separate mesolimbic dopaminergic pathways mediate the ... · separate mesolimbic dopaminergic...
TRANSCRIPT
Separate mesolimbic dopaminergic pathways mediate the
opposing motivational effects of acute caffeine
by
Mandy Yee
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Institute of Medical Science
University of Toronto
© Copyright by Mandy Yee 2018
ii
Separate mesolimbic dopaminergic pathways mediate the opposing
motivational effects of acute caffeine
Mandy Yee
Master of Science
Institute of Medical Science
University of Toronto
2018
Abstract
Caffeine is the most commonly consumed psychoactive drug in the world, yet little is
known about the neural substrates that underlie its rewarding and aversive properties. Using male
Wistar rats in a place conditioning procedure, we showed that systemic caffeine at a low
intraperitoneal dose of 2 mg/kg (or 100µM injected directly into the rostral, but not caudal, ventral
tegmental area) induced reward. By contrast, high doses of systemic caffeine (10 and 30 mg/kg)
produced aversions that were not recapitulated by a caffeine analog restricted to the periphery. We
demonstrated that pharmacological blockade of dopamine receptors using α-flupenthixol injected
into the nucleus accumbens shell, but not core, blocked caffeine reward. Conversely, α-
flupenthixol injected into the nucleus accumbens core, but not shell, blocked caffeine aversions.
Thus, our findings reveal two dopamine-dependent and functionally dissociable mechanisms for
processing caffeine motivation that are segregated between nucleus accumbens subregions.
iii
Acknowledgments
I would like to acknowledge my friends and family for supporting me through all the ups and
downs of research. First and foremost, I would like to thank my parents for their loving support. I
would also like to thank my siblings – Lance, Angela, Holly, and Kingly – for their support.
A great big thanks to my graduate supervisor and mentor, Dr. Derek van der Kooy, whose
combination of excellent guidance, thoughtful critiques, and vast knowledge inspired me to grow
and learn as a scientist. Thank you to my program advisory committee members, Drs. Cindi
Morshead and Paul Fletcher, for their insights, advice, and patience.
I would also like to thank my lab mentors, Taryn and Geith, who patiently taught me everything I
needed to know for my project. A huge thanks to our lab staff extraordinaire, Brenda and Monica,
for being incredibly kind and helpful. I also cannot stress enough the importance of my amazing
fellow lab members that I have worked with over the years. I want to especially thank Isabel,
Michael, and the other undergraduates in the motivation group for keeping me company in the
necropsy and surgery room those long days and nights. Additionally, I would like to thank Justin,
Daniel, Ken, Sylvia, and the other lab members for always being in the lab even at the increasingly
odd hours that I came in to do lab work. Even though we were not working on the same projects,
you guys are excellent company and made the evenings far more interesting. I would also like to
thank the staff at the Division of Comparative Medicine for taking excellent care of the animals,
and the Zandstra lab members who patiently helped me with my secondary project.
A special thank you to Marco for putting up with all my complaining – and there was a lot of it! I
am especially grateful for the time you spent giving me the push I needed to finish this thesis.
I dedicate this thesis to my family and friends who have supported me every step of the way.
iv
Statement of Contributions
Mandy Yee (author) solely prepared this thesis. All aspects of this body of work were performed
by the author in whole or in part.
The following contributions by other individuals is formally acknowledged below:
Dr. Derek van der Kooy (primary supervisor)
- Mentorship, including guidance in planning experiments and interpreting results
- Provision of laboratory materials and equipment
- Preparation of manuscript and thesis
Dr. Cindi Morshead (program advisory committee member)
- Mentorship, including guidance in planning experiments and interpreting results
Dr. Paul Fletcher (program advisory committee member)
- Mentorship, including guidance in planning experiments and interpreting results
Dr. Taryn Grieder
- Mentorship, including guidance in planning experiments and interpreting results
Geith Maal-Bared
- Mentorship, including guidance in planning experiments and interpreting results
- Assistance in executing experiments
Dr. Ryan Ting-A-Kee
- Assistance in executing experiments and laying foundation of project
Michal Chwalek
- Assistance in executing experiments and laying foundation of project
Isabel MacKay-Clackett
- Assistance in executing experiments
Michael Bergamini
- Assistance in executing experiments
v
Table of Contents
Abstract .................................................................................................................................... ii
Acknowledgments .................................................................................................................... iii
Statement of Contributions ......................................................................................................... iv
Table of Contents ....................................................................................................................... v
List of Abbreviations............................................................................................................... viii
List of Figures and Tables ......................................................................................................... xi
Chapter 1 - Literature Review ................................................................................................. 1
1.1 Introduction to motivation ..................................................................................................... 2
1.2 Introduction to drug addiction .............................................................................................. 4
1.3 Measurements of motivation ................................................................................................. 7
Operant conditioning (self-administration)....................................................................... 8
Classical conditioning (place conditioning) .................................................................... 11
1.4 Models and theories of drug motivation ............................................................................... 16
The mesolimbic dopamine reward hypothesis ............................................................... 16
The error prediction model ............................................................................................ 19
The incentive-sensitization theory .................................................................................. 22
The non-deprived/deprived hypothesis........................................................................... 23
The opponent process theory ......................................................................................... 26
1.5 Neuroanatomy of motivation ............................................................................................... 32
Anatomy of the ventral tegmental area ........................................................................... 32
Anatomy of the nucleus accumbens .............................................................................. 38
vi
1.6 Neurobiology of caffeine motivation .................................................................................. 41
Introduction to caffeine consumption ............................................................................. 41
1.7 Research hypotheses and objectives .................................................................................... 43
Chapter 2 - Materials and Methods ....................................................................................... 46
2.1 Animal subjects and drugs ................................................................................................... 47
2.2 Experimental procedures .................................................................................................... 49
Surgical cannulation ...................................................................................................... 49
Place conditioning ......................................................................................................... 49
2.3 Histology ............................................................................................................................ 51
Verification of cannula placements ............................................................................... 51
2.4 Statistical methods .............................................................................................................. 52
Chapter 3 - Results ................................................................................................................. 53
3.1 Systemic caffeine dose-response ......................................................................................... 54
Systemic caffeine elicits reward at low doses and aversion at higher doses ................... 54
3.2 Intra-rVTA caffeine reward ................................................................................................. 59
Caffeine injections into the rostral VTA elicit reward .................................................... 59
Intra-rVTA caffeine reward is mediated through the NAc shell, but not core ................. 65
3.3 High dose systemic caffeine aversion .................................................................................. 69
Systemic caffeine aversion is mediated centrally through the NAc core, not shell .......... 69
vii
Chapter 4 - General Discussion ............................................................................................. 72
4.1 Overview............................................................................................................................. 73
Neuroanatomy of caffeine aversion................................................................................ 73
Neuroanatomy of caffeine reward ................................................................................. 75
Caffeine and other drugs of abuse ................................................................................. 77
Caffeine and the role of adenosine receptors ................................................................. 78
Caffeine pharmacokinetics and metabolism ................................................................... 79
Caffeine use by people ................................................................................................... 80
4.2 Conclusions ........................................................................................................................ 83
4.3 Future Directions ................................................................................................................ 84
References ............................................................................................................................... 89
viii
List of Abbreviations
6-OHDA 6-hydroxydopamine
8-SPT 8-(p-sulfophenyl)theophylline
α-flu α-flupenthixol (also known as α-flupentixol)
A1R adenosine receptor subtype-1
A2AR adenosine receptor subtype-2A
ACC anterior cingulate cortex
ANOVA analysis of variance
CeA central amygdala
cm centimeter
CNS central nervous system
CPA conditioned place aversion
CPP conditioned place preference
CR conditioned response
CS conditioned stimulus
cVTA caudal ventral tegmental area
D1R dopamine receptor subtype-1
D2R dopamine receptor subtype-2
DA dopamine
DR dorsal raphe
DStr dorsal striatum
DSM-5 diagnostic and statistical manual of mental disorders-fifth edition
EEG electroencephalogram
ix
GABA γ-aminobutyric acid
GAD glutamic acid decarboxylase
GP globus pallidus
Glu glutamate
HPLC high-pressure liquid chromatography
Hz hertz
i.c. intracranial
ICD-10 international classification of diseases and related health problems-tenth revision
i.p. intraperitoneal
KO knockout
LH lateral hypothalamus
LHb lateral habenula
MFB medial forebrain bundle
mg/kg milligrams per kilogram
mm millimeter
mPFC medial prefrontal cortex
ms millisecond
MSN medium spiny neuron
mV millivolt
NAc nucleus accumbens
NAc core nucleus accumbens core
NAc shell nucleus accumbens shell
NMDA N-methyl-D-aspartate
x
NS neutral stimulus
PAG periaqueductal grey
PBS phosphate-buffered saline
PFA paraformaldehyde
PPTg pedunculopontine tegmental nucleus
rVTA rostral ventral tegmental area
s second
s.c. subcutaneous
SEM standard error of the mean
TH tyrosine hydroxylase
TPP tegmental pedunculopontine nucleus
tVTA tail of the ventral tegmental area
UR unconditioned response
US unconditioned stimulus
VP ventral pallidum
VTA ventral tegmental area
VTAR ventral tegmental area-rostral subregion
WT wild-type
xi
List of Figures
Figure 1.1 - Schematic of the place conditioning paradigm (Page 12)
Figure 1.2 - Schematic of the mesolimbic dopamine system in the rat brain (Page 17)
Figure 1.3 - Schematic illustrating the opponent process theory of motivation (Page 27)
Figure 1.4 - Selected input and output projections of the VTA (Page 37)
Figure 1.5 - Schematic of the nucleus accumbens shell and core (Page 39)
Figure 3.1 - Systemic caffeine produces DA-dependent rewarding effects at low doses, and DA-
dependent aversive effects at high doses that are centrally mediated (Page 56)
Figure 3.2 - Caffeine reward is mediated through the rostral, not caudal, VTA (Page 61)
Figure 3.3 - Verification of bilateral cannula placements (Page 66)
Figure 3.4 - Intra-rVTA caffeine reward is DA-dependent and mediated centrally through the
NAc shell, but not the core (Page 67)
Figure 3.5 - Caffeine aversion is mediated centrally through the NAc core, but not the shell (Page
70)
List of Tables
Table 1 - Estimated percentage of cell types in the VTA (Page 34)
Table 2 - Projections to and from the VTA (Page 36)
1
Chapter 1 - Literature Review
2
1 Literature Review
1.1 Introduction to motivation
In the evolutionary struggle for survival and reproduction, a group of organisms are
successful if they can contribute to the gene pool of the next generation. To influence evolutionary
(phylogenetic) success of a species, certain key features must be obtained over the developmental
(ontogenetic) history of each organism. One of these ontogenetic features is motivation, which can
be broadly defined as the process in which an organism responds to stimuli to maintain goal-
oriented behaviours. Generally, motivation encompasses physiological, psychological, and social
processes that can manifest as a set of approach and avoidance behaviours. For example, organisms
can learn to approach the necessities of life such as food and water to reduce hunger and thirst,
while avoiding potential harm or dangers such as poisons and predators. Over time, organisms can
learn to associate previously neutral cues or environments with either positive (rewarding or
beneficial to the organism in some way), negative (aversive or detrimental), or neutral (neither
rewarding or aversive) outcomes. Repeated associations result in reinforcement of behaviours that
ensure a selection of optimal behaviours for the well-being and survival of the organism.
Therefore, the study of motivation and its underlying mechanisms is crucial for understanding
behaviour.
Integral to the study of motivation is what happens when motivational processes go awry
and become maladaptive. In the extreme case where there is a complete absence of motivation,
organisms would be unlikely to meet their needs, and may perish early. However, a more intriguing
example of maladaptive motivation is drug addiction, in which there is a large range in severity.
3
An individual that actively and compulsively engages in drug-seeking and drug-taking behaviour
despite the negative consequences is colloquially termed a “drug addict.” Individuals may
consume one or many types of drugs of abuse – including nicotine, cocaine, ethanol, cocaine, and
amphetamine – that produce subjective feelings of pleasure or reward. As more of the drug is
consumed, the pleasure of taking the drug is positively reinforced and leads to continued
consumption and drug dependence (also known as addiction). Upon halting consumption of a drug
in a dependent/addicted individual, the aversive feelings of withdrawal typically lead to further
drug-seeking behaviour to alleviate the aversive feelings. The studies described within this thesis
focus on the neurobiological substrates and possible pathways that mediate the rewarding and
aversive responses to acute caffeine given to drug-naïve rats.
4
1.2 Introduction to drug addiction
According to the Diagnostic and Statistical Manual of Mental Disorders – Fifth Edition
(DSM-5), a substance use disorder or addiction can be described as a pattern of drug use that results
in impairment or distress in daily life. It is a brain disease characterized by compulsive drug-
seeking behaviour, despite adverse consequences (American Psychiatric Association, 2013). The
International Classification of Diseases and Related Health Problems (ICD-10) by the World
Health Organization (10th revision, 1992) provides a classification of mental and behavioural
disorders with a similar definition. Along with addiction, three other common terms that are often
used in the literature (reviewed in Nestler, 1992) to describe different states of substance use
disorder, include:
1) Tolerance, defined as “a reduced effect upon repeated exposure to a drug at constant
dose, or the need for an increased dose to maintain the same effect;”
2) Dependence, defined as “the need for continued exposure to a drug so as to avoid a
withdrawal syndrome,” and;
3) Withdrawal, defined as “physical or psychological disturbances when the drug is
withdrawn.”
The DSM-5 defines several diagnostic criteria for substance-related disorder that fall into four
major categories, including:
1) Impaired control, described as use for longer than intended periods of time or in larger
amounts than intended;
5
2) Social impairment, evidenced by continued use of the drug, causing problems with
work, school, or other social obligations;
3) Risky use, demonstrated by drug use in conjunction with dangerous situations (e.g.
drinking and driving) or continued use despite knowing the harmful physiological side
effects, and;
4) Pharmacological indicators, which include tolerance of the drug such that increased
amounts of the drug are required to achieve the same pleasurable effect, and withdrawal
where cessation of the drug leads to unpleasant symptoms.
An individual that meets at least two of these criteria can be diagnosed with substance use disorder,
of which the severity is dependent on how many of the criteria are met.
Caffeine dependence and withdrawal did not meet these criteria in the previous editions of
the DSM, but numerous preclinical and clinical studies have supported the addition of a caffeine
use disorder category (Dingle et al., 2008; Silverman et al., 1992; Kendler & Prescott, 1999;
Ogawa & Ueki, 2007; reviewed in Juliano & Griffiths, 2004). The DSM criteria that would be
most applicable to caffeine dependence are the categories of social impairment and
pharmacological indicators. While short-term caffeine use is associated with enhance alertness and
mood, prolonged use of caffeinated products has been associated with stress, anxiety, and
depression (Pettit & DeBarr, 2011; Richards & Smith, 2015). While these relationships are not
causal, caffeine may indirectly impair social activities or obligations by placing mental or
emotional strain on the consumer. A more direct effect of caffeine is the development of tolerance,
a prominent pharmacological indicator listed in the DSM-5. Caffeine tolerance is well-documented
in rodents and humans (Chou et al., 1985; Finn & Holtzman, 1986; Evans & Griffiths, 1992).
