) (Grieder et al., 2010; Grieder
et al., 2012). Brain punches of the PVN, CeA, and VTA (Figure 4.1a) were sampled in saline-
treated mice, in dependent mice with intact minipumps (dependent mice), or 8 h after removal of
the minipump (withdrawn mice). CRF mRNA levels were 7-15 times lower in the VTA than in
the CeA and PVN (Figure 4.1b), in accordance with the fact that the CeA and PVN contain large
populations of cell bodies that contain CRF mRNA, whereas similar neurons have never been
reported in the VTA (Swanson et al., 1983). However, we detected CRF mRNA in saline-treated
groups that could not be attributed to experimental noise, suggesting that a very small amount of
CRF mRNA is present in the VTA of nondependent animals. Chronic exposure to nicotine
selectively increased CRF mRNA levels only in the VTA in both the dependent and withdrawn
groups of mice compared with saline-treated mice (F4,100 = 2.7, p = 0.034), without altering CRF
expression in the PVN or CeA (Figure 4.1b). CRF neurons project to and synapse with both DA
and γ-aminobutyric acid (GABA) neurons in the VTA (Tagliaferro and Morales, 2008), and CRF
release in the VTA is potentiated after repeated but not acute cocaine exposure (Hahn et al.,
2009), suggesting that chronic exposure to abused drugs may either lead to axonal transport of
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Figure 4.1. Nicotine dependence increases CRF mRNA levels in the VTA.
(a) Location of the brain tissue samples in the CeA, VTA, and PVN. (b) rtPCR measurement of
CRF mRNA levels (delta Ct) in the CeA, VTA, and PVN relative to the housekeeping gene
GAPDH, expressed on a logarithmic scale, in mice chronically exposed to saline or nicotine or
withdrawn from chronic nicotine. In saline-treated mice, CRF mRNA levels were approximately
10 times lower in the VTA than in the CeA and PVN, in accordance with the fact that the CeA
and PVN contain large populations of cell bodies that contain CRF mRNA, whereas similar
neurons have never been reported in the VTA. However, a very small amount of CRF mRNA
was detected in saline-treated groups, suggesting that a very small amount of CRF mRNA is
present in the VTA of nondependent animals. A significant increase in CRF mRNA was
observed in the VTA but not the CeA or PVN in both nicotine-dependent and -withdrawn groups
of mice (*p < 0.05), suggesting that the induction of nicotine dependence leads to an
approximately two-fold increase in CRF mRNA selectively in the VTA.
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CRF mRNA to the VTA, or to the recruitment of a small population of VTA neurons that
synthesize CRF mRNA.
Nicotine dependence recruits a population of CRF neurons in the pVTA
To test the hypothesis that chronic nicotine recruits a small population of CRF-
expressing neurons in the VTA, we next performed CRF mRNA in situ hybridization in the
anterior and posterior VTA (aVTA and pVTA respectively; Figure 4.2a, b) in a separate cohort
of nicotine-dependent mice. As expected by the lack of previous reports in the literature of CRF
neurons in the VTA, we observed very few CRF mRNA-containing cells in the VTA in saline-
treated mice (Figures 4.2c and 4.3). However, after exposure to chronic nicotine, a significant
population of CRF neurons with dense CRF mRNA in cell bodies could be detected bilaterally in
the VTA. This increase was associated with a significant two-fold increase in CRF mRNA
density in the pVTA (t42 = -2.43, p = 0.0097; Figure 4.2c) as well as a non-significant increase in
the aVTA (t32 = -1.30, p = 0.1008; Figure 4.2c). Nicotine dependence also increased the number
of neurons per section that contained CRF mRNA in the pVTA (t42 = -1.9, p = 0.029) but not
aVTA (t32 = 0.75, p = 0.77; Figure 4.3). These results confirm the findings obtained with rtPCR
and further demonstrate that exposure to chronic nicotine recruits a population of CRF mRNA-
expressing neurons in the VTA, specifically in the pVTA. Interestingly, a recent study
demonstrated that nicotine selectively activates dopaminergic neurons in the pVTA, but not
aVTA, suggesting that newly CRF-expressing neurons in the pVTA may be dopaminergic
(Zhao-Shea et al., 2011).
CRF mRNA is expressed in dopamine neurons
To test whether CRF neurons in the pVTA were also dopaminergic, we performed double
labeling of CRF mRNA and DA neurons using CRF mRNA radioactive and fluorescent in situ
hybridizations coupled with tyrosine hydroxylase (TH) immunohistochemistry (Figure 4.4). CRF
mRNA positive neurons were located bilaterally in the pVTA (Figure 4.4a) in TH enriched
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Figure 4.2. Nicotine dependence and withdrawal recruits and activates the CRF system in
the VTA.
(a) Location of the aVTA (left: bregma range: -2.92 to -3.16) and pVTA (middle: bregma range:
-3.28 to -3.80) and density of CRF mRNA signal in the aVTA and pVTA in saline- and chronic
nicotine-treated mice (right). Nicotine-dependent mice showed an increase in CRF mRNA
density compared with saline control (*p < 0.05) in the pVTA but not aVTA. (b) Photographic
representation of Nissl-stained sections that validate the proper anatomical location for
corresponding in situ hybridization. Scale bars = 100 µm. (c) Representative CRF mRNA in situ
hybridization sections of the aVTA and pVTA (box) in mice chronically exposed to nicotine or
saline. The number of CRF mRNA-containing neurons (arrows) was increased in nicotine-
dependent mice in the pVTA (inset).
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112
Figure 4.3. Nicotine dependence increased the number of cells that contain CRF mRNA in
the pVTA but not aVTA.
The average number of CRF mRNA-positive cells counted per section across the entire aVTA or
pVTA in saline-treated or chronic nicotine-treated groups of mice is shown. Nicotine
dependence increased CRF-positive cells in the pVTA but not the aVTA.
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Figure 4.4. Double labeling of DA neurons and CRF mRNA using CRF in situ
hybridization and TH immunohistochemistry.
(a) CRF mRNA radioactive in situ hybridization (black) demonstrates CRF-positive cell bodies
(arrows) in the pVTA. (b) TH immunohistochemistry (red) on the same section of the pVTA
shows that dopaminergic cell bodies are located bilaterally in the pVTA, in TH immunoreactive
areas. (c) Double fluorescent labeling of CRF mRNA (red) and TH protein (green) in the pVTA
at high magnification shows that the majority of CRF mRNA-positive neurons also express TH
(CRF+/TH+ neuron: arrow, CRF-/TH+ neuron: arrowhead). (d) Confocal image of a single VTA
neuron co-expressing TH (green) and CRF mRNA (red).
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regions (Figure 4.4b), and significantly colocalized with TH-positive neurons with an estimated
87.5% (14/16) of CRF mRNA-expressing neurons being TH-positive (Figure 4.4c, d). The
induction of CRF mRNA in dopaminergic neurons in the pVTA after the development of
nicotine dependence suggests that a pool of neurons in the pVTA can either synthesize CRF
mRNA de novo, or that it reflects a prominent upregulation over baseline levels in naive animals
that were too low to be easily detected using classical detection methods (Kovacs and
Sawchenko, 1996). Our results showing very low levels of CRF mRNA in the VTA using rtPCR
and in situ hybridization in naive rats support the latter hypothesis. Nevertheless, the
upregulation of CRF mRNA suggests a gain of CRF function in VTA dopaminergic neurons.
Withdrawal depletes CRF peptide in the pVTA
Increased CRF release during drug withdrawal is associated with decreased
immunodensity of CRF peptide, which has been proposed to reflect CRF depletion from synaptic
vesicles subsequent to a local increase in CRF release (Merlo-Pich et al., 1995). Thus, we
examined if recruitment of CRF neurons in the pVTA was also associated with a local decrease
in CRF peptide density during withdrawal from chronic nicotine using CRF
immunohistochemistry in the aVTA and pVTA (Figure 4.5a). Densitometry analysis of the VTA,
CeA, and PVN revealed that both nicotine dependence and withdrawal from chronic nicotine
decreased CRF peptide density compared with saline-treated controls in the pVTA (F2,31 = 4.4, p
= 0.02; Figure 4.5b) and CeA (F2,23 = 1.1, p = 0.36; Figure 4.6), but not aVTA (F2,23 = 0.03, p =
0.97; Figure 4.5b) or PVN (F2,21 = 0.75, p = 0.48; Figure 4.6). Background immunoreactivity
outside of, but surrounding, the VTA was not significantly different between groups (F2,31 =
3.08, p > 0.05; data not shown), demonstrating the specificity of these effects. Our combined
observations that chronic nicotine exposure increases CRF mRNA expression and decreases
CRF peptide density in the pVTA suggest that newly induced CRF-synthesizing neurons are
functional and locally release CRF in the pVTA. The decreased immunodensity observed in the
CeA replicates results obtained previously with alcohol-dependent and -withdrawn rats (Funk et
al., 2006) and extends them to nicotine-dependent and -withdrawn mice. The finding that CRF
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Figure 4.5. Withdrawal from chronic nicotine depletes CRF peptide in the pVTA.
(a) Representative CRF immunohistochemistry sections of the areas containing the aVTA (left)
and pVTA (middle; pVTA enclosed in box), and close-up of the pVTA (right) in mice
chronically exposed to saline (upper sections) or nicotine (lower sections). Scale bars = 100 µm.
(b) CRF peptide density in the aVTA and pVTA in mice given saline (Sal), chronic nicotine
(Nic), or chronic nicotine and withdrawal (WD). The density of CRF peptide was decreased was
decreased after chronic nicotine and withdrawal from chronic nicotine (*p < 0.05, vs. saline),
corresponding to a proposed increase in CRF release.
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117
Figure 4.6. Nicotine dependence and withdrawal decreases CRF peptide density in the CeA
but not the PVN.
Densitometry analysis on CRF immunohistochemical labeling of the CeA and PVN revealed that
both chronic nicotine exposure and withdrawal from chronic nicotine decreased CRF peptide
density compared with saline-treated controls (*p < 0.05, vs. saline) in the CeA (F2,23 = 1.1, p =
0.36) but not the PVN (F2,21 = 0.75, p = 0.48) .
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density was decreased in the pVTA, but not aVTA or PVN, suggests that increased CRF release
not only in the CeA but also in the pVTA may contribute significantly to the motivational effects
of nicotine withdrawal in dependent subjects.
Blocking upregulation of CRF mRNA in the pVTA prevents the aversive effects of withdrawal
To test whether CRF mRNA-expressing neurons in the VTA play a role in nicotine
dependence, we tested the causal relationship between upregulation of CRF mRNA in the VTA
of nicotine-dependent and -withdrawn animals and the expression of conditioned place aversion
to nicotine withdrawal and anxiety-like behavior. We injected an adeno-associated viral vector
that encodes a short-hairpin RNA-targeting CRF mRNA (AAV2-shCRF) or a non-targeting
sequence (AAV2-shSCR) bilaterally in the pVTA 3-4 weeks before exposure to chronic or acute
nicotine to chronically decrease CRF mRNA expression during exposure to nicotine and
withdrawal (Figure 4.7a, b). Quantification of CRF-positive neurons in withdrawn mice injected
with the AAV2-shCRF in the pVTA and subjected to in situ hybridization showed a significant
downregulation (-31%; Figure 4.7c) of the number of CRF mRNA-containing cells in the pVTA
compared with withdrawn mice injected with the AAV2-shSCR vector (t11 = 2.737, p = 0.0193;
Figure 4.7d). Moreover, in the remaining CRF-expressing pVTA cells, AAV2-shCRF vector-
injected mice exhibited an 11% decrease in CRF mRNA content (normalized optical density:
88.6 ± 5.5 for AAV2-shCRF vs. 99.9 ± 3.4 for AAV2-shSCR; p = 0.1128). Notably, the mice
were sacrificed approximately 4 weeks into withdrawal after the end of behavioral testing,
demonstrating that the upregulation of CRF mRNA in nicotine-withdrawn mice and CRF
silencing by the viral vector were both long-lasting. In the place conditioning paradigm, a two-
way analysis of variance (ANOVA) revealed a significant interaction between virus injection and
nicotine history (F1,23 = 4.976, p = 0.0357; Figure 4.7e). Nicotine-dependent and -withdrawn
mice infused with the AAV2-shSCR control vector in the pVTA showed an aversive
motivational response to the withdrawal-paired environment (p < 0.05), which was not observed
in mice infused with the AAV2-shCRF vector (p > 0.05). Furthermore, nondependent mice given
either the control or silencing vector in the pVTA and an acute aversive injection of nicotine
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Figure 4.7. Preventing upregulation of CRF mRNA in the VTA by a viral vector prevents
the aversive motivational response to withdrawal from chronic nicotine.
(a) Timeline of experiment. Groups of mice were injected with AAV2-shCRF or AAV2-shSCR
in the VTA and allowed 3-4 weeks to recover. Osmotic minipumps containing 7 mg/kg/d
nicotine were implanted (D1) and left in place for 12 days. On D13, pumps were removed for
withdrawn groups, or left in place for dependent groups, and conditioning was performed 8 hours
later. Five days later, on D18, mice were preference tested, and then tested in the open field on
D38. Subsequent to testing, all mice were sacrificed and their brains subjected to in situ
hybridization. (b) DNA construct used to produce vectors for CRF silencing (AAV2-shCRF) and
control vectors (AAV2-shSCR). ITR, inverted terminal repeat; CMV, cytomegalovirus; EGFP,
enhanced green fluorescent protein; hGH polyA, human growth hormone polyadenylation signal.
(c) Atlas showing location of sections and representative images of VTA CRF mRNA-containing
cells. Scale bars = 100 µm. (d) The absolute number of CRF mRNA-positive cells after AAV2-
shCRF was significantly decreased compared to mice injected with AAV2-shSCR (*p < 0.05).
(e) AAV2-shCRF blocked the conditioned place aversion to nicotine withdrawal (*p < 0.05) in
nicotine-dependent and withdrawn mice given AAV2-shSCR, but not the aversion to acute
nicotine in nondependent mice. (f) Nicotine-dependent and -withdrawn mice injected with
AAV2-shSCR spent significantly less time spent in the central area of the open field than mice
injected with AAV2-shCRF (*p < 0.05).
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(1.75 mg/kg) showed an aversive response to the nicotine-paired environment, demonstrating
that the lack of aversion to withdrawal in dependent mice infused with the silencing vector was
not due to a general impairment of conditioned place aversion but was specific to withdrawal in
dependent mice. Mice that were injected with the AAV2-shCRF vector outside of the pVTA
showed an aversive response to nicotine withdrawal similar to control mice (t10 = 0.2101, p =
0.8378; Figure 4.8), demonstrating the anatomical specificity of this effect. Altogether, these
results suggest that CRF mRNA in the pVTA does not mediate the aversive response to acute
nicotine, but specifically mediates aversion to nicotine withdrawal in dependent subjects. These
results establish a causal relationship between the recruitment of CRF mRNA-expressing
neurons in the VTA during withdrawal from chronic nicotine and the aversive motivational
response to nicotine withdrawal.