6
However, despite the support and recommendation for the addition of caffeine dependence and
withdrawal disorder into the DSM-5, caffeine will not be listed as a substance use disorder on par
with the other established disorders, including the use of alcohol, cannabis, nicotine, etc., without
further research (Hasin et al., 2013).
Though the further investigation of caffeine use and other licit drugs, including nicotine
and alcohol, can be performed in humans, the pervasive use of caffeine worldwide means that it is
difficult to find a naïve population for studying caffeine’s acute motivational effects. Further, as
addiction is primarily mediated by neural mechanisms, research can be more easily performed in
rodent models. Therefore, this present thesis uses a rodent model to study the acute motivational
effects of caffeine as a foundation for future studies on the interdependent effects of caffeine with
other drugs of abuse.
7
1.3 Measurements of motivation
Over the past century, the use of rodent models in neuroscience has increased dramatically.
The two most common types of laboratory rodents – mice (Mus musculus) and rat (Rattus
norvegicus) – share many advantages: they are purposefully bred to reduce biological variation,
their relatively short life cycles and prolific breeding ensure that they remain affordable, they are
well-characterized in numerous studies as bridges between in vitro and in vivo experiments, and
they share homologous traits with humans. Though the mouse is a powerful transgenic model, the
use of rats is more advantageous in neurobiological experiments requiring targeting of specific
brain regions due to their larger brain sizes. Therefore, rats are especially useful to model mental
disorders or diseases common to humans, including the studies of drug addiction presented here.
As there are no complete animal models of drug addiction and studies in people are heavily
constrained by the number of available samples to manipulate and investigate, rodents that model
different aspects of drug addiction remain the best alternative for unraveling its complexities.
A fundamental task of the mammalian brain is to assign motivational valence to stimuli to
ensure an organism’s survival and well-being. Though this process can be affected by any number
of psychological, physiological, or social factors, motivation can nonetheless be quantified with
behavioural paradigms that measure an organism’s response to specific stimuli. Studies focusing
on the neurophysiological aspect of motivation, as in this present thesis, generally require
preclinical models. The standard preclinical behavioural models used to study the motivational
effects of drugs can be largely divided into two major categories with a basis in either operant or
classical conditioning.
8
Operant conditioning (self-administration paradigm)
Operant conditioning (also known as instrumental conditioning) is a learning process that
is characterized by the modification of behaviour through reinforcement or punishment. An action
(or operant) is initially instinctual or spontaneous (e.g. accidentally pressing on a bar when the
animal is freely roaming in an enclosure), but the consequence of that action reinforces or inhibits
the likelihood of the action’s reoccurrence. Early observations by Edward Thorndike (1898) serve
as a basis for our current understanding of this type of animal learning. He measured how long it
took for hungry cats to perform simple actions (including lever pressing, turning buttons, and
pulling on string) to escape from enclosures for a food reward (Thorndike, 1898). He found that
the time it took for the animal to perform the action shortened after numerous trials and generally,
actions that bore fruitful reward were strengthened (reinforced) while those that did not were
weakened. Burrhus Frederic (B.F.) Skinner (often referred to as the father of operant conditioning)
rejected the notion of intangible mental states referenced by his predecessor, Thorndike, in favour
of only observable behaviours and its subsequent observable consequences (reviewed in Skinner,
1950). Skinner’s experiments where he carefully controlled what stimuli the animals were exposed
to and limiting his response criterion to simple repeatable responses, laid the groundwork for the
notion of modifying behaviour. This “behaviour shaping” by delivering rewards and/or
punishments would spur an animal to behave in a desired, and more complex, way (reviewed in
Skinner, 1950).
When an animal performs an observable action and receives a rewarding stimulus, the
action is positively reinforced such that it is more likely to be repeated. The rate or quantity of
response at which the animal performs the action is inferred to be a measurement of how rewarding
9
the stimulus is. This action can be varied widely across experiments and could have animal subjects
pressing a lever or nose-poking for a reward. These rewards can come in many forms – ranging
from access to food or water, pleasurable electrical stimulation to the brain, or administration of a
pleasurable drug. The effects of experimental manipulations can then be performed to determine
its impact on the subjects’ response. These manipulations can include brain lesions,
pharmacological pretreatment with chemical agonists or antagonists, optogenetic activation or
silencing of specific neurons, and many other types of manipulations. Reinforcement schedules
also can be manipulated (Machado, 1989). The reward can simply be provided on a continuous
schedule such that one unit of action (e.g. a nose poke) delivers one unit of reward or be provided
after a constant amount of time (fixed interval) or amount of actions (fixed ratio). The reward can
also be provided on a variable interval or variable ratio schedule such that the amount of time or
actions, respectively, is reinforced at random (Yukl et al., 1972).
The advantage of operant conditioning is that it more closely mirrors the reward-seeking
and reward-using behaviours of people who compulsively take drugs. This self-administering
aspect of operant conditioning makes it suitable for studying the rewarding properties of drugs,
however, it does not come without its limitations. Most troubling, both an increase or decrease in
the rate of self-administration can be interpreted as rewarding. An increase in rate of response
could indicate that the animal finds the drug pleasurable and therefore, increases responding for
more of the drug. A decrease in rate of response could be interpreted as the animal finding each
subsequent drug administration more rewarding and thus, they do not have to respond as often to
reach a similar state. Further, this dependency on the animal engaging in a motor response must
be considered when studying drugs that are known to potentially affect locomotion. For example,
a drug that increases locomotion like caffeine can overestimate the number of positive responses
10
in bar-pressing or nose-poking tests (Kuzmin et al., 2000). Dopamine receptor antagonists that
decrease animal locomotion to near paralysis also would have obvious confounding ramifications
(Vezina & Stewart, 1989). If the subject cannot respond at high dosages of rewarding yet motor-
depressing drugs, then the lack of response could be mistakenly interpreted as unrewarding or even
misconstrued as aversive. Additionally, standard self-administration paradigms generally lack the
ability to sensitively measure aversive responses, as animals tend to avoid performing an action if
it leads to an aversive consequence no matter the severity of the aversion. This can be overcome
by negatively reinforcing a behaviour (i.e. animals learn to perform an action to remove an aversive
consequence) though interpretations of the results from these procedures can be difficult to
generalize, as positive and negative reinforcement may be mediated by different neural substrates
(Namburi et al., 2015). Most important, the reinforcing properties of caffeine are inconsistent in
the self-administration paradigm. Griffiths and Woodson (1988) compared the results of six studies
that attempted to demonstrate caffeine self-administration in non-human primates and found that
three failed to observe caffeine self-administration, while the other three observed erratic patterns
of self-administration.
Therefore, though operant conditioning (and the self-administration paradigm) has been a
great tool for modeling and measuring rewarding properties of drugs, the nature of caffeine and
the paradigm itself make it difficult to study the aversive properties of drugs or drugs that affect
locomotion. These limitations can be circumvented to an extent using classical conditioning
procedures.
11
Classical conditioning (place conditioning paradigm)
Classical conditioning is a learning process that is characterized by the acquisition of a new
behaviour via association between a cue (stimulus) and an innate bodily reflex or instinct. Ivan
Petrovich Pavlov first developed this theory of learning using an audio cue (neutral stimulus or
NS) paired with presentation of food, which is an unconditioned stimulus (US) because it would
elicit a natural and reflexive unconditioned response (UR) of salivation in dogs (Pavlov, 1927).
During conditioning, as the NS and US are repeatedly paired together, the dog learns to associate
the two such that the NS becomes a conditioned stimulus (CS). After conditioning, the presentation
of the CS alone can produce a new conditioned response (CR) of salivation (Pavlov, 1927).
According to Pavlov (1927), “It is pretty evident that under natural conditions that the normal
animal must respond not only to stimuli which themselves bring immediate benefit or harm, but
also to other physical or chemical agencies – waves of sound, light, and the like – which in
themselves only signal the approach of these stimuli…” Therefore, learning by association has
applications in many aspects of an animal’s survival and well-being, including motivated
behaviour in response to drugs. Based on the principles of classical Pavlovian conditioning, the
place conditioning paradigm relies on repeated exposure of a biologically motivating US (e.g. food
or drug) paired with a neutral environment (acting as the NS), such that the animal learns to
associate the previously neutral environment with the subjective effects of the motivating stimulus,
as described in more detail in Figure 1.1.
12
Figure 1.1 - Schematic of the place conditioning paradigm
In the place conditioning paradigm, animals are first habituated to a neutral environment
to reduce novelty effects before conditioning. During conditioning, animals passively receive an
injection of the drug and then are immediately placed in a chamber with distinct features for a
fixed amount of time. For example, in the schematic, the animal is conditioned to associate a black-
walled and smooth-floored chamber with the experience of the drug. On alternate conditioning
sessions, the animal is given an injection of vehicle solution in a different chamber. In the
schematic, the animal is given a vehicle injection in a chamber with white walls and a wire grid
floor. The animal receives the drug- and vehicle-pairing over a fixed number of conditioning
cycles, such that there is equal exposure to both drug- and vehicle-pairings. After which, the
animals are given a period of rest to allow for testing in a drug-free state. During the testing phase,
Drug is rewarding or
Vehicle
Drug is aversive
Drug
1) Habituation 2) Conditioning 3) Testing
13
the animal is given free access to roam in a testing apparatus that has the distinct features of both
conditioning chambers on either end. The amount of time that the animal spends on each side of
the testing apparatus is recorded. If the animal spends relatively more time in the previously drug-
paired side, then the animal exhibits a conditioned place preference (CPP) and we can infer that
the animal finds the drug pleasurable or rewarding. If the animal spends more time in the vehicle-
paired side and avoids the previously drug-paired side, we can infer that the animal finds the drug
aversive in what is termed a conditioned place avoidance (CPA). If the animal spends
approximately equal amounts of time in both sides, then the drug is inferred to be motivationally
neutral (neither rewarding nor aversive).
14
The advantages of place conditioning are numerous. In comparison to the self-
administration paradigm, the major advantage of the place conditioning paradigm is that it allows
for the investigation of the rewarding, aversive, or motivationally neutral properties of drugs in
one testing phase. Because of this, the inference of motivational valence can be compared across
drugs more easily and with good sensitivity. For example, Mucha and colleagues (1982) were able
to use the place conditioning paradigm in rats to compare the preferences or aversions produced
by many drugs, including morphine (and other opiate agonists), naloxone, cocaine, and lithium
chloride. In addition, since the animals are tested in the absence of the drug, the consequences of
the drugs on motor systems can be circumvented. Rather, the impact on locomotion can be
investigated during conditioning (with the appropriate sensory equipment) as an additional
property of the drug of interest.
However, the place conditioning paradigm is not without its criticisms. One criticism is
that the passive administration of drug means that it does not closely mirror the reward-oriented
behaviours in people who seek out drugs intentionally. However, the place conditioning paradigm
has elements of both classical and operant conditioning (reviewed in Huston et al., 2013).
Inadvertent reinforcement of behaviours that are spontaneous in nature, which then increase in
frequency are hallmarks of operant conditioning but could also occur in place conditioning. For
instance, an animal that is again confronted with a place/context (and associated environmental
cues) where it experienced a pleasurable drug may re-engage in behaviours that encourage the
animal to seek out or stay in that particular place. This approach behaviour is analogous to
positively reinforced bar-pressing behaviour in that it is an increased rate of motor response. Along
the same vein, people who abuse drugs may be conditioned to associate certain environmental cues
15
with the experience of drugs, leading to an increase in drug use and abuse. Thus, the place
conditioning paradigm has great value as a tool to investigate the mechanisms behind drug-seeking
behaviour as well as drug relapse that can occur when an individual finds themselves in the place
where they had previously experienced the rewarding effects of drugs. Other criticisms target the
protocol of the paradigm. The requirement of pairing a drug or neutral experience to distinct
environmental contexts means that the animal may have an innate baseline preference for one
environment over the other. As in the present thesis, this concern can be addressed by testing a
separate group of animals in the same cohort as the experimental groups. Scenting the preferred
side with varying percentages of acetic acid can help to balance the two environments.
Additionally, rats can be handled prior to experimentation to reduce stress and novelty effects from
experimenter manipulation.
Therefore, although the place conditioning paradigm has its own advantages and
drawbacks, it can provide a simpler measure of both the rewarding and aversive properties of
drugs. The behavioural studies presented in this thesis utilized fully unbiased and counterbalanced
place conditioning procedures with caffeine as the main drug of interest in rats.
16
1.4 Models and theories of drug motivation
Early demonstrations of rats self-administering electrical stimulation in the brain by James
Olds and Peter Milner (1954) revealed that there was a rewarding circuit in the brain that could be
identified anatomically. Since then, many theories and rodent models of drug addiction have arisen
to investigate the neural mechanisms underlying the motivationally rewarding effects of different
stimuli, including natural rewards (e.g. food and mating) or drug rewards.
The numerous studies on drug addiction gave rise to different theories of drug motivation,
including the mesolimbic reward hypothesis, the error prediction model, the incentive-
sensitization theory, the non-deprived/deprived hypothesis, and the opponent process theory,
which will be discussed below.
The mesolimbic dopamine reward hypothesis
An early proposal for drug addiction that sought to provide a unified theory underlying the
mechanism of how drugs worked to produce their interoceptive rewarding effects was the
“dopamine hypothesis” (reviewed in Wise, 1987; Di Chiara & Imperato, 1988). The dopamine
(DA) hypothesis, elegant in its simplicity, suggested that drugs that led to an increase in the release
of the neurotransmitter, dopamine, would lead to a pleasurable/hedonic effect as experienced by
an individual, thus compelling the user to seek out more of the same drug. Many studies suggested
that a key area responsible for drug and brain stimulation reward was in the medial forebrain
bundle (MFB), which is a neural pathway containing fibres connecting the limbic forebrain and
17
the mesencephalon/midbrain regions (Millhouse, 1969; Gallistel et al., 1985). The MFB also
contains fibres from the mesolimbic dopaminergic (DAergic) brain reward circuit, which mainly
projects from the ventral tegmental area (VTA) DAergic cell bodies in the midbrain to the nucleus
accumbens (NAc) in the ventral striatum. Studies showed that rewarding activation of this
mesolimbic DAergic circuit by drugs of abuse (including ethanol, nicotine, morphine) could also
be disrupted by lesions or pharmacological DAergic antagonism to the same area (Gessa et al.,
1985; Corrigall et al., 1994; Roberts & Koob, 1982).
Figure 1.2 - Schematic of the mesolimbic dopamine system in the rat brain
Schematic illustrating the approximate location of the mesolimbic dopaminergic (DA)
reward pathway in a sagittal section of the rat brain. The mesolimbic pathway is comprised of
DAergic neurons in the midbrain VTA that project to and terminate in the NAc and olfactory
tubercle of the ventral striatum (only the NAc is shown in the schematic for simplicity). One of
the earlier versions of the DA hypothesis posits that these structures compose the final common
pathway for drug reward, although we now know that there are other DA-independent reward
pathways in the brain.
VTA NAc DA
18
Although the hypothesis that an increase in dopamine release is responsible for the
rewarding effects of drugs may be appealing, it cannot account for the complete story of drug
addiction as there is a compelling body of evidence suggesting that DA-independent reward can
also be elicited in rodents (Sturgess et al., 2010; Laviolette et al., 2002; reviewed in Fibiger, 1978).
For example, brain regions that do not have known DAergic innervation (e.g. the tegmental
pedunculopontine nucleus or TPP) were found to mediate the motivationally rewarding effects of
both morphine and amphetamine (Bechara & van der Kooy, 1989).
Tolerance (reduced response to drugs upon repeated exposure) poses another problem to
the DA hypothesis. For instance, the increased presentations of drug lead to decreased DAergic
neuron activation, but drug-seeking may persist. However, as this present thesis focuses on the
acute, and not chronic, effects of caffeine, the mesolimbic DA hypothesis is, nonetheless, the most
relevant theory to this thesis.
In contrast to the simplest version of the mesolimbic DA hypothesis, later work has
revealed that DA is important for mediating signals other than reward. For example, DA mediates
aversive responses to foot-shocks in rats, as evidenced by an increase in DA release following
foot–shock measured using microdialysis by Young and colleagues (1993). DA also has been
found to be an error prediction signal – a model developed and refined by Schultz and colleagues.
19
The error prediction model
Common to the mesolimbic DA reward hypothesis, Wolfram Schultz and colleagues
proposed that an increase in DA release plays a key role in reward. However, in this proposed error
prediction model, phasic DA is initially released in response to any potentially rewarding stimulus
(even if they turn out to be neutral or aversive) and then, evolves to be an established signal for a
rewarding value (Schultz et al., 1993; reviewed in Schultz, 2016). This response could be split into
two temporally dissociable components: the initial detection of a stimulus, followed by the main
valuation component.
Initial detection component
In the initial detection component, a brief activation of DA neurons (demonstrated by an
increase in neuronal impulse frequency) occurs in response to a broad range of stimuli perceived
by any of the sensory modalities that have potential to predict an outcome that may be rewarding,
neutral, or aversive in nature. However, because there exist innumerable stimuli of varying
intensities, not all stimuli are strong enough to evoke this initial activation. Though this activation
appears to be unselective and prone to errors during reward prediction, the probability of activation
to these stimuli can be enhanced by several factors that are related to potential reward availability.
For instance, pairing its presentation with a reward or reducing the number of stimulus
presentations (as the response of DA neurons decrease with repetition) can increase its salience.
Stronger reactions to presentations of novel stimuli prior to interaction also mean that the chances
of missing a potential reward are lower, as any novel stimulus could be a potential reward until its
value is established. For example, in Schultz and colleagues’ experiments with monkeys, they
20
found that unpredicted rewards or conditioned, reward-predicting stimuli were sufficient to induce
DA neuron activation (reviewed in Schultz, 2016). They found that a liquid reward by itself, or a
cue predicting a food reward could induce an increase in DA neuron firing, which is consistent
with the mesolimbic DA hypothesis (Ljungberg et al. 1992; Schultz et al.1993; Mirenowicz &
Schultz, 1996).
Valuation component
The main valuation component temporally evolves from the initial detection of effective
stimuli (reviewed in Schultz, 2016). Conceptually, the transition process from initial detection to
valuation sharpens the unselective detection of a stimulus to more specific identification of the
stimulus and its corresponding value. While the initial detection causes a transient spike in
neuronal activation, the subjective valuation component persists during the animal’s behaviour
until the reward is received. It is difficult to directly measure the subjective value of a stimulus.
Instead, one can offer a choice of two relatively equal rewards that differ by a single factor to find
the relative rewarding value (e.g. an animal choosing orange juice over the same amount of apple
juice can be assumed to subjectively find orange juice more rewarding). The neuronal DAergic
response to a correctly predicted outcome of reward or no reward results in a return to baseline
activity, which indicates that no prediction error had occurred. If a prediction error does occur,
then the second response component reflects the error. For instance, a predicted reward that is
unrewarded, results in depression of DA neuron activity while conversely, an unpredicted reward
that is rewarded results in a spike of DA neuron activation. The importance of this subjective
valuation component is its role in modifying subsequent behaviour. As this main valuation
component occurs within 10 ms, Schultz (2016) proposed that stimulant drugs, which are known
21
to increase DA concentrations, may be prolonging the initial detection component such that DA
surges overlap with the second valuation component, resulting in a false reward value. After
persistent consumption of stimulant drugs, neuronal adaption could result in the erroneous
attribution of a reward value to unrewarded stimuli.
The error prediction model is conceptually appealing – correctly predicted outcomes are
signaled by the lack of DA neuron activation, which indicate that no modification of behaviour is
required, while incorrectly predicted outcomes elicit DA neuron activation or depression that can
modify behaviour via downstream signaling to postsynaptic neurons. However, a direct criticism
of this theory, similar to the mesolimbic DAergic hypothesis, is the evidence for aversive stimulus-
induced DA activation. It can be argued that the removal of an aversive stimulus could be
experienced as rewarding, which would then be signaled by DA neuron activation. However, in
this study, where high intraperitoneal (i.p.) doses of caffeine (eliciting aversion) is experienced by
the animals over a 40-minute conditioning period every other day, it is difficult to determine the
moment when animals would be experiencing ‘rewarding’ relief from caffeine aversion. Another
disadvantage of this theory is that it does not make strong mechanistic predictions about how acute
drug reward experiences can evolve to drug addiction in a subset of individuals. The incentive-
sensitization theory, to be discussed below, partially addresses this concern.
22
The incentive-sensitization theory
The incentive-sensitization theory was first proposed by Berridge, Robinson, and
colleagues (1989, 1993). The primary difference between this theory and the others is that this
theory dissociates the incentive salience or attractiveness of a drug (inducing ‘wanting’) and its
subjective pleasurable/hedonic effects (inducing ‘liking’). Subjective ‘liking’ can be measured by
the taste reactivity test that had been developed by Grill and Norgren (1978) in which they recorded
and assessed videoframes of rat orofacial expressions denoting pleasure (e.g. rhythmic mouth
movements, rhythmic tongue protrusions, and lateral tongue movements) and aversion (e.g.
gaping, chin rubbing, lateral head shake, face washing, paw pushing). In an early experiment,
Berridge and colleagues (1989) lesioned the mesostriatal DA system with 6-hydroxydopamine (6-
OHDA) in rats that had produced hedonic orofacial expressions to sugar reward and found that
taste reactivity did not change. These results suggested that DA mediated the ‘wanting’ component
of reward by imbuing the drug and its associated cues with incentive salience but did not mediate
the ‘liking’ component. Unlike the other theories that assumed that drugs are ‘wanted’ by
individuals because they ‘like’ the pleasurable sensations produced by rewarding drugs, this theory
argues that ‘liking’ a drug does not explain why ‘wanting’ of a drug increases while less pleasure
is derived from repeated drug use (reviewed in Berridge & Robinson, 2016).
The incentive-sensitization theory is useful in that it addresses three fundamental questions
regarding drug craving: why an addict may crave drugs, what could be the underlying mechanisms
of craving drugs, and why do cravings persist after prolonged abstinence. The three items posited
by the incentive-sensitization theory help to address these questions:
23
1) Addictive drugs increase DA neuron activity in the mesotelencephalic (mesostriatum,
mesolimbic, and mesocortical) regions of the brain (Hefco et al., 2003)
2) One consequence of increased DA neuron activity is the attribution of ‘incentive
salience’ (imbuing attractiveness or ‘wanting’) to events that are associated with DA
neuron activation
3) ‘Wanting’ turns into craving due to incremental neuroadaptation, leading to
hypersensitivity of the DAergic neural systems to drugs and its associated stimuli.
However, despite these strong predictions, this theory was criticized for its stance against
the traditional models of drug reward, including the mesolimbic DA hypothesis, because it
suggested that DA was not necessary for a drug’s hedonic effects despite the abundant evidence
suggesting otherwise (Roberts & Koob, 1982; reviewed in Wise, 1987; Di Chiara & Imperato,
1988). However, we now know that DA is not necessary for expression of every type of drug-
seeking behaviour and may play a key role in aversion.
The non-deprived/deprived hypothesis
The non-deprived/deprived hypothesis, developed by van der Kooy and colleagues,
proposes that the brain substrates and mechanisms underlying motivation are dependent on the
motivational state of the animal for both natural (e.g. food) and drug (e.g. opiates) rewards
(Bechara & van der Kooy, 1992; Nader et al., 1994; Nader & van der Kooy, 1997; Laviolette et
al. 2002; Ting-A-Kee R et al., 2009). This hypothesis makes strong predictions on the neural
systems responsible for drug-seeking behaviour, as a function of the animal’s history of drug
exposure.
24
1) Non-deprived animals are in one of three states: drug-naïve, drug-dependent and not in
withdrawal, or recovered from a state of dependence. Upon drug exposure, the expression
of drug-seeking behaviour was found to be dependent on a brainstem region called the
TPP, which is also known as the pedunculopontine tegmental nucleus (PPTg) (Bechara et
al, 1992; Nader & van der Kooy, 1997; Olmstead et al. 1998). For example, in the place
conditioning paradigm, animals that find a drug rewarding would spend more time in the
previously drug-paired compartment. While TPP lesions could reduce the rewarding
effects of the drugs, antagonism of DA had no effect on this drug-seeking behaviour.
2) Deprived animals are drug-dependent and withdrawn. In other words, they have a history
of drug use that renders them addicted to continued drug consumption, such that abstinence
from the drug induces a state of aversive withdrawal. For example, in the place
conditioning paradigm, animals that are drug-dependent and withdrawn tend to avoid the
compartment that was previously paired with the absence of drug (withdrawal). In direct
contrast to non-deprived animals, this behaviour can be blocked by a widespread DA
receptor antagonism but is unaffected by TPP lesions (Bechara et al, 1992).
Derived from the double dissociation outlined above (between the motivational state of the
animals (non-deprived or deprived) and its underlying mechanisms (TPP- or DA-dependent), this
hypothesis suggests that drug history is crucial for our understanding of how the brain processes
motivation. Nader and van der Kooy (1997) showed that this double dissociation could be
reproduced via microinjections of morphine directly into the VTA eliciting reward (indicated by
25
CPP), which could be blocked by TPP lesions or DA antagonism depending on the motivational
state of the animal.
In contrast to the other theories of motivation in which the brains of individuals are
sensitized (the incentive-sensitization theory) or go through incremental and erroneous
neuroadaptation (the error prediction model), the work supporting the non-deprived/deprived
hypothesis demonstrates that drug motivational states are more transient, meaning that switching
between the two motivational states can be accomplished in rodent models (Bechara & van der
Kooy, 1992; reviewed in Bechara et al, 1998). This hypothesis would suggest that clinical
application would have to account for drug history and arguably presents a more complete picture
than other theories of motivation regarding how an individual that has become addicted to drugs
can revert to a state of non-dependence.
Despite the advantages of this hypothesis for explaining opiate motivation, this hypothesis
does not appear to hold true for caffeine motivation. Sturgess and colleagues (2010) lesioned the
TPP of rats that were non-deprived and found that there was no effect on caffeine motivation.
However, it is possible that DA-independent reward exists elsewhere in the brain. Additionally,
this hypothesis does not account for the DAergic rewarding effects of cocaine or amphetamine in
drug-naïve rodents (Hurd et al., 1989; Ventura et al., 2003; Sellings & Clarke, 2003). Further, a
study by Stuber and colleagues (2002) demonstrated that amphetamine reward can be sensitized
after food deprivation in animals that were not drug-deprived. This suggests that the motivational
states of deprived and non-deprived are not limited to the drug being tested, and that any change
in motivational state (e.g. deprived from food) can affect which neural substrates mediate the drug
26
reward. Another limitation of the non-deprived/deprived hypothesis is that the different
motivational states may affect drugs of abuse differently. The opponent process theory, described
next, proposes that motivation can be looked at more holistically as a homeostatic process.
The opponent process theory
First proposed by Solomon and Corbit (1973, 1974), the opponent process theory of
motivation is a counter-adaptive process, wherein an affective (rewarding or aversive) stimulus
generates a corresponding sharp perturbation to the homeostatic state of an animal (the ‘a-
process’), followed by a gradual recruitment of opposing systems that allow for restoration of
homeostasis (the ‘b-process’).
Characteristics of the a-process
The a-process is thought to be a simple, short-acting response that closely follows exposure
to the drug stimulus. An a-process could apply to both reinforcing or aversive stimuli. For example,
the intense pleasure (‘rush’ or ‘high’) following consumption of an opiate drug would reflect a
hedonic a-process, as would the aversion (indicated by CPA) seen in drug-naïve rodents after
nicotine administration (Koob et al., 1989; Grieder et al., 2010).
Characteristics of the b-process
In contrast, b-processes have a longer latency in the opposing direction of the a-process.
The b-process is also slow to build up and slow to decay (Solomon & Corbit, 1974). For rewarding
drugs, this means that the aversive effects of withdrawal (provided that the rewarding drug is not
27
taken continuously without break) would persist even after the rewarding effects of the drug wear
off. The neurobiological mechanisms of the b-process are thought to be the manifestation of a
combination of two factors: the decrease in function of the brain’s reward system and the
recruitment of the brain’s stress or anti-reward system (Koob & Le Moal, 2005; Koob & Le Moal,
2008a; Koob & Le Moal, 2008b).
Figure 1.3 - Schematic illustrating the opponent process theory of motivation
The opponent process theory proposes that any affective (rewarding or aversive) stimulus
would generate a corresponding a-process of fast onset and short duration. It would then be
followed by a counter-adaptive b-process (anti-rewarding or anti-aversive) of later onset and
longer duration to gradually restore the homeostatic state of the animal. This figure was adapted
from Grieder et al., 2010.
Reward (+) a-process
Anti-reward (-) b-process
Time → c
Aversion (-) a-process
Anti-aversion (+) b-process
Time → c
Response to a rewarding stimulus Response to an aversive stimulus
Adapted from Grieder et al., 2010
28
Building upon the work of Solomon and Corbit (1973, 1974), Koob, Le Moal, and
colleagues (reviewed in Koob & Bloom, 1988; Koob & Le Moal, 2001; Koob & Le Moal, 2005;
Koob & Le Moal, 2008a; Koob & Le Moal, 2008b) expanded the opponent process theory to an
allostatic model of the brain motivational system. Koob and Le Moal (2001) hypothesized that
infrequent drug use would allow for sufficient time for individuals to experience the hedonic a-
process, followed by the counter-adaptive b-process to appropriately restore a homeostatic state.
Upon repeated drug use, the b-process is unable to restore homeostasis before each repeated drug-
taking session, leaving the individual in an allostatic state of drug dependence. Therefore, Koob &
Le Moal (2008a) had conceptualized drug addiction as a cycle of impulsive (early stage) and
compulsive (late stage) drug-taking, comprised of three stages:
1) Binge/intoxication, in which the pleasant effects of the drug lead to a positively
reinforced increase in drug intake and tolerance;
2) Withdrawal/negative affect, characterized by the emergence of an aversive emotional
state following the loss of drug euphoria, leading to negatively reinforced drug intake
to reduce the aversive state;
3) Preoccupation/anticipation (craving), defined as “the memory of the rewarding
effects of a drug superimposed upon a negative state,” which is hypothesized to be the
key factor in chronic relapse and reversion to the binge/intoxication stage of drug
addiction.