Activation of the brain CRF-CRF1R system is associated with increased anxiety-like
behavior in humans and animals (Holsboer and Ising, 2008) and is hypothesized to be
responsible for the negative emotional states after protracted abstinence (George et al., 2007). To
test this hypothesis, we used the same nicotine-dependent and -withdrawn mice injected with the
AAV2-shCRF and AAV2-shSCR vectors and measured open field activity to evaluate anxiety-
like behavior during protracted abstinence (3-4 weeks). A one-way ANOVA revealed a
significant effect of the viral vector on the duration of time spent in the central open area (F3,31 =
4.148, p = 0.015; Figure 4.7f). Mice injected with the AAV2-shSCR vector spent significantly
less time in the central open area of the open field than mice injected with the AAV2-shCRF
vector (p < 0.05). These results demonstrate that upregulation of CRF mRNA in the pVTA is
required for the anxiogenic-like effects of protracted nicotine abstinence.
CRF1R receptor blockade prevents withdrawal aversions
The present results revealed a depletion of CRF peptide in the pVTA during nicotine
withdrawal, which is thought to reflect a local increase in CRF release (Merlo-Pich et al., 1995).
We therefore hypothesized that activation of CRF1Rs in the pVTA would be necessary for the
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Figure 4.8. The silencing vector must be injected in the VTA to block withdrawal aversions.
Nicotine-dependent and -withdrawn mice that were injected with AAV2-shCRF silencing vector
outside of the VTA showed an aversive motivational response to nicotine withdrawal similar to
AAV2-shSCR control vector-injected mice, demonstrating the anatomical specificity of this
effect.
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conditioned aversive motivational response to a withdrawal-paired environment shown by
nicotine-dependent and -withdrawn mice (Grieder et al., 2010; Grieder et al., 2012). Based on
the AAV2-shCRF results above, we further hypothesized that neither the aversive response to
acute nicotine nor the rewarding response to chronic nicotine (Grieder et al., 2010) would depend
on CRF1R signaling. To test these hypotheses, we first systemically administered the CRF1R
antagonist N,N-bis(2-methoxyethyl)-3-(4-methoxy-2-methylphenyl)-2,5-di-methyl-pyrazolo[1,5-
a]pyrimidin-7-amine (MPZP; 20 mg/kg, s.c.) (Richardson et al., 2008) prior to place
conditioning in nondependent mice that were given saline or acute nicotine, nicotine-dependent
mice (7 mg/kg/d), or mice withdrawn from chronic nicotine (Figure 4.9a). A two-way ANOVA
showed a significant effect of nicotine history (F3,93 = 25.39, p < 0.0001; Figure 4.9b).
Withdrawn mice pretreated with vehicle showed a conditioned place aversion to a withdrawal-
paired environment (p < 0.05) that was blocked by MPZP pretreatment (p > 0.05), demonstrating
that activation of CRF1Rs during withdrawal is required for the aversive motivational response
to nicotine withdrawal. In contrast to withdrawn mice, nicotine-dependent mice that were not in
withdrawal and pretreated with saline showed a conditioned place preference for a nicotine-
paired environment (p < 0.05) that was not blocked by MPZP pretreatment (p < 0.05; Figure
4.9b). This result suggests that activation of CRF1Rs is not required for the rewarding
motivational response to chronic nicotine in dependent mice. Furthermore, nondependent mice
given acute nicotine and pretreated with saline showed a conditioned place aversion to the
nicotine-paired environment (p < 0.05) that was not blocked by MPZP pretreatment (p < 0.05;
Figure 4.9b), similar to the effect of CRF mRNA vector silencing. Mice treated with chronic
saline and pretreated with vehicle or MPZP showed no motivational response to a novel
environment or MPZP (p > 0.05), confirming the specificity of these results. Additional groups
of nondependent mice given acute nicotine and pretreated with saline showed an opponent
rewarding process that occurred 8 h after acute aversive nicotine (Grieder et al., 2010 and see
Chapter 2) that also was not blocked by MPZP pretreatment (t12 = 0.0917, p = 0.8653; Figure
4.10). Altogether, these results suggest that activation of CRF1Rs is required only to mediate the
motivational response to nicotine withdrawal in dependent rats, but not the initial aversive or
opponent rewarding motivational response in nondependent mice, or the rewarding motivational
response in nicotine-dependent mice.
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Figure 4.9. CRF1R antagonism prevents the aversive motivational response to withdrawal
from chronic nicotine.
(a) Timeline of chronic nicotine treatment, withdrawal, place conditioning, and testing. Nicotine-
dependent and -withdrawn mice were given chronic nicotine and underwent 8 h of spontaneous
withdrawal prior to conditioning. Dependent (not withdrawn) mice were given chronic nicotine
and conditioned with intact minipumps. All of the groups were preference tested 5 days after
conditioning in a drug- and withdrawal-free state. (b) Nicotine-dependent and -withdrawn mice
pretreated with vehicle showed a conditioned place aversion to the withdrawal-paired
environment that was blocked by pretreatment with the CRF1R antagonist MPZP (*p < 0.05).
Conversely, nicotine-dependent mice that were not experiencing withdrawal showed a
conditioned place preference for the nicotine-paired environment that was not blocked by MPZP
pretreatment. Nondependent mice given acute nicotine and pretreated with vehicle showed an
aversion to the nicotine-paired environment that was not blocked by MPZP pretreatment. These
results suggest that CRF1Rs participate in mediating only the aversive motivational response to
withdrawal in dependent mice, but not nicotine reward in dependent mice or aversion to acute
nicotine in nondependent mice. Saline-treated mice given MPZP showed no motivational
response to the drug. (c) Schematic of the pVTA showing placements of the intra-pVTA
cannulae. (d) Nicotine-dependent and -withdrawn mice implanted with intra-VTA cannulae that
delivered vehicle showed an aversive motivational response to the withdrawal-paired
environment that was blocked in mice that received intra-VTA CRF1R antagonist MPZP (*p <
0.05).
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126
Figure 4.10. The opponent motivational process occurring after acute nicotine is not
blocked by CRF1R antagonism.
Nondependent mice given acute nicotine (1.75 mg/kg) and pretreated with saline showed an
acute opponent rewarding process 8 hours after nicotine administration that was not blocked by
CRF1R antagonist MPZP pretreatment.
0 20 40 60 80
100 120 140 160 180
Vehicle MPZP
Diff
eren
ce sc
ore
(s)
Pretreatment
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To test whether activation of CRF1Rs specifically in the pVTA mediate the conditioned
aversive motivational response to nicotine withdrawal, we measured place conditioning during
withdrawal after infusion of MPZP or vehicle prior to conditioning, through cannulas surgically
implanted in the pVTA (Figure 4.9c). Nicotine-withdrawn mice pretreated with vehicle showed
an aversive response to the withdrawal-paired environment that was blocked in mice pretreated
with intra-pVTA MPZP (t15 = -2.7, p = 0.00818; Figure 4.9d). These results demonstrate that
CRF1R activation, specifically in the pVTA, is necessary for the expression of the aversive
motivational response to nicotine withdrawal in dependent mice.