29
Our lab has also refined and applied the opponent process theory to rodent models of acute
and chronic nicotine substance use disorders (Grieder et al., 2010; Grieder et al., 2014). Notably,
Grieder and colleagues (2014) demonstrated a direct link between the VTA reward system and the
brain’s stress system by identifying a population of VTA DAergic neurons in both rodents and
humans that express corticotropin-releasing factor, a peptide hormone involved in the stress
response.
Although the opponent process theory accounts for both the hedonic/aversive effects of
drugs and subsequent restoration to baseline, it is not without its criticisms. For instance, the b-
process is unlikely to require months to build up and decay, yet individuals who have ceased
taking drugs for months, or even years, may relapse (Brown et al., 1989; Xie et al., 2005).
30
To briefly summarize the theories discussed above,
1) The mesolimbic DA hypothesis proposes that increased DA signals hedonic reward and its
acquisition (e.g. reviewed in Wise, 1987; Di Chiara & Imperato, 1988; Roberts & Koob,
1982).
2) The error prediction model proposes that DA signals are split into two components, in
which the initial detection of unselective stimuli evolve to a subjective valuation
component. Overlapping DA activity may falsely signal reward, leading to erroneous
neuroadaptation (proposed by Schultz et al. 1993).
3) The incentive-sensitization theory proposes that increased DA signals the subjective
reward attractiveness (or ‘wanting’). Subsequent neuroadaptation to drugs and associated
stimuli could lead to hypersensitivity to drugs transforming the ‘wanting’ into a drug
craving (proposed by Robinson & Berridge, 1993)
4) The non-deprived/deprived hypothesis proposes that the neurobiological mechanisms
underlying drug addiction and its development is dependent on the motivational state of
the animal (proposed by van der Kooy and colleagues. e.g. Bechara & van der Kooy, 1992).
5) The opponent process theory proposes that addictive drugs perturb the homeostatic state of
the animal, followed by a more gradual counter-adaptive process that restores homeostasis.
Drug dependence occurs as a cycle of impulsive and compulsive drug-taking such that an
allostatic state persists (proposed by Solomon & Corbit, 1973, and later refined by Koob
& Le Moal, 2001).
31
While none of these theories can account for the complete picture of drug addiction, each
of these theories target different facets of drug addiction and can be thought of as complementary.
For example, as individuals experience drugs for the first time (i.e. in a non-deprived state), both
DA-dependent and DA-independent mechanisms (e.g. through the TPP) could simultaneously
signal to any number of separate downstream pathways, indicating the physical acquisition of the
reward, the attractiveness of the reward, the context in which the reward was received, the reward’s
subjective value, etc. This perturbation of the motivational systems, having also widely activated
other subsequent pathways, could lead to a counter-adaptive negative feedback system that restores
homeostasis. As individuals persist in their consumption of addictive drugs and become dependent,
incremental neuroadaptation could conceivably occur such that the DAergic neural system
becomes hypersensitive to drugs and associated stimuli. Other brain regions could also be recruited
to mediate the rewarding or aversive experience of drugs. The neural systems mediating a
dependent and withdrawn (deprived) state would, then, be different than those that mediate a non-
deprived motivational state.
32
1.5 Neuroanatomy of motivation
Although the theories described previously have many contradictions, the DAergic neurons
have a significant role that cannot be discounted. As the VTA contains cell group A10 (the largest
group of DAergic neurons in rodents and primates), it has been heavily implicated as a key reward
processing centre in the brain. As the results of caffeine reward in this thesis project aligns closely
with the mesolimbic DAergic hypothesis, a description of the anatomy of the VTA and NAc will
follow.
Anatomy of the ventral tegmental area
The VTA, also known as the VTA of Tsai, is a component of the medial forebrain bundle,
that passes through the lateral hypothalamus and the basal forebrain (Veening et al., 1982).
Anatomically, the VTA is a group of neurons located near the midline on the floor of the midbrain.
It is bordered rostrally by the mammillary bodies and the posterior hypothalamus, and caudally by
the pons and hindbrain (reviewed in Oades & Halliday, 1987). The VTA contains a large
population of DA and γ-aminobutyric acid (GABA) neurons in the brain, with emerging evidence
suggesting that there are glutamatergic neurons as well (Yamaguchi et al., 2007). DAergic and
GABAergic cells have been traditionally identified through various methods (of which some are
listed below in Table 1), including immunohistochemistry, electrophysiology, and pharmacology.
For example, Steffensen and colleagues (1998) found that GABA neurons in the VTA can be
distinguished from DAergic neurons by their rapid-firing, non-bursting activity (~19 Hz), short
duration action potentials (~310 µsec), and small action potentials (~68 mV). However, identifying
DA neurons alone through electrophysiological means has proven more difficult. Margolis and
33
colleagues (2006) measured widely accepted electrophysiological criteria for DA neuron activity,
including spike duration and spontaneous firing rate, and concluded that these criteria are not
reliable. Generally, DAergic and GABAergic neurons are distinguished in the VTA based on
immunohistochemical staining. DAergic neurons can be stained for tyrosine hydroxylase (TH),
which is the rate-limiting enzyme of catecholamine biosynthesis (Olson & Nestler, 2007). Some
receptor types located on the VTA DAergic neurons are D1, D2, GABAA, GABAB, N-methyl-D-
aspartate (NMDA), and cholinergic (Westerink et al., 1996). GABAergic neurons can be stained
for glutamic acid decarboxylase (GAD), which is the rate-limiting enzyme in the conversion of
glutamate (Glu) to GABA (Olson & Nestler, 2007).
34
Table 1 - Estimated percentage of cell types in the VTA
DA GABA Glu Co-labelled Species Method Source
- - ~5% - Rat VGluT2 radioactive
antisense riboprobes
Yamaguchi
et al., 2007
~66% ~35% ~2-3% ~0.58% GABA
(GAD) /DA (TH) Rat
Immunohistochemistry
(TH) and in situ
hybridization (GAD and
VGlut2 mRNA)
Nair-
Roberts et
al., 2008
~39
to
~72%
- - - Rat Immunohistochemistry
(TH)
Margolis et
al., 2006
35
The VTA is a highly heterogenous region of the midbrain proposed to have four or five
subdivisions (reviewed in Ikemoto, 2007 and Sanchez-Catalan et al., 2014). In an early
cytoarchitectural definition, the lateral subdivisions contained two nuclei – the paranigral nucleus
and the parabrachial pigmented nucleus (also known as the parabrachial pigmentosus nucleus).
The medial subdivisions included three nuclei – the interfascicular nucleus, the rostral linear
nucleus, and caudal linear nucleus. However, as the three medial subdivisions have been proposed
to belong to the raphe nuclei instead of the VTA, Ikemoto (2007) proposed that the VTA has only
four lateral subdivisions: the paranigral nucleus, the parabrachial pigmented nucleus, the
parafascicular retroflexus area, and the tail of the VTA. Additionally, there is rostrocaudal
heterogeneity in the VTA (reviewed in Sanchez-Catalan et al., 2014), however this will be
discussed further in the thesis discussion in conjunction with the experimental results.
Because of the heterogeneity of the VTA, the input and output projections of the VTA have
been investigated by many research groups (Beckstead et al., 1979; Swanson, 1982; Geisler &
Zahm, 2005; Poller et al., 2013; Beier et al., 2015). Selected projections to and from the VTA are
outlined in Table 2 and Figure 1.4).
36
Table 2 - Projections to and from the VTA
Afferent (inputs) Efferent (outputs)
Glutamatergic
• Lateral Hypothalamus°
• Lateral Habenula-
• Prefrontal cortex@
• Medial forebrain bundle&
• Fasciculus retroflexus&
GABAergic
• Nucleus accumbens$
• Ventral pallidum$
• Diagonal band of Broca$
• Rostromedial tegmental nucleus/Tegmental pedunculopontine nucleus!
Telencephalon
• Nucleus accumbens*,#
• Anterior limbic cortex#
• Lateral septal nucleus*,#
• Amygdala*,#
• Entorhinal Area*
• Hippocampal formation#,~
• Cingulate gyrus^
• Prefrontal cortex^
• Dorsal striatum+
Diencephalon
• Lateral Habenula*,#,°
• Thalamus*
Brain stem
• Parabrachial nucleus#
• Locus coeruleus*,#
+ Balleine et al., 2007
* Beckstead et al., 1979 - Beier et al., 2015 @ Carr & Sesack, 2000
~ Gasbarri et al, 1994 & Geisler & Zahm, 2005 ! Jhou et al., 2009a; Jhou et al., 2009b $ Kalivas et al., 1993 ^ Loughlin & Fallon, 1984 ° Poller et al., 2013 # Swanson, 1982
37
Figure 1.4 - Selected input and output projections of the VTA
Schematic illustrating selected input and output projections to and from the VTA based on
information listed in Table 1 and Table 2. Intra-VTA circuitry shown in more detail with three cell
type populations (DA, GABA, and Glu). Anterior cingulate cortex (ACC), dorsal striatum (DStr),
ventral pallidum (VP), globus pallidus (GP), lateral hypothalamus (LH), lateral habenula (LHb),
dorsal raphe (DR), medial prefrontal cortex (mPFC), central amygdala (CeA), tegmental
pedunculopontine nucleus (TPP), and nucleus accumbens (NAc).
38
Anatomy of the nucleus accumbens
The NAc is a brain region that is comprised of >90% GABAergic medium spiny neurons
(MSNs) (reviewed in Kourrich et al., 2015). The NAc receives a major input from the DAergic
projections of the VTA with mainly non-overlapping medial-lateral topography (Phillipson &
Griffiths, 1985). Aside from the DAergic inputs from the VTA, the NAc also receives excitatory
glutamatergic inputs from the prefrontal cortex (PFC), hippocampus, and amygdala (reviewed in
Kourrich et al., 2015). The NAc projects to the globus pallidus, dorsal striatum, bed nucleus of the
stria terminalis, septum, preoptic region, thalamus, lateral habenula, and ventral pallidum (Nauta
et al., 1978).
Neuroanatomically, the NAc can be divided into three sub-regions: the lateral NAc shell,
the medial NAc shell, and the lateral NAc core. For the purposes of this thesis project, the NAc
shell notation refers to the medial NAc shell only (as demonstrated by the verification of cannula
placements shown in Figure 3.3). The NAc shell and core also have differential inputs and outputs:
the NAc shell shares afferents with the extended amygdala but has stronger outputs towards the
cortex, while the NAc core is related more closely to the caudate-putamen (nigrostriatal system)
and is thought to be more involved in locomotion (Zahm, 1999; Deutch & Cameron, 1992). More
specifically, although both NAc subregions are reciprocally connected to the VTA, the shell
strongly projects to the ventromedial part of the subcommissural VP and the pre-optic area/lateral
hypothalamus, while the core projects to the dorsolateral VP and the medial substantia nigra (Zahm
& Heimer, 1990; Heimer et al., 1991; Zahm & Heimer, 1993; Zahm, 1999). Anterograde tracing
performed by Zahm and colleagues (1993) also showed that the rostral pole of the NAc has
differing connectivity: the lateral NAc core-like area projects to the GP, VP, lateral VTA, dorsal
39
pars compacta, entopeduncular nucleus, LH, lateral mesencephalic tegmentum, and central grey,
while the NAc shell-like area projects to the VP, the lateral preoptic regions, LH, VTA, dorsal-
most pars compacta, retrorubral field, and central grey. To distinguish the NAc shell and core,
different methods can be used. For example, baseline concentrations of DA are greater in the NAc
shell than the NAc core (Deutch & Cameron, 1992). Immunoreactivity to substance P
demonstrates NAc core, while calbindin 28 kD demonstrates NAc shell (Zahm & Heimer, 1993).
Figure 1.5 - Schematic of the nucleus accumbens shell and core
Schematic illustrating the approximate location of the NAc core and shell in a coronal
section of the rat brain. The NAc core is bordered by the medial and lateral components of the
NAc shell.
Medial shell
Lateral shell
Lateral core
40
Although the NAc shell and core sub-regions have been established as separate
subdivisions for neuroanatomical reasons, these sub-regions have received increased attention due
to their functional differences in mediating the motivational properties of drug stimuli, including
alcohol, cocaine, morphine and, amphetamine (Pontieri et al., 1995; Ito et al. 2004; Chaudhri et
al., 2010). For example, intravenous cocaine, morphine, and amphetamine preferentially increased
DA in the NAc shell over the NAc core in the rat (Pontieri et al., 1995). Additionally, 6-OHDA
lesions of the NAc shell reduced nicotine conditioned place preferences, while lesions of the NAc
core abolished nicotine conditioned taste aversions (Sellings et al., 2008). The present thesis also
finds separable roles in the two sub-regions mediating the opposing motivational effects of
caffeine.
41
1.6 Neurobiology of caffeine motivation
Introduction to caffeine consumption
The history of caffeine use has firm roots in mythology. From Islamic mythology, it is said
that the archangel Gabriel offered the prophet Muhammad a “heavenly brew” to help him
overcome sleepiness. This “brew” was a cup of coffee so potent that just one sip would enable him
to “unhorse 40 men and make 40 women happy.” Though some of the myths are rather outlandish,
they all share a common feature where caffeine was found to have stimulating properties
(Fredholm, 2011). Today, caffeine is the most extensively consumed psychoactive drug in the
world. It is readily available in food, drugs, dietary supplements, and beverages (Fredholm et al.,
1999; Persad, 2011).
As a central nervous system (CNS) stimulant, caffeine has a broad range of effects on the
brain including increased arousal, improved mood, and heightened attention (Lazarus et al., 2011;
Smith, 2009; Brice & Smith, 2002; Lorist et al., 1996). The bulk of these effects have been
attributed to its role as a competitive adenosine receptor antagonist (Lazarus et al., 2011; Higgins
et al., 2007, Fredholm et al.,1999). Previous work has further suggested that caffeine’s
motivationally rewarding or aversive effects also could be acting through antagonism of the
adenosine receptors in the brain, more specifically the A1 receptors (A1Rs) located throughout the
brain and A2A receptors (A2ARs) located mainly in the ventral striatum (Fredholm et al., 1999).
However, our group has demonstrated that the motivational effects of caffeine demonstrated in a
place conditioning paradigm is spared in mouse knockouts (KOs) of A1Rs and/or A2ARs (Sturgess
et al., 2010). A1R KO, A2AR KO, and double KO mice showed similar aversion to wild-type (WT)
42
C57BL/6 mice after caffeine (10 mg/kg i.p.) administration in a place conditioning paradigm. This
indicates that neither the two adenosine receptors individually nor in tandem are necessary for the
motivational effects of caffeine under those conditions. Rather, the DAergic receptors are more
likely to be critical for the motivated response to caffeine as pharmacological blockade of DAergic
receptors was sufficient to block or reduce caffeine motivation (Sturgess et al., 2010). Of note, a
radioligand competitive binding assay conducted by Watanabe and Uramoto (1986) demonstrated
that caffeine does not bind directly to DA receptors, yet their measures of DA stimulation showed
that caffeine was able to mimic the activity of a DA agonist.