Discussion
The consequences of upregulation of CRF mRNA in dopaminergic neurons in the pVTA
and activation of CRF1Rs in the pVTA on DA cell firing, and on DA and CRF release locally
and in VTA projection areas are still unknown, and will undoubtedly be the focus of many future
investigations. However, previous studies using similar experimental procedures to those in the
present work have shown that withdrawal from chronic nicotine both decreased tonic
dopaminergic activity in the VTA (Grieder et al., 2012) and increased CRF levels in the basal
forebrain (George et al., 2007; Slawecki et al., 2005), and that intra-VTA CRF decreased DA
metabolism in the prefrontal cortex (Kalivas et al., 1987). Other studies have found that acute
intra-VTA CRF increases the firing rate of VTA DA neurons in non-dependent animals (Wanat
et al., 2008), but the effect of chronic increases of CRF mRNA and peptide and CRF1R
activation in the VTA in dependent animals experiencing withdrawal is essentially unknown.
Moreover, increasing evidence suggests that a neurobiological switch occurs in the VTA during
the transition from a nondependent to drug-dependent motivational state (Grieder et al., 2012;
Nader and van der Kooy, 1997; Vargas-Perez et al., 2009), suggesting that the effect of CRF1R
activation in dependent vs. non-dependent animals may be opposite. Our results showing a
specific role for CRF in the pVTA in mediating aversions to withdrawal in dependent but not to
acute nicotine in nondependent mice is consistent with this hypothesis.
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The transition to drug dependence and drug addiction have been hypothesized to be
driven and maintained by two relatively independent systems: Downregulation of DA function in
the VTA, a within-system neuroadaptation leading to a reward deficit disorder (Volkow et al.,
2007), and upregulation of CRF function, a between-system neuroadaptation leading to a stress
surfeit disorder (Koob and Le Moal, 2008). Here we demonstrate that CRF mRNA is expressed
in the core of the brain reward system, in dopaminergic neurons of the pVTA, and that
upregulation of CRF mRNA and activation of CRF1R locally in the pVTA occur after the
induction of nicotine dependence and withdrawal that mediate the anxiogenic and aversive
motivational responses to withdrawal. This within-system neuroadaptation links the brain reward
and stress systems in the same neurobiological substrate, providing evidence that both DA and
CRF in the VTA mediate the aversive motivational effects of nicotine withdrawal.
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Chapter 5
General Discussion
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5.1 Overview
Understanding the neurobiological substrates mediating the negative experiences of
smokers during nicotine withdrawal has important implications for improving smoking
cessation. The fundamental questions set forth in this thesis concerned the identification of the
involvement of VTA DA and CRF in the mediation of the opponent motivational responses
occurring after acute and chronic nicotine. Specifically, I proposed to examine how VTA
dopaminergic activity at DA receptors differentially mediates acute nicotine aversions in
nondependent animals and the aversive motivational response to withdrawal in nicotine
dependent animals. I have further attempted to elucidate how CRF and the CRF1 receptor in the
VTA are involved in mediating the expression of nicotine withdrawal aversions.
In chapter 2, I tested whether the DA system and the D2R specifically are involved in the
opponent motivational responses to nicotine and in the somatic withdrawal syndrome
demonstrated during nicotine abstinence. I did this by pharmacologically blocking DA signaling
at dopaminergic receptors as well as genetically deleting the D2R and observing the effect on the
expression of a somatic nicotine abstinence syndrome and on the conditioned opponent
motivational responses to acute and chronic nicotine modeled in the place conditioning
paradigm.
I first characterized the somatic abstinence syndrome in mice and rats. Consistent with
previous work using antagonist-precipitated withdrawal (Isola et al., 1999; Malin et al., 1992),
both mice and rats showed a significant nicotine abstinence syndrome after spontaneous
withdrawal from chronic nicotine, demonstrating similar somatic signs to those described
previously (Epping-Jordan et al., 1998; Isola et al., 1999; Malin et al., 1992; Watkins et al.,
2000) and similar plasma levels of nicotine to those observed in previous human and rodent
studies after chronic nicotine administration (Guillem et al., 2005; O’Dell et al., 2006). I then
showed that the time course of the somatic withdrawal syndrome coincides with the motivational
response to nicotine withdrawal, such that the largest amount of abstinence signs occurred when
the aversive response to withdrawal could be place conditioned. Previous work has shown that
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opiate withdrawal aversions can be blocked without attenuating somatic withdrawal signs
(Bechara et al., 1995), and similarly, pretreatment with the broad-spectrum DA receptor
antagonist α-flu, or genetic deletion of the D2R, blocked the aversive motivational response to
nicotine withdrawal but not the somatic signs of withdrawal. These results suggest that somatic
and motivational withdrawal occur coincidentally, but are not causally related.
I also showed that acute aversive nicotine in nondependent mice and chronic nicotine and
withdrawal in dependent mice would produce opponent motivational processes that could be
modeled and measured using the place conditioning paradigm. The initial motivational processes
of acute nicotine aversion in nondependent animals and chronic nicotine reward in dependent
animals have been previously demonstrated in place conditioning paradigms (Acquas et al.,
1989; Laviolette and van der Kooy, 2004; Sellings et al., 2008; Wilkinson et al., 2008). I showed
that these initial motivational a-processes would both stimulate an opponent motivational b-
process that could be modeled in the place conditioning paradigm 8 hours after the onset of the
a-process. To my knowledge, this was the first demonstration of a rewarding opponent b-process
occurring after acute aversive nicotine that could be modeled by place conditioning.
Additionally, previous studies have successfully shown a conditioned place aversion to a
nicotine withdrawal-paired environment, however these studies used nicotine antagonists to
precipitate withdrawal in dependent rats (Laviolette et al., 2008; Watkins et al., 2000). The
spontaneous withdrawal observed in my studies more closely models human nicotine
withdrawal. Additionally, there is no possibility of a nicotinic antagonist having any motivational
effects on its own, therefore the withdrawal aversions demonstrated in my studies are a more
pure measurement of the motivational effect of nicotine withdrawal.
To show the differential involvement of the DA system in the opponent motivational
processes occurring after acute and chronic nicotine, I blocked DA receptors using a broad-
spectrum antagonist and utilized D2R KO mice in the place conditioning paradigm. I
demonstrated that DA receptor signaling, specifically at the D2R, is required for the opponent b-
process modeled by the aversive motivational response to nicotine withdrawal, but not for the
expression of the rewarding motivational response to chronic nicotine in dependent subjects. By
contrast, I showed that DA receptor activation is required for the expression of the initial
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aversive motivational a-process but not the rewarding opponent motivational b-process occurring
after acute nicotine administration in non-dependent subjects. Therefore I showed that the initial
aversive a-process occurring after acute nicotine as well as the initial rewarding and opponent
aversive withdrawal process occurring after chronic nicotine are DA-mediated. Only the
opponent rewarding process occurring after acute aversive nicotine is not DA-dependent.