The mechanisms and pathways involved in the midbrain reward circuitry have mainly been
investigated through its response after administration of strong stimulants, including amphetamine
and cocaine (Ashok et al., 2017; Ito & Hayen, 2011). Though less than 2% of the general
population are direct consumers, the motivational effects of these highly addictive drugs are
predictable upon consumption (Ashok et al., 2017). For example, acute and short-term
administration of cocaine is generally self-reported to be followed by euphoria, increased
confidence, and mental alertness (Walsh et al., 2009; van der Poel et al., 2009). Conversely,
approximately 90% of people consume caffeine with a large variance in global consumption
practices, making it more suitable for investigating the opposing rewarding and aversive
behaviours possibly mediated by the mesolimbic pathway (Fredholm et al., 1999).
43
1.7 Research hypotheses and objectives
The mesolimbic DAergic projection – from the VTA to the NAc – has received great
attention as the reward centre of the mammalian brain with mounting evidence for its involvement
in processing aversive stimuli (Sellings et al., 2008; Lammel et al., 2012; Kim et al., 2004;
Carlezon & Thomas, 2009). How the same area can elicit opposing approach and avoidance
behaviours has been poorly described for caffeine. The downstream NAc is composed of the
anatomically and functionally dissociable medial shell and lateral core subregions; these
subregions provide one conceivable way by which information encoding approach and avoidance
behavior can be separated.
Therefore, it was hypothesized that the separation of the downstream NAc into the shell
and core subregions could provide a way for information encoding caffeine reward and aversion
as these two contending behaviours can be elicited by other drugs, including nicotine and
amphetamine (Sellings et al., 2008; Ito & Hayen, 2011).
Therefore, the overarching objectives of this project were to:
1) Characterize a reliable rodent model of caffeine reward and aversion using the conditioned
place preference paradigm as a basis
2) Target the mesolimbic DAergic pathway with DA receptor antagonists to see which brain
regions were necessary for expression of caffeine reward and aversion
44
Objective 1: Characterizing a rodent model of caffeine reward and aversion
In this study, I generated a dose-response curve for systemic caffeine ranging from 1 mg/kg
to 60 mg/kg i.p. in rats using the place conditioning paradigm. Systemic caffeine was found to
elicit reward (indicated by CPP) at a dosage of 2 mg/kg i.p., which could be blocked by prior
systemic treatment of α-flupenthixol (α-flu) i.p., suggesting that the behavioural expression of
caffeine reward was dependent on DA transmission. Of note, injections of caffeine (100 µM)
directly into the rostral VTA was sufficient to induce a comparable CPP magnitude. Systemic
caffeine also was found to elicit aversion (indicated by CPA) at tested doses equal or greater than
10 mg/kg i.p, but less than 60 mg/kg i.p. Caffeine aversion could also be blocked by prior systemic
treatment of α-flu i.p., suggesting that the behavioural expression of caffeine aversion was also
dependent on DA transmission. Of note, injections of caffeine (1000 µM) into the caudal VTA
produced aversion, which did not reach statistical significance. However, combined with the
aversion seen in the high dose systemic caffeine aversion, it suggests that testing higher
concentrations in future studies could potentially yield an expression of aversion of comparable
magnitude to systemic caffeine aversion.
Objective 2: Investigating the brain regions responsible for caffeine reward and aversion
Given the essential role that the VTA plays in both drug reward and aversion, I sought to
investigate the effects of targeting the VTA with a broad-spectrum DA receptor antagonist by
applying it to the rodent model of caffeine and aversion characterized under the first objective. I
found that direct injections to the downstream targets of the VTA projections – the NAc shell and
core – could separately block caffeine reward and aversion, respectively. Notably, DA antagonism
45
of the NAc shell had no effect on caffeine aversion and DA antagonism of the NAc core had no
effect on caffeine reward. This suggests that the two caffeine pathways are separately mediated by
the two sub-regions of the NAc.
46
Chapter 2 - Materials and Methods
47
2.1 Animal subjects and drugs
Subjects
All animals were handled in accordance with the regulations and guidelines of the
University of Toronto Animal Care Committee, the Animals for Research Act in Ontario, and the
Canadian Council on Animal Care. Adult male Wistar rats (weighing 250-350 g) were purchased
from Charles River (Montreal, Canada) and maintained in the animal facilities of the University
of Toronto Division of Comparative Medicine. Female rats were not used due to sex differences
(e.g. of the gonadal hormones) that have been demonstrated to impact drug-seeking behaviors in
the place conditioning paradigm (Russo et al., 2003). The rats were double-housed with
environmental enrichments (plastic tunnel, wooden block, and chew toy) in clear Plexiglas cages
at a constant temperature of 22°C on a controlled 12-hour light/dark cycle (lights on at 7am). Rats
were given ad libitum access to food and water. Rats did not receive any drugs prior to the first
day of conditioning.
Drugs
Caffeine (C0750, Sigma-Aldrich) was dissolved in warm saline (pH adjusted to 7.4). A
dose-response curve for the motivational effects of i.p. caffeine injections was generated in the
present study with doses ranging from 1 mg/kg to 60 mg/kg. Bilateral intracranial (i.c.) injections
of caffeine (100 µM) were performed through surgically implanted cannulas (Plastics One)
targeting the VTA at a total volume of 0.5 µl over 1 minute. The injector tip was kept in place for
an additional minute to ensure complete diffusion of the drug from the tip of the injector. The
48
caffeine analog, 8-(p-sulfophenyl)theophylline (8-SPT, sc-217511, Santa Cruz), was dissolved in
warm saline and administered via i.p injections at a dose of 10 mg/kg only. The DA receptor
antagonist, α-flupenthixol (α-flu, F114, Sigma-Aldrich), was dissolved in saline and administered
as a pretreatment via i.p. injections (0.8 mg/kg, 2.5 hours prior to conditioning) or bilateral i.c.
injections (3 µg/0.5 µl per hemisphere in the NAc, 15 minutes prior to conditioning). The doses
and timings of injection were selected based on previous studies showing that α-flu could
antagonize postsynaptic DA (both D1 and D2 subtypes) receptors (Creese et al., 1976), but without
producing any motivational effects of its own (Laviolette & van der Kooy, 2003).
49
2.2 Experimental procedures
Surgical cannulation
To target specific brain regions, rats were surgically cannulated under anesthesia (2-5%
isoflurane) and injected with analgesic subcutaneously (ketoprofen, 5 mg/kg s.c.). Rats were
shaved and then placed in a stereotaxic frame for craniotomy. Small burr holes were drilled into
the skull based on coordinates (in mm from Bregma) for the rostral VTA (AP: -5.3, ML: ± 0.5,
and DV: -7.6), NAc shell: +1.3, ML: ±1.0, and DV:-7.4) or NAc core (AP:+1.3, ML:± 2.4, and
DV:- 7.4) (Olson et al., 2005; Paxinos & Watson, 2005; Olson & Nestler, 2007). Stainless steel
guide cannulas (C313G(2)-G11/SP, Plastics One) were fixed permanently into position using
dental acrylic and jeweler’s screws (0-80 X 3/32, Plastics One) implanted in the skull, with the
tips positioned 1 mm above the target brain region. Removable dummy cannulas (C313DC/1/SPC,
Plastics One) were inserted into the guide cannulas to prevent occlusion over the course of surgical
recovery and behavioral experiments. Rats were given at least one week to recover from their
surgeries before further experimentation. Following which, removable internal cannulas
(C313I/SPC, Plastics One) were inserted into the guide cannulas with the tips of the internal
cannulas extending 1 mm ventral to the guide cannula tips during conditioning.
Place conditioning
Prior to place conditioning, the rats were habituated for 20 minutes in a neutral, grey
chamber (41 cm x 41 cm x 38 cm box) to reduce novelty effects. Rats that had received surgical
cannulations received i.c. injections of saline vehicle on the following day to habituate the rats to
50
i.c. injections. The rats then underwent an unbiased place conditioning procedure, which was
counterbalanced in terms of drug-place pairing and day of first drug exposure. Conditioning
alternated between two place conditioning boxes (41 cm x 41 cm x 38 cm boxes) that were distinct
in colour, floor texture, and scent. One box had black walls, a smooth black Plexiglas floor, and
was scented with ~0.3 mL of glacial acetic acid (AX0073, EMD) solution. The other box had white
walls, a metal grid wire floor over a smooth metallic surface, and remained unscented. The amount
of glacial acetic acid was determined by testing a separate group of rats from the same cohort to
ensure that there was no initial baseline preference. During the conditioning period, rats were
pretreated with α-flu or vehicle solution (administered i.p. or i.c. where appropriate). Following
pretreatment, caffeine was administered to the rats i.p. or i.c. immediately before exposure to a
conditioning box for 40 minutes, which is a sufficient period to capture peak caffeine levels in the
brain following i.p. injections (Latini et al., 1978; Lukas et al., 1971). The drug and vehicle
solutions were administered on alternate days over a period of 8 days for a total of 4 drug-place
pairings and 4 vehicle-place pairings. At least 48 hours following the final conditioning session,
rats were tested in a rectangular testing box consisting of two compartments with features identical
to the conditioning boxes separated by a neutral, grey zone. On testing day, rats were placed on
the centre grey zone and allowed to roam freely in the testing box for 10 minutes. All rats were
tested in a drug-free state. The amount of time spent on each side of the testing box was recorded.
A greater amount of time spent in the previously drug-paired compartment indicates a conditioned
place preference, while a greater amount of time spent in the previously vehicle-paired side
indicates a conditioned place aversion.
51
2.3 Histology
Verification of cannula placements
Following testing, rats were deeply anesthetized with 54 mg/kg i.p. of sodium pentobarbital
(Ceva Santé Animale). Rats were perfused with ~100ml of phosphate-buffered saline (PBS),
followed by ~100ml of 4% paraformaldehyde (PFA, P6148, Sigma-Aldrich) dissolved in PBS.
Brains were removed and preserved in 4% PFA solution overnight, and then transferred to a 30%
sucrose (BioShop) solution stored at 4°C. Coronal sections of flash-frozen brains were cut at a
thickness of 40 µM with a freezing microtome (Cryostat model Hm525 NX, Thermo Scientific).
The sectioned slices were mounted on slides and stained with Cresyl violet (C5042, Sigma-
Aldrich). The slides were viewed under a light microscope to verify the bilateral cannula
placements in the NAc and VTA.
52
2.4 Statistical Methods
Statistical analysis
For the systemic caffeine dose-response curve, a two-way analysis of variance (ANOVA)
followed by post hoc Bonferroni-Dunn multiple comparison tests were conducted using GraphPad
Prism. Comparisons of the mean difference scores to zero were conducted with one-sample t-tests
with p-values adjusted using the Bonferroni correction. Results are displayed as mean difference
scores (time spent in caffeine-paired minus vehicle paired environment) ± the standard error of the
means (SEMs). A two-way ANOVA followed by post hoc Bonferroni-Dunn multiple comparison
tests were conducted for the experiment comparing the effects of 8-SPT and caffeine, with results
displayed as the mean amount of time that animals spent on each side of the testing apparatus ±
SEM. For the experiments comparing intra-VTA injections with 100 µM and 1000 µM of caffeine,
three-way ANOVAs (Type III tests) were conducted using R statistical software, followed by post
hoc Bonferroni-Dunn multiple comparison tests. Results are displayed as the mean amount of time
that animals spent on each side of the testing apparatus ± SEM. Two-way ANOVAs followed by
post hoc Bonferroni-Dunn multiple comparison tests also were conducted using GraphPad Prism
for the experiments comparing the effects of vehicle and α-flu in the NAc core and in the NAc
shell, with results displayed as the mean amount of time that animals spent on each side of the
testing apparatus ± SEM. Animals with mistargeted cannula placements (e.g. mistargeted in one
hemisphere and complete misses, n = 8 in total) were excluded from statistical analyses.
Differences were considered significant if the p-value < 0.05.
53
Chapter 3 - Results
54
3.1 Systemic caffeine dose-response curve
Systemic caffeine elicits reward at low doses and centrally-mediated aversion at higher doses
To examine the behavioral response to systemic caffeine in male rats, a place conditioning
paradigm was used (Figure 3.1a). First, a separate group of adult male rats were tested for baseline
bias towards either conditioning box type to ensure that changes in preference were due only to
drug-place conditioning (Figure 3.1b). Following this, a dose-response curve for systemic i.p.
injections of caffeine using doses ranging from 1 mg/kg to 60 mg/kg was generated (Figure 3.1c).
A two-way ANOVA indicated that there was an interaction between caffeine dose and
pretreatment, confirming that α-flu pretreatment can disrupt the motivational effects of caffeine
(Figure 3.1c). A low dose of caffeine (2 mg/kg i.p.) produced conditioned place preferences, while
caffeine doses of 10 and 30 mg/kg i.p. produced conditioned place aversions (Figure 3.1c). The
difference scores between the time that the animals spent in the caffeine-paired side minus the
saline-paired side at doses of 10 and 30 mg/kg i.p. were not significantly different (Figure 3.1c).
Therefore, the dose of 10 mg/kg i.p. was used for the remainder of this study when examining the
aversive effects of caffeine.
We hypothesized that caffeine aversions would diminish in response to DA receptor
antagonism based on our previous work showing this to be the case in mice (Sturgess et al., 2010).
Comparable to the results seen in mice, a DA receptor antagonist (α-flu, 0.8 mg/kg i.p.) blocked
the conditioned place aversions produced by 10 and 30 mg/kg i.p of caffeine in rats (Figure 3.1c).
The pretreatment with α-flu also blocked the conditioned place preferences when a low dose of 2
55
mg/kg i.p. of caffeine was administered (Figure 3.1c). Thus, acute caffeine reward and aversion in
rats as elicited by systemic administrations of low and high doses, respectively, are dependent on
DAergic transmission.
Next, we asked whether the aversive effects of high doses of systemic caffeine were solely
mediated by the CNS or if caffeine-responsive adenosine receptors in the peripheral nervous
system also contributed. A quaternary caffeine analog (8-SPT) that is unable to cross the blood-
brain barrier was administered at 10 mg/kg i.p. in an identical place conditioning paradigm as the
systemic caffeine experiment described above. Despite 8-SPT having greater binding affinity to
adenosine receptors than theophylline, a methylxanthine with similar binding properties to
caffeine (Tao & Abdel-Rahman, 1993), there was no significant difference in the time that the rats
spent in the vehicle- or 8-SPT-paired chambers at testing, suggesting that peripheral activation of
adenosine receptors alone at 10 mg/kg i.p. does not contribute to the behavioral expression of
caffeine aversions (Figure 3.1d).