Although I showed here that acute nicotine stimulated an initial aversive DA-dependent
a-process, other groups have demonstrated DA-dependent acute nicotine reward (Acquas et al.,
1989; Lecca et al., 2006; Merlo Pich et al., 1999; Pak et al., 2006; Sellings et al., 2008; Spina et
al., 2006). Acute nicotine administered directly into the brain of nondependent rats produces
both rewarding and aversive effects (Laviolette et al., 2002; Sellings et al., 2008) that can be
segregated within the nucleus accumbens (Sellings et al., 2008). These results suggest that
different neurons within the same neurobiological substrate may mediate both rewarding and
aversive motivational effects, and demonstrate that acute nicotine stimulates both rewarding and
aversive motivational responses depending on the dose administered directly in to the VTA
(Laviolette et al., 2002). Thus, in the case of acute nicotine, both a rewarding and aversive initial
a-process would be stimulated after acute nicotine administration, which would be hypothesized
to lead to aversive and rewarding opponent b-processes, respectively. However, in my studies
using the place conditioning paradigm to model the a- and b-processes occurring after acute
nicotine, I only observed an aversive motivational response. The Laviolette et al. (2002) results
suggest that this aversive response is DA-mediated, results that I replicated in my studies in
chapter 2. This previous data also suggested that the rewarding response to acute nicotine could
be revealed if DA receptor signaling is blocked by use of a broad-spectrum antagonist, as acute
nicotine reward is not DA-mediated, instead being mediated by the VTA GABAergic projection
to the TPP (Laviolette et al., 2002). Therefore it appears that acute nicotine in a nondependent
animal will stimulate both rewarding and aversive motivational responses that occur at the same
time. These simultaneously occurring a-processes can be revealed separately by blocking DA-
mediated aversion with a broad-spectrum DA receptor antagonist or TPP-mediated reward by
lesioning the TPP (Laviolette et al., 2002). Indeed, in unpublished observations I have shown
that non-dependent mice pretreated with α-flu and given a low dose of nicotine (0.35 mg/kg) will
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show a conditioned place preference for the nicotine-paired environment, as well as an aversive
motivational response to a higher dose of acute nicotine (1.75 mg/kg; the same acute nicotine
dose used in the experiments detailed in this thesis). Using β2 nAChR KO mice with specific
reintroduction of the β2 receptor on DA or GABA VTA neurons, I have shown that acute
nicotine reward is GABA- but not DA-mediated and aversion is DA- but not GABA-mediated
(unpublished observations). However, in my model of the opponent motivational processes using
the place conditioning paradigm detailed in this thesis, I did not use DA receptor antagonist
pretreatment, thus only the initial aversive motivational a-process was observed.
I showed that both genetic deletion of the D2R and broad-spectrum antagonism of DA
receptors could block the aversive motivational response to spontaneous nicotine withdrawal,
similar to previous work showing that D1R and D2R antagonists block conditioned aversions to
pharmacologically-precipitated nicotine withdrawal (Laviolette et al., 2008). Withdrawal from
chronic nicotine has been shown to change the activity of DA neurons in the VTA and the
release of DA in the NAc (Hildebrand et al., 1998; Liu and Jin, 2004; Rada et al., 2001), which
suggests that the modification of DA signaling prevents a specific pattern of activity that may
signal nicotine withdrawal. When I pharmacologically and genetically modified the specific
dopaminergic signal that occurs during withdrawal, the aversive motivational response to
withdrawal was blocked, a result that supports the hypothesis that the specific pattern of DA
signaling mediates the aversive motivational response to nicotine withdrawal (the b-process of
chronic rewarding nicotine). I tested this idea in chapter 3.
In chapter 3, I directly investigated the specific pattern of DA signaling that mediates
nicotine motivation by electrophysiologically measuring the effects of acute nicotine and
withdrawal from chronic nicotine on tonic and phasic VTA DA activity. I then examined
whether the specific pattern of activation of D1Rs, D2Rs, or A2ARs mediates the conditioned
motivational responses to nicotine withdrawal and acute nicotine by using D1R-, D2R-, and
A2AR-specific agonists and antagonists, as well as D1R, D2R, and A2AR KO mice, in the place
conditioning paradigm. The WT mice in every experiment in this thesis were C57, and the KO
mice used were backcrossed a minimum of ten generations to a C57 background. This type of
mouse was used to ensure consistency across all the studies, a method which preferable due to
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previous research showing that different strains of mice have different levels of anxiety and
motivational responses to drugs of abuse (Dockstader and van der Kooy, 2001). However, the
consistent use of the C57 strain of mice may not very accurately represent the human population,
which has a diverse genetic background.
I first showed in chapter 3 that both increasing or decreasing dopaminergic signaling at
receptors prevented nicotine withdrawal aversions. Pretreatment with either a DA receptor
agonist or antagonist blocked the aversive motivational response to withdrawal from chronic
nicotine, results similar to previous work suggesting that a specific pattern of DA receptor
activation signals opiate withdrawal aversions (Laviolette et al., 2002). This data supports the
idea that a specific pattern of signaling at and/or activation of DA receptors mediates the
expression of nicotine withdrawal aversions. These results suggest that either increasing or
decreasing DA receptor activation modified the specific pattern that signals nicotine withdrawal,
thereby preventing the expression of withdrawal aversions in the place conditioning paradigm.
I next demonstrated that different patterns of VTA DA signaling occur during acute and
chronic nicotine administration and withdrawal using in vivo electrophysiology in rats. Previous
research has shown that DA neurons exhibit burst- and population-firing activity that leads to
phasic and tonic DA release, respectively (Floresco et al., 2003; Goto and Grace, 2005; Grace,
2000), and that acute nicotine affects phasic VTA DA activity (Mameli-Engvall et al., 2006)
while chronic nicotine affects tonic VTA DA activity (Tan et al., 2009). When I administered a
dose of acute nicotine that produces conditioned place aversions in nondependent animals, I
observed a change in phasic VTA DA activity. However, there was not a significant change in
tonic VTA DA activity, suggesting that similar to previous reports, acute nicotine selectively
affects phasic DA neuronal firing (Mameli-Engvall et al., 2006) while leaving the baseline tonic
DA signal intact. Conversely, when I made rats nicotine dependent, I replicated the previous
results from Tan et al. (2009), showing that chronic nicotine treatment decreased tonic VTA DA
activity without modifying phasic DA firing. Furthermore, I showed for the first time that
spontaneous withdrawal from chronic nicotine at a dose and time that produces a conditioned
place aversion to a withdrawal-paired environment in nicotine-dependent and -withdrawn rats
(see chapter 2) would further modify the tonic firing rate of VTA DA neurons. Tonic VTA DA
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firing was significantly decreased compared to saline-treated, acute nicotine-treated, and chronic
nicotine-treated groups in a group of rats that was chronically exposed and spontaneously
withdrawn from nicotine, suggesting that nicotine withdrawal leads to a decrease in tonic DA
release in VTA projection sites, and this modified DA release participates in the signaling of the
motivational effects of nicotine withdrawal. A criticism of this idea could be that the amount of
DA released from tonic DA firing is very small in comparison to large scale phasic release,
however, a previous study has shown that tonic DA activity can indeed provide sufficient DA to
engage behavior (Zweifel et al., 2009), thus it is plausible that a tonic DA signal mediates the
behavioral response to nicotine withdrawal.
In this thesis, I have suggested that relapse to nicotine use is driven by the negative
experience of withdrawal (many others have suggested this as well, for example see Koob and
Le Moal, 2006). If a decrease in tonic activity is signaling the negative experience of withdrawal,
which was suggested by the electrophysiology results, and a single injection of acute nicotine
leads to an increase in phasic DA activity (see chapter 3) and the large-scale phasic release of
DA (Rice and Cragg, 2004; Zhang et al., 2009), then it is plausible that nicotine-dependent
subjects who are experiencing the negative motivational effects of withdrawal are feeling these
effects because of a decrease in tonic DA release in VTA projection sites, and thus renew
nicotine use as a way to quickly increase DA levels in the brain through phasic activation of
VTA DA neurons. In this way, the dependent subject could quickly restore DA levels in the
brain and alleviate the aversive effects of nicotine withdrawal.
Because acute nicotine at a dose that leads to conditioned place aversions modifies phasic
DA activity and chronic nicotine withdrawal modifies tonic DA activity, I hypothesized that
antagonist drugs that selectively block phasic DA activity without effecting tonic DA activity
would prevent the expression of acute nicotine aversions but not aversions to chronic nicotine
withdrawal in a place conditioning paradigm. I showed that these antagonist drugs did indeed
selectively block acute nicotine aversions in nondependent mice, while withdrawal aversions in
dependent mice were not affected, providing support for the idea that a specific pattern of phasic
activity mediates acute nicotine aversions and for the overall hypothesis that reward and aversion
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can be signaled by the same neurobiological substrate through the use of different patterns of
signaling.