56
A)
B)
C)
*
* *
57
*
D)
Figure 3.1 - Systemic caffeine produces DA-dependent rewarding effects at low doses, and
DA-dependent aversive effects at high doses that are centrally mediated
a) Schematic of place conditioning paradigm and timeline.
b) Initial preference for each side of the testing apparatus was found to be similar in a separately
tested group of animals (t-test, p = 0.7795, n = 12). These animals only received saline injections
during the conditioning phase. Bars represent mean amount of time animals spent on each side of
the testing apparatus ± SEM.
c) In the systemic caffeine dose-response curve, a two-way ANOVA revealed a significant dose
by pretreatment interaction, confirming that α-flu pretreatment (gray bars) disrupted the
motivational effects of caffeine (F4,109 = 7.990, p = 0.0001). At a dose of 2 mg/kg i.p of caffeine,
multiple comparison tests (Bonferroni) of the mean difference scores to zero indicated that the
non-pretreated animals (black bars) experienced a significant caffeine reward (p = 0.001, n = 13),
58
which was blocked by α-flu pretreatment (p = 0.9999, n = 13). At 10 mg/kg i.p. (p = 0.001, n =
24) and 30 mg/kg i.p. (p = 0.001, n = 11), caffeine produced significant aversions, which were not
significantly different from each other (p = 0.9999). These aversions could also be blocked by α-
flu pretreatment (10mg/kg: p = 0.001, n = 24; 30mg/kg: p = 0.001, n = 11). Bars represent
difference scores between the times animals spent in the caffeine-paired side minus vehicle-paired
side in the testing apparatus ± SEMs. * indicates p < 0.05 for comparisons of mean difference
scores compared to zero.
d) Animals treated with caffeine (10mg/kg i.p.) showed aversions that were not recapitulated by
the administration of 8-SPT (10 mg/kg i.p.), as indicated by an interaction between the type of
drug administered (caffeine or 8-SPT) and drug-pairing to each side of the testing apparatus (two-
way ANOVA, F1,26 = 20.1, p = 0.0001). In the caffeine group, animals spent more time in the
vehicle-paired side compared to the drug-paired side (p = 0.0002, n = 8), indicating an aversion to
caffeine. In the 8-SPT group, the amount of time that the animals spent in each side was not
significantly different (p = 0.8485, n = 7). Bars represent mean amount of time animals spent on
each side of the testing apparatus ± SEMs. * indicates a significant difference between the time
spent in the vehicle-paired and drug-paired sides of the testing apparatus (p < 0.05)
59
3.2 Intra-rVTA caffeine reward mediated through NAc shell
Caffeine injections into the rostral VTA elicit reward
To determine where caffeine motivation may be mediated within the brain, the VTA was
chosen for its well-established role as part of a drug motivation circuit (Nader & van der Kooy,
1997; Laviolette & van der Kooy, 2003; Nestler, 2005). Caffeine was directly injected into the rat
VTA immediately preceding place conditioning. Preliminary results suggested that the rostral, but
not caudal VTA, produced reward so these regions were targeted separately. Rostral VTA (rVTA)
and caudal VTA (cVTA) were defined as in Olson and colleagues’ study (2005) showing that there
is an inflection point between -5.5mm and -5.6 mm Bregma where cocaine is found to be rewarding
rostrally and aversive caudally. This rVTA area is defined to include the anatomical VTA
subregions, composed of the rostral subregion and the rostral portion of the parabrachialis
pigmentosus (Paxinos & Watson, 2005). Indeed, we found that intra-VTA caffeine injections
produced conditioned place preferences when specifically targeted to the rVTA (Figure 3.2a).
Given that the VTA is comprised of a majority of DAergic neurons and that systemic
caffeine at 2 mg/kg i.p. was able to produce DA-dependent reward, we injected rats with α-flu (0.8
mg/kg i.p) to determine if DA activity was also required for intra-rVTA caffeine reward. α-flu
blocked the rewarding effects of caffeine produced by intra-rVTA injections, suggesting that rVTA
caffeine at a 100 µM concentration is behaviorally and mechanistically equivalent in the place
conditioning paradigm to 2 mg/kg i.p. of caffeine. It seems unlikely that this blockade is due to
non-specific effects of α-flu, as this same dose of α-flu does not block the rewarding effects of
60
opiates or food in previously drug-naïve or food-sated animals, respectively (Laviolette et al.,
2002; Bechara et al., 1992).
Unlike the intra-rVTA caffeine reward, injections of 100 µM caffeine directly into the
cVTA had no effect on motivation with or without pretreatment of α-flu i.p. (Figure 3.2a).
However, injections of a higher concentration of caffeine (1000 µM) into the cVTA produced a
trend towards caffeine aversion (Figure 3.2b). Though this result was not significant, it is
consistent with the mounting evidence for the role of the cVTA in mediating aversive motivational
responses (Balcita-Pedicino et al., 2011; Bourdy & Barrot, 2012; Sanchez-Catalan et al., 2017).
61
A)
B)
62
*
C)
D)
63
Figure 3.2 - Caffeine reward is mediated through the rostral, not caudal, VTA
a) Schematic of place conditioning paradigm and timeline.
b) Animals given caffeine at three concentrations of 10, 100, and 1000 µM nonspecifically in the
VTA did not result in any rewarding or aversive effects as indicated by the lack of CPP and CPA
expression, respectively (p > 0.05).
c) A significant interaction (three-way ANOVA, F1,68 = 6.0274, p = 0.01665) of pretreatment
(vehicle i.p. vs. α-flu i.p.) by drug pairing (vehicle- vs. caffeine-paired) by VTA subregion (rVTA
vs. cVTA) was revealed when caffeine was injected at a concentration of 100 µM. Multiple
comparison tests indicated that caffeine (100 µM) directly injected into the rVTA only could elicit
a significant reward (p = 0.0009, n = 10) that could be disrupted by α-flu pretreatment (p = 0.9999,
n = 12). In the cVTA groups, animals spent similar amounts of time in both sides of the testing
apparatus without (p = 0.9999, n = 9) or with (p = 0.9999, n = 7) α-flu pretreatment. Bars represent
mean amount of time animals spent on each side of the testing apparatus ± SEMs. * indicates a
significant difference between time spent in the vehicle-paired and caffeine-paired sides of the
testing apparatus (p < 0.05).
d) Conversely, when caffeine was injected at a higher dose of 1000 µM in the VTA, there was no
interaction between pretreatment, drug pairing and VTA subregion (three-way ANOVA, F1,62 =
2.0414, p = 0.1581). Multiple comparison tests indicated that the time that animals spent in each
side of the testing apparatus was similar in the rVTA groups without (p = 0.9999, n = 13) or with
(p = 0.9999, n = 6) α-flu i.p. pretreatment. In the cVTA groups, animals that had received caffeine
64
without α-flu i.p. pretreatment showed a nonsignificant aversion to caffeine (p = 0.1510, n = 7),
whereas those with α-flu i.p. pretreatment spent similar amounts of time in both the vehicle- and
caffeine-paired sides (p = 0.9999, n = 9). Bars represent mean amount of time animals spent on
each side of the testing apparatus ± SEMs.
65
Intra-rVTA caffeine reward is mediated through the NAc shell, but not core
Having determined that rVTA DA neurotransmission is necessary for caffeine reward, the
roles of the downstream NAc subregions were investigated. While increased DA levels in the NAc
shell are associated with increased caffeine locomotion and arousal (Solinas et al., 2002; Lazarus
et al., 2011), the role of both NAc subregions in the DA-mediated motivational effects of caffeine
is unknown. Cannulas were implanted in the rVTA and NAc, targeting either the shell or core
(placements shown in Figure 3.3). To determine which NAc subregion is necessary for the
manifestation of caffeine reward, we injected α-flu into either the shell or core, followed by an
injection of caffeine into the rVTA. Intra-NAc shell injections of α-flu blocked the intra-rVTA
caffeine reward (Figure 3.4a). Conversely, this effect was not observed when α-flu was injected
into the NAc core (Figure 3.4b). This suggests that the rVTA-NAc shell pathway is necessary for
DA-dependent caffeine reward.
66
Figure 3.3 - Verification of bilateral cannula placements
Bilateral cannula tips targeted at the medial NAc shell (●), lateral NAc core (○), and rostral
VTA for data shown in Figure 4. The outlines shown in the rVTA slices represent the anatomical
VTA subregions, composed of the rostral VTA (VTAR) and the rostral portions of the
parabrachialis pigmentosus. For Figure 3.4a) and b), caffeine was injected in the rVTA following
α-flu or vehicle pretreatment in the intra-NAc shell or intra-NAc core. For Figure 3.4c) and d),
caffeine was administered i.p. following α-flu or vehicle pretreatment in the NAc shell or NAc
core. Shown in Figure 3.3 are coronal slice reconstructions based on the stereotaxic atlas of
Paxinos and Watson (2005) with reference to Bregma. Numbers indicate the approximate distance
from Bregma in millimetres (mm).
NAc rVTA
67
A)
B)
68
Figure 3.4. Intra-rVTA caffeine reward is DA-dependent and mediated centrally through
the NAc shell, but not the core
a) A two-way ANOVA indicated that animals treated with intra-rVTA caffeine (100µM)
experienced caffeine reward that could be blocked by α-flu pretreatment within the NAc shell
(NAcSh in graph), as indicated by an interaction of pretreatment and drug pairing (F1,26 = 5.31, p
= 0.0294). Multiple comparison tests revealed that animals that had received caffeine in the rVTA
without α-flu pretreatment spent more time in the caffeine-paired side than the vehicle-paired side
(p = 0.0051, n = 8). In the intra-NAc shell α-flu pretreatment group, animals spent similar amounts
of time in both sides of the testing apparatus (p = 0.9999, n = 7). Bars represent mean amount of
time animals spent on each side of the testing apparatus ± SEMs. * indicates a significant
difference between time spent in the vehicle-paired and caffeine-paired sides of the testing
apparatus (p < 0.05).
b) In separate groups of animals treated with intra-rVTA (100µM) caffeine, a two-way ANOVA
did not reveal an interaction between pretreatment and drug pairing (F1,26 = 0.4572, p = 0.5049).
Rather, animals that had received intra-rVTA caffeine exhibited preference for caffeine regardless
of α-flu pretreatment within the NAc core (NAcC in graph), as indicated by a main effect of
caffeine administration (F1,26 = 70.30, p = 0.0001). Multiple comparison tests confirmed that
animals spent more time in the caffeine-paired than the vehicle-paired side in the vehicle
pretreatment group (p = 0.0001, n = 7) and the α-flu pretreatment group (p = 0.0001, n = 8). Bars
represent mean amount of time animals spent on each side of the testing apparatus ± SEMs. *
indicates a significant difference between time spent in the vehicle-paired and caffeine-paired sides
of the testing apparatus (p < 0.05).
69
3.3 High dose-systemic caffeine aversion mediated by NAc core
Systemic caffeine aversion is mediated centrally through the NAc core, but not shell
Given the evidence above showing that the NAc core is not necessary for caffeine reward
in the conditioned place preference paradigm but is implicated in nicotine taste aversions (Sellings
et al., 2008), we hypothesized that the core would mediate caffeine aversions. As higher doses of
systemic caffeine produced conditioned place aversions that were blocked by systemic α-flu, rats
were pretreated with injections of α-flu targeting the NAc core prior to place conditioning. α-flu
blocked the aversions produced by 10mg/kg i.p. of caffeine, indicating that the NAc core
specifically mediates caffeine aversion in a DA-dependent manner (Figure 3.4d). Additionally, we
tested whether the rVTA-NAc shell reward pathway influenced the NAc core-mediated aversive
pathway. We did not observe a block of conditioned place aversions to systemic caffeine in rats
pretreated with α-flu targeted to the NAc shell (Figure 3.4c). As our earlier findings indicated that
caffeine reward may be mediated by the rVTA-NAc shell pathway, and not the NAc core, the data
in this section serve to double dissociate functionally segregated caffeine reward and aversion
pathways in the NAc shell and core, respectively.
70
A)
B)
71
Figure 3.5. Caffeine aversion is mediated centrally through the NAc core, but not the shell
a) For animals treated with systemic caffeine (10 mg/kg i.p.), a two-way ANOVA did not reveal
an interaction between intra-NAc shell α-flu pretreatment and drug pairing (F1,26 = 0.02237, p =
0.8822). Rather, animals that were given systemic caffeine experienced significant aversions to
caffeine regardless of pretreatment condition, as indicated by a main effect of caffeine
administration (F1,26 = 45.37, p = 0.0001). Multiple comparison tests confirmed that animals spent
more time in the vehicle-paired side than the caffeine-paired side in the vehicle pretreatment group
(p = 0.0004, n = 7) and α-flu pretreatment group (p = 0.0003, n = 8). Bars represent mean amount
of time animals spent on each side of the testing apparatus ± SEMs. * indicates a significant
difference between time spent in the vehicle-paired and caffeine-paired sides of the testing
apparatus (p < 0.05).
b) Animals treated with caffeine i.p. (10mg/kg) exhibited caffeine aversions that could be blocked
by pretreatment with intra-NAc core α-flu, as indicated by an interaction between pretreatment
and drug-pairing to each side of the testing apparatus (two-way ANOVA, F1,28 = 15.21, p =
0.0005). Multiple comparisons tests revealed that the animals that had received systemic caffeine
in the intra-NAc core vehicle pretreatment group spent significantly less time in the caffeine-paired
side than the vehicle-paired side (p = 0.0001, n = 6). In the intra-NAc core α-flu pretreatment
group, animals did not spend significantly different amounts of time in the two sides of the testing
apparatus (p = 0.9999, n = 10). Bars represent mean amount of time animals spent on each side of
the testing apparatus ± SEMs. * indicates a significant difference between time spent in the vehicle-
paired and caffeine-paired sides of the testing apparatus (p < 0.05).
72
Chapter 4 - General Discussion
73
4.1 Overview
A fundamental task for the mammalian brain is to assign motivational valence to stimuli
to determine whether to approach a reward or avoid an aversive stimulus. Although VTA DA
neurons are known to play a key role in processing various motivational stimuli, the roles of
mesolimbic DA regarding acute caffeine’s motivational effects are not yet understood. We have
separately elicited conditioned caffeine place preferences through low dose systemic injections
and intra-rVTA caffeine, or conditioned caffeine place aversions through higher dose systemic
injections by performing in vivo administration of caffeine and pharmacological blockade of DA
receptors in a place conditioning paradigm. These two motivational effects were blocked by α-flu
in the NAc shell or core, suggesting that there are two separate and double dissociated DA-
dependent pathways that mediate caffeine reward and aversion, respectively.
Neuroanatomy of caffeine aversion
Previous work in mice demonstrated that doses of 10 and 30 mg/kg i.p. of caffeine
produced high aversions (Sturgess et al., 2010), which was recapitulated in rats in the present
study. The present study demonstrates that caffeine aversion is mediated within the CNS, as a
quaternary caffeine analogue restricted to the peripheral nervous system did not have any
motivational effect despite its greater binding affinity to adenosine receptors (Tao & Abdel-
Rahman, 1993). Further, this aversion is mediated by DA activity in the NAc core, as both systemic
and direct intra-NAc core injections of α-flu were sufficient to block the aversions.