Previous research examining phasic and tonic activity at various DA receptors showed
that phasic DA release mainly activates D1Rs and tonic DA release mainly activates D2Rs
(Floresco et al., 2003; Goto and Grace, 2005). Furthermore, nicotine dependence leads to
decreases in D2R availability in humans (Fehr et al., 2008) and D1R antagonism blocks acute
nicotine motivation in nondependent mice (David et al., 2006). These results, combined with my
results showing that both agonism and antagonism of DA receptors would block the aversive
response to nicotine withdrawal, led me to hypothesize that modifying D1R activity would
prevent the expression of acute nicotine aversions in nondependent mice and modifying D2R
activity would prevent aversions to withdrawal from chronic nicotine in dependent mice. I
demonstrated that increasing or decreasing D1R activity using D1R agonists or antagonists,
respectively, and genetic deletion of the D1R using D1R KO mice would prevent acute nicotine
aversions in nondependent mice, but not nicotine withdrawal aversions in dependent mice. I also
showed that increasing or decreasing D2R activity using D2R agonists and antagonists, or
genetic deletion of the D2R using D2R KO mice, would prevent aversions to withdrawal from
chronic nicotine in dependent mice but not to acute nicotine in nondependent mice. With these
results I doubly dissociated the role of the D1R versus the D2R in nicotine motivation, such that
the motivational response to withdrawal in dependent mice is D2R-mediated and acute nicotine
motivation is D1R-mediated, but not vice versa. The nondeprived/deprived hypothesis posits that
a neurobiological switch occurs during the transition from a drug-naive to a drug-dependent
motivational state (Bechara et al., 2002). My results show that a switch does indeed occur when
a mouse moves from an acute, nondependent (nondeprived) motivational state to a nicotine-
dependent and -withdrawn (deprived) motivational state, whereby nondependent motivation is
D1R-mediated and dependent and withdrawn motivation is D2R-mediated. Furthermore, the
VTA DA activity that mediates the motivational response in nondependent vs. dependent and
withdrawn animals also switches from phasic DA- to tonic DA-mediated.
In chapter 3 I also showed that modulation of adenosine A2ARs prevents the expression
of nicotine withdrawal aversions, without affecting the expression of aversions to acute nicotine.
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A2ARs colocalize with D2Rs on neurons in the mesolimbic system (Tozzi et al., 2011) acting
antagonistically to the D2R in the nucleus accumbens (Fuxe et al., 2010; Tanganelli et al., 2004).
My work has demonstrated that both increasing or decreasing D2R activation prevented nicotine
withdrawal aversions but not acute nicotine aversions. Because A2ARs and D2Rs act
antagonistically (Fuxe et al., 2010; Tanganelli et al., 2004), an A2AR agonist would decrease
D2R activity, and an A2AR antagonist would increase D2R activity. Thus I hypothesized that
modifying A2AR activity would prevent aversions to chronic nicotine withdrawal in dependent
mice, but not to acute nicotine in nondependent mice, and tested this idea using specific A2AR
agonists and antagonists as well as A2AR KO mice. I showed that pharmacological and genetic
modulation of A2AR activity did not prevent the expression of acute nicotine aversions in
nondependent mice, results that are similar to a previous study showing that conditioned taste
aversions to nicotine in nondependent mice are not blocked in A2AR KO mice (Castañé et al.,
2006). Interestingly, similar to the D2R results described above, A2AR agonism and antagonism,
as well as genetic deletion of the A2AR, prevented the aversive motivational response to nicotine
withdrawal in dependent mice. These results support the idea that any modification of the
specific pattern of signaling at D2Rs will prevent the expression of the aversive motivational
response to nicotine withdrawal, and imply that drug therapies that modulate D2R activity may
be helpful in the treatment of nicotine addiction and withdrawal. However, directly increasing
neural DA activity could potentially produce schizophrenic-like symptoms, and decreasing DA
activity may induce Parkinson-like symptoms. This makes the A2AR results very attractive as a
potential drug therapy for nicotine addiction, since modulating A2AR activity appears to be an
indirect method of disrupting the specific pattern of activity that signals the aversive
motivational response to nicotine withdrawal.
Taken together, my results from chapter 3 show that the mechanisms signaling the
aversive motivational responses to acute nicotine and withdrawal from chronic nicotine can be
doubly dissociated, such that nondependent nicotine aversions are mediated by a phasic VTA
DA signal through D1Rs, and nicotine-dependent and -withdrawn aversions are mediated by a
tonic VTA DA signal through D2Rs and indirectly, A2ARs. Considered with my results from
chapter 2, I have identified the role of phasic and tonic VTA DA activity and signaling at DA
138
receptors in mediating the opponent motivational responses occurring after acute and chronic
nicotine.
In chapter 4, I shifted focus to identify the role of VTA CRF in the opponent motivational
responses of acute and chronic nicotine. Although CRF neurons had not previously been shown
to exist in the VTA, many previous studies suggested a CRF/DA connection in the VTA. Very
few of these studies examined the effects of nicotine on CRF activity, and none had investigated
a CRF/DA link in nicotine dependence. My work detailed in chapters 2 and 3, as well as a large
number of previous studies, have shown that VTA DA signaling mediates nicotine’s acute
aversive and chronic motivational effects. This change in VTA DA function is a within-system
neuroadaptation (Volkow et al., 2007) because it occurs locally within the VTA. The
involvement of CRF activity in nicotine motivation has been less well studied, but previous
results have suggested that nicotine withdrawal leads to increased CRF release in the amygdala
(George et al., 2007). This upregulation of CRF activity is a between-system neuroadaptation
(Koob and Le Moal, 2008) because the acute motivational effects of nicotine are not mediated in
the amygdala, and CRF has not been shown to be involved in the motivational response to any
drug of abuse in nondependent animals. Studies showing that CRF-BP is present in DA and
GABA neurons in the VTA (Wang and Morales, 2008) led me to hypothesize that a within-
system neuroadaptation could be possible that may have effects on the aversive motivational
response to nicotine withdrawal. Therefore I examined the involvement of the CRF system in the
VTA in nicotine motivation, and showed for the first time that chronic nicotine administration
led to an upregulation of CRF mRNA in VTA neurons by using both in situ hybridization and
rtPCR. A recent study showed that nicotine selectively activates DA neurons in the pVTA, but
not aVTA (Zhao-Shea et al., 2011), which led me to hypothesize that the observed CRF-
expressing neurons would be preferentially located in the pVTA and may be dopaminergic.
Indeed, I showed that CRF mRNA-expressing neurons were in the pVTA and were TH-positive.
This newly discovered within-system neuroadaptation links the brain DA reward and CRF stress
systems in the same neurobiological substrate, the VTA, a structure that has been implicated in
processing the motivational effects of almost all drugs of abuse (Koob and Le Moal, 2006). To
investigate whether the upregulation of CRF mRNA in the VTA played a role in the aversive and
139
anxiogenic motivational responses to nicotine withdrawal, I blocked CRF mRNA upregulation
by using a siRNA and then subjected separate groups of nicotine-dependent and -withdrawn
mice given a silencing or a control viral vector to place conditioning and open field testing. I
demonstrated that preventing the upregulation of CRF mRNA that occurs in pVTA DA neurons
upon the induction of nicotine dependence prevented the expression of the anxiogenic and
aversive motivational effects of nicotine withdrawal.
I also showed that withdrawal from chronic nicotine depleted the amount of CRF peptide
in the VTA using immunohistochemistry. Previous studies have shown that depletion of
neuronal CRF peptide is associated with increased CRF release during drug withdrawal (Merlo-
Pich et al., 1995), that the VTA expresses both CRF1Rs and CRF2Rs (Sauvage and Steckler,
2001; Ungless et al., 2003), and that CRF1R antagonists can block the anxiogenic effects of
withdrawal from chronic nicotine (George et al., 2007; Tucci et al., 2003). I thus hypothesized
that CRF released in the VTA during nicotine withdrawal would act at CRF1Rs to signal the
aversive motivational response to nicotine withdrawal, and tested this idea by subjecting
nicotine-dependent and -withdrawn mice to place conditioning after intra-pVTA and systemic
administration of a CRF1R antagonist. I showed that antagonism of pVTA CRF1Rs prevented
the expression of the aversive motivational response to nicotine withdrawal. Taken together with
the siRNA results, I demonstrated that upregulation of CRF mRNA and activation of CRF1Rs
locally in the pVTA occur after the induction of nicotine dependence and withdrawal; effects that
mediate the expression of the anxiogenic and aversive motivational responses to nicotine
withdrawal.
I also examined the involvement of CRF activity in the opponent motivational processes
occurring after the administration of acute and chronic nicotine. The results discussed above
showed that CRF activity mediates the aversive b-process of withdrawal from chronic nicotine in
dependent mice. However, by using the place conditioning paradigm that models the opponent
motivational processes of acute aversive and chronic rewarding nicotine (discussed in chapter 2),
I showed that CRF is not involved in the initial aversive a-process or the rewarding opponent b-
process occurring after acute aversive nicotine. I also showed in nicotine-dependent mice that
CRF activity is not involved in the initial rewarding motivational response, or a-process, to
140
chronic nicotine. Therefore I have demonstrated that CRF activity selectively mediates the
opponent motivational response to chronic nicotine, which is modeled by the conditioned
aversive response to a withdrawal-paired environment in nicotine-dependent and -withdrawn
mice. Taken with the results from chapter 2 where I showed that the initial rewarding and
aversive opponent motivational processes occurring after chronic nicotine as well as the initial
acute aversive process in nondependent subjects are DA-mediated, but the opponent rewarding
response after acute aversive nicotine does not involve DA, I have demonstrated that only the
aversive motivational response to withdrawal from chronic nicotine (the chronic nicotine b-
process) is both DA- and CRF-mediated (Figure 5.1). Although I did not examine the
GABA/TPP-mediated rewarding a-process after acute nicotine administration in nondependent
animals (Laviolette et al., 2002; also see discussion above) in this thesis, I have shown that non-
dependent mice pretreated with α-flu and given a low dose of acute nicotine (0.35 mg/kg) will
demonstrate a rewarding motivational a-process (unpublished observations, Figure 5.1b).
My overall hypothesis that nicotine dependence upregulates CRF and modifies DA
activity in the VTA, and that both the increase in CRF and the specific pattern of DA activity in
the VTA are necessary for the experience of the aversive motivational response to nicotine
withdrawal, has been supported by the results detailed above. Nicotine dependence and
withdrawal indeed upregulated CRF mRNA in the VTA and also changed the mediation of
nicotine’s motivational effects from phasic to tonic, and from D1R to D2R. If the increase in
CRF was prevented by RNA interference or CRF1R antagonism, or the specific pattern of DA
activity was modified by D2R agonism or antagonism, then the aversions to withdrawal from
chronic nicotine were no longer expressed in my place conditioning paradigm. A previous study
showed that CRF1R antagonism significantly increased DA neuron tonic population activity
without affecting phasic burst firing, average firing rate, or NAc DA levels (Lodge and Grace,
2005), suggesting that modulating CRF activity may have effects on DA neuronal firing activity
and DA receptor activation. In chapter 3, I showed that withdrawal from chronic nicotine
decreased tonic VTA DA activity and that a specific pattern of DA receptor activation mediated
the expression of nicotine withdrawal aversions. Furthermore, in chapter 4 I showed that CRF1R
antagonism prevented nicotine withdrawal aversions in dependent mice. Taken with the Lodge
141
Figure 5.1. Summary of the involvement of DA and CRF in the opponent motivational
responses occurring after acute and chronic nicotine.
a) In nicotine-dependent mice, I have shown that chronic nicotine leads to a rewarding a-process
that is DA- but not CRF-mediated. Withdrawal from chronic nicotine in nicotine-dependent mice
leads to an aversive opponent b-process that is both DA- and CRF-mediated. In nondependent
mice, acute aversive nicotine will lead to an aversive a-process that is DA- but not CRF-
mediated. This acute aversive a-process triggers and is followed by a rewarding opponent b-
process that is neither DA- nor CRF-mediated. b) In non-dependent mice, I have shown in
unpublished results that acute nicotine will stimulate an initial rewarding motivational response
(a-process) that is revealed when the DA system is pharmacologically blocked. This acute
rewarding a-process is mediated through a TPP connection with GABAergic VTA neurons. I
have not examined the opponent motivational b-process to acute rewarding nicotine, and do not
know which neurobiological substrates mediate its expression.
142
a
b
143
and Grace (2005) results, it appears that the aversive response to withdrawal is prevented
because CRF1R antagonism increases tonic DA activity, thereby modifying the specific pattern
of DA activity that signals nicotine withdrawal aversions and preventing the expression of this
motivational response. As previously mentioned, it is difficult to modify DA activity without
producing adverse side effects in human subjects, therefore these results indicate that CRF1R
antagonism may prove a very useful therapeutic treatment for nicotine addiction and withdrawal.
5.2 Overall conclusion
Using a place conditioning paradigm to model the opponent motivational effects
occurring after acute and chronic nicotine, as well as pharmacological, genetic and viral vector
approaches, electrophysiology, rtPCR and in situ hybridization, I found that tonic DA activity
and a newly identified CRF system in the VTA are both necessary for the expression of the
aversive motivational response to withdrawal from chronic nicotine. Only DA but not CRF was
necessary for both the initial aversive a-process in nondependent mice and the rewarding
motivational a-process in dependent mice, and neither CRF nor DA were involved in the acute
nicotine rewarding opponent b-process in nondependent mice. The involvement of both CRF and
DA in the VTA in nicotine withdrawal motivation represents a within-system neuroadaptation
that occurs when an animal becomes nicotine dependent.
The transition from a nondependent to a dependent motivational state leads to a switch in
the neurobiological substrates that mediate nicotine’s motivational effects. In a nondependent
state, acute nicotine aversion is mediated by phasic VTA DA activity and the D1R, while in a
dependent motivational state, aversions to withdrawal from chronic nicotine are mediated by
tonic VTA DA activity and the D2R. Similarly, the upregulation of CRF and CRF1R activity are
not involved in nondependent nicotine motivation, but are necessary for nicotine withdrawal
motivation in dependent mice. The tonic VTA DA activity and possibly CRF1R activity produce
a specific pattern of D2R activation that signals the aversive motivational effects of nicotine
144
withdrawal, and modifying this specific activity pattern by either increasing or decreasing D2R
activation will prevent the expression of nicotine withdrawal aversions.
I demonstrated that chronic nicotine administration and withdrawal increase CRF mRNA
selectively in dopaminergic neurons in the pVTA, a region heavily implicated in drug reward
that was previously thought to be devoid of CRF. I showed that upregulation of CRF mRNA and
activation of CRF1Rs locally in the pVTA occur after the induction of nicotine dependence and
withdrawal and that these CRF activities mediate the anxiogenic and aversive motivational
responses to nicotine withdrawal.