74
Given that the lipid solubility of caffeine allows it to pass through membranes easily and
that systemic caffeine should reach both subregions of the NAc, it is unclear why the NAc core-
mediated aversion is the effect that prevails at higher caffeine doses. It may be that the 10 and 30
mg/kg i.p. doses of caffeine reach higher concentrations in the VTA than does 100 µM of caffeine
injected directly into the VTA and thus, overshadow the rewarding effects of rVTA activation,
resulting in an overall aversion. This could be through a cVTA-mediated mechanism, which
includes the tail of the VTA (also known as the rostromedial tegmental nucleus). The tail of the
VTA (tVTA) is composed of a larger proportion of GABA neurons than DA neurons and is
reported to act as a brake to the midbrain DA systems, which is important for avoidance behaviors
elicited by the LHb (Balcita-Pedicino et al., 2011; Bourdy & Barrot, 2012; Lammel et al., 2012;
Sanchez-Catalan et al., 2017). Higher doses of caffeine could preferentially activate the tVTA,
leading to a brake on the DA-mediated caffeine reward elicited at lower doses in the proposed
rVTA-NAc shell reward pathway (Balcita-Pedicino et al., 2011; Bourdy & Barrot, 2012).
Consistent with this notion, Kaufling and colleagues (2010) quantified the number of
FosB/ΔFosB-positive nuclei (early gene indicating cell activation) following caffeine exposure
and found that doses equal or higher than 10 mg/kg i.p. induced a significant increase in recruited
FosB/ΔFosB-positive nuclei in the tVTA as compared to saline exposure. Given that the GABA-
rich neurons in the tail of the VTA provide inhibitory input to VTA DA cells (Matsui & Williams,
2011), this would also predict a lower activation of FosB/ΔFosB in the rVTA. This suggests that
caffeine reward elicited through the rVTA-NAc shell reward pathway would be inhibited when
caffeine is administered at high doses. Given that aversions (though not statistically significant)
were seen with caffeine injections into the cVTA, it also is possible that caffeine may work in
75
other areas than the VTA; areas which then project to the NAc core to interact with DA
mechanisms there.
However, at the highest dose of 60 mg/kg i.p. of caffeine, rats did not experience reward
or aversion. A secondary rewarding process could be counteracting the expected higher aversion,
but a more likely explanation is that higher caffeine doses may be more stressful to the animal. A
high dose of caffeine is known to cause the body temperature of an animal to rise, which indicates
physiological stress (Pechlivanova et al., 2010). In response to stress, animals become anhedonic,
reduce locomotor activity, and reduce exploratory activity (Rygula et al., 2005). It is conceivable
that these behavioral changes that represent a loss of interest may interfere with learning about the
drug-place cues during conditioning.
Neuroanatomy of caffeine reward
In contrast to the acute caffeine aversions that were elicited systemically by a wide range
of high doses, caffeine reward was only seen at a restricted low dose range, as reported by others
(Brockwell et al, 1991). Further, caffeine reward is dependent on activity localized to a specific
subregion of the VTA. There is ample evidence for heterogeneity along the rostrocaudal axis of
the VTA from anatomical and behavioural studies (Olson et al., 2005; Ikemoto, 2007; Olson &
Nestler; 2007; Yamaguchi et al., 2007). Our findings that caffeine can elicit reward only in the
rVTA align closely with the work of Olson and colleagues (2005) who administered cocaine to
the rostrocaudal subregions of the VTA and found that only the rVTA could elicit cocaine reward
76
in the place conditioning paradigm. As caffeine reward was blocked by α-flu in the NAc shell, the
rVTA DA neurons are the most likely candidates for mediating reward.
The functional distinction along the rostrocaudal axis could be explained if the rVTA DA
neurons project more to the NAc shell and the cVTA neurons project more to the NAc core.
However, it remains unclear whether this is the case as anatomical labeling studies often tend to
examine the NAc or VTA, without specifically distinguishing subregions in both at the same time.
Brog and colleagues (1993) did find that the VTA projects to the NAc with mediolateral
topography, evidenced by retrograde Fluoro-Gold injections in the medial NAc shell resulting in
dense labeling of the more medial portions of the VTA subnuclei. However, a more recent
retrograde labeling study conducted by Rodriguez-Lopez and colleagues (2017) distinguished both
VTA and NAc subregions more clearly. Of the retrograde tracer injections that targeted the NAc
shell or core without overlap, they found that more cells with initial injections in the NAc shell
than the NAc core were positive for TH (marker for DA) in the rostral VTA (VTAR) – an
anatomical VTA subregion that, along with the rostral parabrachialis pigmentosus, comprises the
rVTA defined in the present study. However, further studies will be required to anatomically
determine more quantitatively the strength of the specific connections from the rVTA to NAc shell
versus NAc core.
The functional heterogeneity of the accumbens is arguably more well-defined than that of
the VTA (Pontieri et al., 1995; Jones et al., 1996; Ikemoto & Panksepp,1999; Di Chiara, 2002;
Solinas et al., 2002; Carlezon & Thomas, 2009). For example, the rewarding and aversive effects
of nicotine have been found to also be segregated in the NAc shell and core, respectively (Sellings
77
et al., 2008). Intravenous cocaine self-administration also preferentially increased levels of DA in
the NAc shell over the NAc core (Pontieri et al., 1995). Although these functional studies support
our findings for caffeine, it remains to be seen whether these functional distinctions arise due to
dissimilar patterns of connectivity between the rVTA and the cVTA.
Caffeine and other drugs of abuse
Caffeine consumption is further complicated by its co-occurrence with other licit
substances with addictive properties, including tobacco and alcohol (Istvan & Matarazzo, 1984).
Istvan and Matarazzo (1984) found that the use of both caffeine and nicotine (the active ingredient
in tobacco) is more strongly correlated than caffeine paired with alcohol. There is an abundance
of evidence implicating the mesolimbic DA neurons in nicotine use with DA loss-of-function
manipulations (via lesions, pharmacological blockade, or genetic knockouts) disrupting expression
of motivated behaviors (Clarke & Pert, 1985; Corrigall et al., 1992; Picciotto et al., 1998;
Laviolette & van der Kooy, 2003; Sellings et al., 2008). More specifically, Picciotto and colleagues
(1998) found that activation of the β2 subunit-containing nicotinic acetylcholine receptors is
necessary for the reinforcing properties of nicotine. However, as caffeine has a low binding affinity
to nicotinic receptors (Reavill et al., 1988), caffeine’s motivational effects are unlikely to be
produced through the same mechanism.
In contrast to nicotine, acute ethanol reward in rodents is more similar to acute caffeine
reward in that both are mediated by mesolimbic DA neurons in a place conditioning paradigm
(Ting-A-Kee et al., 2013). However, for ethanol, the state of drug dependence must also be
78
considered. When rodents become dependent on ethanol and are then deprived, rodents given
ethanol are thought to experience a rewarding effect due to alleviation from the negative/aversive
feelings of deprivation (Ting-A-Kee et al., 2013). The acute ethanol reward is thought to be
mediated through the mesolimbic DA pathway before ‘switching’ to a DA-independent
mechanism mediated by the TPP when rodents experience ethanol reward in a deprived state
(Ting-A-Kee et al., 2013). Though Sturgess and colleagues (2010) found that the TPP is not
involved in acute caffeine reward in mice, this does not rule out the possibility that the TPP may
be necessary for the reward experienced by caffeine-deprived rodents. Further investigation into
the shared mechanisms between caffeine, nicotine, and alcohol are required to shed light on
whether these mechanistic similarities confer additive addictive properties that could help to
explain their prevalent use in contemporary society.
Caffeine and the role of adenosine receptors
In addition to the functional heterogeneity within the VTA and NAc, the specific role of
receptors and mechanisms for caffeine motivation remains unknown. Caffeine can act through a
variety of biochemical mechanisms (reviewed in Fredholm et al., 1999). Low millimolar
concentrations of caffeine causes direct release of intracellular calcium and influences 5’-
nucleotidases and alkaline phosphatases, while higher concentrations (greater than that achieved
during human consumption) inhibit cyclic phosphodiesterases (McPherson et al. 1991; Smellie et
al., 1979; reviewed in Nehlig & Debry, 1994). At the levels generally assumed to be reasonable
for human consumption patterns of caffeine, caffeine likely acts as an adenosine receptor
antagonist (reviewed in Nehlig & Debry, 1994). For example, caffeine’s widespread arousal
effects can be attributed to its role as an adenosine receptor antagonist. However, the adenosine
79
receptors are unlikely to be directly involved in caffeine motivation as A1R KO, A2AR KO, and
double KO mice behave similarly to WT mice in an analogous caffeine place conditioning
paradigm (Sturgess et al., 2010). Adenosine receptors form heterodimers with DA receptors and
directly inhibit DA receptors in the striatum (Chen et al., 2001). Therefore, the loss of adenosine
receptor inhibition likely results in greater DA receptor activation and upregulation of DA
receptors in the ventral striatum (Chen et al., 2001; Volkow et al., 2015). As caffeine does not
directly bind to DA receptors (Watanabe & Uramoto, 1986), caffeine may act through a
downstream mechanism that affects DA transmission. The present results also provide evidence
for the specific involvement of the DA mesolimbic pathway in caffeine motivation. However, the
present results do not rule out the possibility of an unknown receptor mediating caffeine
motivation.
Caffeine pharmacokinetics and metabolism
In humans and animals, the hydrophobic caffeine molecules can freely bypass through all
biological membranes (reviewed in Fredholm, 1999). It is rapidly absorbed from the
gastrointestinal tract with peak levels at around 30-60 minutes in plasma (Marks & Kelly, 1973;
Kaplan et al., 1997; reviewed in Fredholm, 1999). Caffeine (also known as 1,3,7-
trimethylxanthine) is metabolized by the liver to form dimethylxanthines (e.g. theophylline,
theobromine, and paraxanthine), monomethylxanthines, and other derivatives with lower
pharmacological activity (Kaplan et al., 1997; reviewed in Fredhom, 1999). Salivary and plasma
levels of caffeine can be measured using high-pressure liquid chromatography (HPLC) or through
a radioimmunoassay procedure (Cook et al., 1976). In HPLC, the sample mixture containing
80
caffeine is pumped through a column that is filled with a known solid adsorbent material. Each
component in the sample mixture interacts differently with the solid material, leading to separation
of components based on varying flow rates. Cook and colleagues (1976) found that both
procedures were comparable with caffeine being equally distributed between saliva and plasma.
Additionally, they found that caffeine had a half-life of about 4 hours (Cook et al., 1976).
Subjective effects of caffeine can also be quantified using electroencephalogram (EEG), as doses
of 250 mg and 500 mg were able to produce reductions in alpha waves, beta waves, theta waves,
and total wave amplitudes (Kaplan et al., 1997). However, these measures are more variable than
HPLC and radioimmunoassay. It is known that lower doses of caffeine in humans have pleasant
stimulant effects and higher doses produce aversive anxiogenic effects (Kaplan et al., 1997). This
also is seen with the rats in the present study, so it would be of interest to use these methods to
measure caffeine in a comparative study between animals and humans.
Caffeine use by people
The human consumption patterns of caffeine, for which caffeine reward is likely
heightened by its general pairing with other rewarding substances like sugar, social environments,
tobacco, or alcohol (Istvan & Matarazzo, 1984; Keast et al., 2015), might explain some of the
human versus non-human species differences found in substance abuse studies. Direct
comparisons of caffeine consumption between humans and rodents ought to be made with caution
and with consideration of the higher metabolic function in rodents. Nonetheless, Fredholm and
colleagues (1999) estimated that a dose of “10 mg/kg in a rat represents about 250 mg of caffeine
in a human weighing 70 kg (3.5 mg/kg), and that this could correspond to about 2 to 3 cups of
81
coffee.” Lao-Peregrin and colleagues (2016) had a similar estimate of 5 to 7 mg/kg of caffeine
representing 1 energy drink with high caffeine content or 3 cups of coffee. Extrapolating from
these two estimates would suggest that 1 cup of coffee is roughly equivalent to 2.3 mg/kg to 5
mg/kg in rodents. Although the lower end of the estimate is higher than the 2 mg/kg seen to
produce reward in rats and many people certainly consume greater amounts than 1 cup of coffee,
the duration in which the caffeine is administered also must be considered. While caffeine was
administered to rats in a single injection, people generally consume their caffeine over a period,
which would arguably result in people experiencing a lower dose of caffeine (over a greater
number of consumption sessions). Therefore, in humans experiencing caffeine reinforcement,
caffeine consumption may be tightly regulated with minor variation in intake amount such that
100µM intra-rVTA caffeine or a 2 mg/kg i.p dosage in rats may be comparable to the level of
caffeine, which is rewarding in humans (Nehlig, 1999; Lawson et al., 2004).
In 1976, Cook and colleagues conservatively estimated that people in the United States
consumed at least 10 million kilograms of caffeine from coffee. In 1999, Fredholm and colleagues
found that over 90% of people consumed caffeine. The prevalence of caffeine consumption
appears to have remained stable as one recent study showed that an astonishing 89% of adults in
the United States still consume caffeine regularly (Fulgoni et al., 2015). Despite this, very little is
known about the neurobiological mechanisms that lead to caffeine consumption. Our data suggest
that caffeine’s motivational effects are mediated by mesolimbic dopaminergic pathways, as is seen
with other psychoactive drugs (Ikemoto & Panksepp, 1999; Laviolette & van der Kooy, 2003;
Grieder et al., 2010, Wise & Morales, 2010; Lammel et al., 2011; Ting-A-Kee et al., 2013; Ashok
et al., 2017). In fact, given the early evidence that caffeine consumption is correlated with tobacco
82
and nicotine use (Friedman, 1974; Istvan & Matarazzo, 1984), a question that warrants further
study is whether caffeine’s reliance on the same neural systems as these drugs of abuse can explain
the extraordinary prevalence of its use.
83
4.2 Conclusions
Due to its ready availability in dietary sources, caffeine is the most commonly consumed
psychoactive drug. Though considerable effort has been made to investigate the mechanisms
underlying caffeine’s arousal and mood-enhancing properties, far less is known about the neural
substrates mediating its motivational properties. Using a rodent place conditioning model, we
demonstrate that caffeine’s rewarding and aversive properties are mediated downstream of the
VTA by two functionally dissociable pathways. Specifically, caffeine reward is contingent upon
DA activity in the NAc shell, whereas caffeine aversion is contingent upon DA activity in the NAc
core.
84
4.3 Future Directions
What could account for the differences seen between mice and rats?