Taken together, the work contained in this dissertation provides novel evidence of a VTA
DA/CRF neuroadaptation occurring during nicotine dependence that mediates nicotine
withdrawal motivation. Both increased CRF expression in pVTA DA neurons and CRF1R
activation, as well as the specific pattern of tonic VTA DA activity and D2R activation are
necessary for the expression of the aversive motivational response to withdrawal from chronic
nicotine. This link between the brain reward and stress systems offers novel and exciting insight
into the neurobiological substrates mediating drug motivation, providing new avenues for
therapeutic treatments of nicotine addiction and withdrawal.
5.3 Future directions
The identification of a link between the mesolimbic DA reward and CRF brain stress
systems in signaling drug withdrawal will undoubtedly lead to a huge amount of future research.
The following are a select few studies that may shed further insight on the mechanisms behind a
DA/CRF link in the VTA.
How does CRF peptide influence phasic and tonic VTA DA neuronal activity?
145
I showed that CRF upregulation and the activation of CRF1Rs are necessary for the
expression of nicotine withdrawal aversions but not acute nicotine aversions, and that tonic DA
activity signals nicotine withdrawal while phasic DA activity signals acute nicotine motivation. I
also demonstrated that CRF peptide is released locally in the VTA in nicotine-dependent and
-withdrawn mice. CRF1R blockade is known to increase tonic VTA DA activity (Lodge and
Grace, 2005), suggesting that CRF1R activation (by CRF) would decrease tonic VTA DA
activity. However, it is unknown how or if CRF peptide modulates the phasic and tonic activities
of VTA DA neurons. Experiments that may address this question involve the use of
electrophysiological recordings of VTA DA neurons after intra-VTA infusion of CRF peptide.
These recordings could be performed in vivo in awake, freely moving rats (as we did in chapter
3), where the tonic and phasic activities of VTA DA neurons are measured after intra-VTA CRF
administration, or in vitro on VTA slices. Most interesting and relevant to this thesis would be
measurement of tonic and phasic activity after CRF administration in both nondependent and
nicotine-dependent and -withdrawn animals. My work showed that CRF is not involved in
mediating the motivational effects of acute nicotine, thus an effect on phasic DA activity is not
expected. However, if the administration of CRF peptide modified tonic DA activity in a
nicotine-withdrawn rat, then the mechanism by which CRF directly modulates tonic VTA DA
activity to signal nicotine withdrawal would be known.
What is the effect of CRF1R and CRF2R antagonism on tonic VTA DA neuronal activity in
nicotine-dependent animals?
Similarly, experiments examining the effect of CRF1R antagonist on phasic and tonic
VTA DA neuronal activity in both nondependent and nicotine-dependent and withdrawn rats
would further our understanding of the neurobiology of nicotine withdrawal and of the CRF/DA
link in mediating nicotine withdrawal motivation. I showed that a specific pattern of signaling at
DA receptors mediates nicotine withdrawal aversions because either increasing or decreasing the
activity at DA receptors would prevent withdrawal aversions. Further, I suggested that tonic
VTA DA activity produces this pattern of signaling at DA receptors that signals withdrawal. It is
known that both CRF1Rs and CRF2Rs are expressed on VTA neurons, and that CRF1R
146
antagonism decreases tonic VTA DA activity (Lodge and Grace, 2005), however it is unknown
how CRF1R or CRF2R antagonism effects tonic VTA DA activity in nicotine-dependent and
-withdrawn animals. This CRF/DA mechanism could be examined electrophysiologically
followed by place conditioning after intra-VTA administration of CRF1R and CRF2R
antagonists. If either antagonist treatment either increased or decreased tonic VTA DA activity,
then the specific pattern of tonic activity signaling nicotine withdrawal would be modified and I
hypothesize that withdrawal aversions would not be demonstrated in the place conditioning
paradigm.
Can a drug naive animal be made nicotine-dependent and -withdrawn by reproducing a tonic
DA activity signal?
I showed that chronic nicotine was rewarding and withdrawal from chronic nicotine was
aversive in a place conditioning paradigm, and that nicotine withdrawal decreased tonic VTA
DA activity. Previous research has shown that tonic VTA DA activity can be increased either by
inhibition of the VP with GABA receptor agonists (Floresco et al., 2003) or by CRF1R
antagonism (Lodge and Grace, 2005), suggesting that the opposite effects of VP excitation with
GABA receptor antagonists, or the administration of CRF, would decrease tonic DA activity. If
nicotine-dependent animals were conditioned after either of these modulations that are
hypothesized to decrease tonic VTA DA activity, they should not show a rewarding response to
the conditioning environment, but an aversive response since the decrease in tonic DA activity
that signals withdrawal was reproduced in those animals during conditioning. This result would
be very interesting because nicotine withdrawal could be caused in a drug naive subject by the
production of a tonic DA activity signal in the brain.
Is CRF mRNA increased in the VTA after chronic exposure to other drugs of abuse?
It is unlikely that the upregulation of CRF mRNA that I observed in nicotine dependent
mice is specific only to chronic nicotine exposure. Thus it is very important to determine whether
147
upregulation of CRF in the brain reward system occurs after dependence to other drugs of abuse.
The measurement of CRF mRNA in drug-dependent animals could be performed using rtPCR or
in situ hybridization, as in this thesis, or by using enzyme-linked immunosorbent assay (ELISA).
If CRF mRNA was indeed upregulated in the VTA in animals dependent on opiates, alcohol,
cocaine, or methamphetamine, then a within-system neuroadaptation would be demonstrated that
occurs upon dependence to most drugs of abuse.
Does the increase in VTA CRF mRNA occur in nicotine-dependent humans?
Research examining the brains of deceased human smokers, comparing the expression of
CRF mRNA in the VTA of smokers to that of non-smokers, could be very interesting and
informative. However, this type of research is beyond the scope of our laboratory.
Is the rest of the CRF mRNA in GABA or glutamate VTA neurons?
DA neurons are the most numerous cells in the VTA, followed by GABA cells and a
small amount of glutamate cells (Dobi et al., 2010; Yamaguchi et al., 2007). I showed that
87.5% of the neurons that upregulated CRF mRNA in nicotine dependent mice were
dopaminergic (see chapter 4), however, the question remains as to which type of neurons the
remaining 12.5% were? The answer to this question could be found by double labeling of CRF
mRNA by in situ hybridization combined with immunostaining for GAD (for GABA neurons) or
vesicular glutamate transporters (for glutamate neurons). Further experiments in this line of
questioning could then examine what effect the upregulation of CRF has on these neurons, and
whether this upregulation then modifies their regulation of VTA DA neuronal activity.
Which neurobiological substrate mediates the rewarding opponent b-process occurring after
acute aversive nicotine?
148
I showed that neither DA nor CRF mediate the b-process after acute aversive nicotine
that is expressed as a rewarding motivational response to the nicotine-paired environment in a
place conditioning paradigm. Previous work has shown that acute nicotine reward is TPP-
mediated (Laviolette et al., 2002), making the TPP a good candidate to mediate this opponent
motivational effect. Lesioning the TPP prior to conditioning for the b-process after acute
aversive nicotine could answer this question.
Undoubtedly, many more questions will arise that will warrant future investigations into
DA and CRF activity in nicotine motivation, and more generally, drug addiction. I hope that my
work detailed in this thesis will lead to future breakthroughs in the drug motivation field, and
that the questions posed here and many others will be answered in such a way that new therapies
for nicotine (and other drug) addiction are discovered.
149
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