Though 10 and 30 mg/kg i.p. doses of caffeine reliably produced caffeine aversion in mice
and rats, caffeine reward was only seen in rats at a low dose of 2 mg/kg i.p. This is consistent with
other rat studies showing that caffeine produces biphasic low dose reward and high dose aversion
(Patkina et al., 1998; Brockwell et al., 1991). However, in the mouse study conducted by Sturgess
and colleagues (2010), they did not observe caffeine reward at lower doses. The discrepancy
between mice and rats could be due to stress or anxiety caused by the lack of mouse handling prior
to experimental procedures and the shorter conditioning time (40 minutes in present rat study
versus 15 minutes in mice) used by Sturgess and colleagues (2010). Prior handling of animals has
been demonstrated to promote reward in animals that do not show reward at the same dosage. For
example, nicotine place conditioning at low doses in mice was rewarding only after the mice were
handled by the experimenter prior to experimentation (Grabus et al., 2006). A shorter conditioning
time would also allow animals less time to overcome the anxiety or stress from injections, which
could be masking the expression of the weak rewarding effects in mice at low doses. This is
consistent with Vautrin and colleagues’ (2005) work, demonstrating that caffeine preferences
appear later in anxious mice than in non-anxious mice. Pechlivanova and colleagues (2010) also
examined the effect of caffeine dose in a chronic unpredictable stress model of depression and
found that caffeine dose-dependently modulated the behaviour of rats in a variety of tests,
including the open field test and the foot shock test. Investigating the effects of handling and
85
conditioning time in rats versus mice could help to resolve the discrepancies between the two
species.
If these experiments were repeated in female rats, would the results be similar?
As systemic caffeine self-administration in rodents is inconsistent (Griffiths & Woodson,
1988) and it is known that there are sex differences related to both caffeine consumption
(Noschang et al., 2009) and place conditioning (Russo et al., 2003), female rats were excluded
from these studies. Nonetheless, the use of female rats would be both generally informative and
could improve on the time course of the place conditioning paradigm. For example, female rats
could acquire a preference or aversion to caffeine with fewer conditioning sessions, as is seen with
cocaine place conditioning (Russo et al., 2003). Female rats also appear to be more vulnerable to
drugs of abuse, as a comparative study of male and female rats showed that a greater percentage
of female rats acquired cocaine and heroin self-administration behavior (Lynch & Carroll, 1999).
Could we elicit caffeine aversion through higher doses of caffeine in the cVTA?
As mentioned previously, caffeine can easily bypass all biological membranes. As such,
systemic caffeine should affect both the NAc core and shell. Therefore, it is unclear why the NAc
core-mediated aversion is the effect that prevails at 10 and 30 mg/kg i.p. doses of caffeine. Though
it was speculated previously that higher systemic doses may correspond to higher than 100 µM of
caffeine in the VTA and could obscure the effects of rVTA activation through a cVTA-mediated
mechanism, this has yet to be determined. Though we have presented data in this thesis using
86
bilateral intra-cVTA injections as high as 1000 µM of caffeine, it appears that even higher doses
may be required to elicit comparable caffeine aversion of that seen using 10 or 30 mg/kg i.p. doses
of caffeine. Additionally, though it was not observed in these current experiments, somatic signs
of caffeine aversion may also be produced with higher doses of caffeine. General somatic signs of
aversion for other drugs of abuse (e.g. amphetamine) and aversive substances (e.g. lithium
chloride) include suppression of grooming, less scratching, and increased time that rats spend lying
on their bellies (Parker et al., 1984; Tuerke et al., 2012).
Is 8-SPT an appropriate proxy as a peripherally-restricted analog of caffeine?
In the present experiments, 8-SPT was injected at a dose of 10 mg/kg i.p. and was found
to produce neither aversion nor reward. While this is consistent with what was seen in mice
(Sturgess et al., 2010), it is possible that 8-SPT may be able to produce rewarding or aversive
effects if given at a different dose. Nonetheless, the interpretation of the present results suggested
that the location of caffeine action was mediated by the CNS. Though it was determined that
caffeine reward and aversion are indeed mediated centrally in the NAc shell and core, respectively,
a more thorough approach would be to repeat some of the experiments previously conducted with
caffeine using 8-SPT instead. For example, if 8-SPT directly injected in the rVTA could elicit
reward that was DA-dependent, then it would suggest that 8-SPT acts in a comparable manner to
caffeine.
87
What other neural substrates mediate DA-independent caffeine reward and aversion?
If activating the tail of the VTA (located in the cVTA) alone is sufficient to produce
caffeine aversion at higher concentrations than 100µM, the LHb (upstream of the tail of the VTA)
would be a good target candidate as Balcita-Pedicino and colleagues (2011) found that the tail of
the VTA is likely an intermediate region between the LHb and the VTA DA neurons. Additionally,
the surrounding regions of the VTA were not definitively ruled out to be involved in caffeine
motivation. To ensure that the targeted intracranial injections into the VTA did not diffuse to
surrounding tissues, small volumes of caffeine were used, and the tissue was examined for
discoloration during histology. However, it is still possible that caffeine was not fully restricted to
the VTA. One way to rule out this possibility is to target the surrounding VTA tissue to see if there
are any behavioral changes. Also, as DA antagonism revealed a DA-independent reward in mice,
it follows that caffeine motivation could also be mediated by other neural substrates and
neurotransmitters. Sturgess and colleagues (2010) showed that lesioning the TPP did not affect
caffeine aversions or DA blockade-induced reward in mice, however, considering other drugs of
abuse could provide valuable insight on which areas are good potential candidates. For example,
injections of morphine into the periaqueductal grey (PAG) has been shown to produce reward (van
der Kooy et al., 1982).
Are the same brain regions mediating acute and chronic caffeine motivation?
As mentioned earlier, the non-deprived/deprived hypothesis developed by van der Kooy
and colleagues suggests that there is a switch of brain regions mediating the effects of opiates
88
depending on the motivational state of the animal. As the work presented in this thesis is based on
the effects of acute caffeine, a future aim could be to test if animals that have become dependent
and withdrawn from caffeine still experience the reward of caffeine through NAc shell mediation
or experience the aversion of caffeine withdrawal if animals learn to associate caffeine withdrawal
with a particular environment. These place conditioning experiments could be repeated in rodents
exposed to caffeine chronically either through daily i.p. injections or simply by administering
caffeine in their drinking water over a long period. After which, blockade of DA in the NAc shell
or NAc core could help determine if these areas are important for mediating caffeine’s motivational
effects in caffeine dependent and withdrawn animals.
89
References
1) American Psychiatric Association (2013) Diagnostic and statistical manual of mental
disorders (5th ed.). Arlington, VA: American Psychiatric Publishing.
2) Ashok AH, Mizuno Y, Volkow ND, Howes OD (2017) Association of stimulants with
dopaminergic alterations in users of cocaine, amphetamine, and methamphetamine: a
systematic review and meta-analysis. Jama Psychiat 74:511-519.
3) Balleine BW, Delgado MR, Hikosaka O (2007) The role of the dorsal striatum in reward and
decision-making. J Neurosci 27:8161-8165.
4) Balcita-Pedicino JJ, Omelchenko N, Bell R, Sesack SR (2011) The inhibitory influence of
the lateral habenula on midbrain dopamine cells: ultrastructural evidence for indirect
mediation via the rostromedial mesopontine tegmental nucleus. J Comp Neurol 519:1143-
1164.
5) Bechara A, Nader K, Harrington F, Nader K, van der Kooy D (1992) Neurobiology of
motivation: Double dissociation of two motivational mechanisms mediating opiate reward
in drug-naïve versus drug-dependent animals. Behav Neurosci 106:798-807.
6) Bechara A, Nader K, van der Kooy D (1998) A two-separate-motivational-systems
hypothesis of opioid addiction. Pharmacol Biochem Behav 59:1-17.
7) Bechara A, van der Kooy D (1989) The tegmental pedunculopontine nucleus: A brain-stem
output of the limbic system critical for the conditioned place preferences produced by
morphine and amphetamine. J Neurosci 9:3400-3409.
8) Bechara A, van der Kooy D (1992) A single brain stem substrate mediates the motivational
effects of both opiates and food in nondeprived rats but not in deprived rats. Behav Neurosci
106:351-363.
90
9) Beckstead RM, Domesick VB, Nauta WJH (1979) Efferent connections of the substantia
nigra and ventral tegmental area in the rat. Brain Res 175:191-217.
10) Beier KT, Steinberg EE, DeLoach KE, Xie S, Miyamichi K, Schwarz L, Gao XJ, Kremer
EJ, Malenka RC, Luo L (2015) Circuit architecture of VTA dopamine neurons revealed by
systematic input-output mapping. Cell 162:622-634.
11) Berridge KC, Robinson TE (2016) Liking, wanting and the incentive-sensitization theory of
addiction. Am Psychol 71:670-679.
12) Berridge KC, Venier IL, Robinson TE (1989) Taste reactivity analysis of 6-
hydroxydopamine-induced aphagia: Implications for arousal and anhedonia hypotheses of
dopamine function. Behav Neurosci 103:36-45.
13) Bourdy R, Barrot M (2012) A new control center for dopaminergic systems: pulling the VTA
by the tail. Trends Neurosci 35:681-690.
14) Brice CF, Smith AP (2002) Effects of caffeine on mood and performance: a study of realistic
consumption. Psychopharmacology 164:188-192.
15) Brockwell NT, Eikelboom R, Beninger RJ (1991) Caffeine-induced place and taste
conditioning: Production of dose-dependent preference and aversion. Pharmacol Biochem
Be 38:513-517.
16) Brog JS, Salyapongse AS, Deutch AY, Zahm DS (1993) The patterns of afferent innervation
of the core and shell in the “accumbens” part of the rat ventral striatum:
Immunohistochemical detection of retrogradely transported Fluoro-Gold. J Comp Neurol
338:255-278.
17) Brown SA, Vik PW, Creamer VA (1989) Characteristics of relapse following adolescent
substance abuse treatment. Addict Behav 14:291-300.
91
18) Carlezon WA, Thomas MJ (2009) Biological substrates of reward and aversion: A nucleus
accumbens activity hypothesis. Neuropharmacology 56:122-132.
19) Carr DB, Sesack SR (2000) Projections from the rat prefrontal cortex to the ventral tegmental
area: Target specificity in the synaptic associations with mesoaccumbens and mesocortical
neurons. J Neurosci 20:3864-3873.
20) Chaudhri N, Sahuque LL, Schairer WW, Janak PH (2010) Separable roles of the nucleus
accumbens core and shell in context- and cue-induced alcohol-seeking.
Neuropsychopharmacol 35:783-791.
21) Chen JF, Moratalla R, Impagnatiello F, Grandy DK, Cuellar B, Rubinstein M, Beilstein MA,
Hackett E, Fink JS, Low MJ, Ongini E, Schwarzchild MA (2001) The role of the D2
dopamine receptor (D2R) in A2A adenosine receptor (A2AR)-mediated behavioral and cellular
responses as revealed by A2A and D2 receptor knockout mice. P Natl Acad Sci USA 98:1970-
1975.
22) Chou DT, Khan S, Forde J, Hirsh KR (1985) Caffeine tolerance: Behavioral,
electrophysiological and neurochemical evidence. Life Sci 36:2347-2358.
23) Clarke PBS, Pert A (1985) Autoradiographic evidence for nicotine receptors on nigrostriatal
and mesolimbic dopaminergic neurons. Brain Res 348:355-358.
24) Cook CE, Tallent CR, Amerson EW, Myers MW, Kepler JA, Taylor GF, Christensen HD
(1976) Caffeine in plasma and saliva by a radioimmunoassay procedure. J Pharmacol Exp
Ther 199:679-686.
25) Corrigall WA, Coen KM, Adamson KL (1994) Self-administered nicotine activates the
mesolimbic dopamine system through the ventral tegmental area. Brain Res 653:278-284.
92
26) Corrigall WA, Franklin BJ, Coen KM, Clarke PBS (1992) The mesolimbic dopaminergic
system is implicated in the reinforcing effects of nicotine. Psychopharmacology 107:285-
289.
27) Creese I, Burt DR, Snyder SH (1976) Dopamine binding predicts clinical and
pharmacological potencies of antischizophrenic drugs. Science 192:481-483.
28) Deutch AY, Cameron DS (1992) Pharmacological characterization of dopamine systems in
the nucleus accumbens core and shell. Neuroscience 46:49-56.
29) Di Chiara G (2002) Nucleus accumbens shell and core dopamine: differential role in
behaviour and addiction. Behav Brain Res 137:75-114.
30) Di Chiara G, Imperato A (1988) Drugs abused by humans preferentially increase synaptic
dopamine concentrations in the mesolimbic system of freely moving rats. Proc Natl Acad
Sci USA 85:5274-5278.
31) Dingle RN, Dreumont-Boudreau SE, Lolordo VM (2008) Caffeine dependence in rats:
effects of exposure duration and concentration. Physiol Behav 95:252-257.
32) Evans SM, Griffiths RR (1992) Caffeine tolerance and choice in humans.
Psychopharmacology 108:51-59.
33) Fibiger HC (1978) Drugs and reinforcement mechanisms: A critical review of the
catecholamine theory. Ann Rev Pharmacol Toxicol 18:37-56.
34) Finn IB, Holtzman SG (1986) Tolerance to caffeine-induced stimulation of locomotor
activity in rats. J Pharmacol Exp Ther 238:542-546.
35) Fredholm BB (2011) Notes on the history of caffeine use. Handb Exp Pharmacol 200:1-10.
93
36) Fredholm BB, Bättig K, Holmén A, Zvartau EE (1999) Actions of caffeine in the brain with
special reference to factors that contribute to its widespread use. Pharmacol Rev 51:83-133.
37) Friedman GD (1974) Cigarettes, alcohol, coffee and peptic ulcer. N Engl J Med 290:469-
473.
38) Fulgoni VL, Keast DR, Lieberman HR (2015) Trends in intake and sources of caffeine in the
diets of US adults: 2001-2010. Am J Clin Nutr 101:1081-1087.
39) Gallistel CR, Gomita Y, Yadin E, Campbell KA (1985) Forebrain origins and terminations
of the medial forebrain bundle metabolically activated by rewarding stimulation or by
reward-blocking doses of pimozide. J Neurosci 5:1246-1261.
40) Gasbarri A, Packard MG, Campana E, Pacitti C (1994) Anterograde and retrograde tracing
of projections from the ventral tegmental area to the hippocampal formation in the rat. Brain
Res Bull 33:445-452.
41) Geisler S, Zahm DS (2005) Afferents of the ventral tegmental area in the rat-anatomical
substratum for integrative functions. J Comp Neurol 490:270-294.
42) Gessa GL, Muntoni F, Collu M, Vargiu L, Mereu G (1985) Low doses of ethanol activate
dopaminergic neurons in the ventral tegmental area. Brain Res 348:201-203.
43) Grabus SD, Martin BR, Brown SE, Damaj MI (2006) Nicotine place preference in the mouse:
influences of prior handling, dose and strain and attenuation by nicotinic receptor
antagonists. Psychopharmacology 184:456-463.
44) Grieder TE, Herman MA, Contet C, Tan LA, Vargas-Perez H, Cohen A, Chwalek M, Maal-
Bared G, Freiling J, Schlosburg JE, Clarke L, Crawford E, Koebel P, Canonigo V, Sanna P,
Tapper A, Roberto M, Kieffer BL, Sawchenko PE, Koob GF, van der Kooy D, George O
(2014) CRF neurons in the ventral tegmental area control the aversive effects of nicotine
withdrawal and promote escalation of nicotine intake. Nat Neurosci 17:1751-1758.
94
45) Grieder TE, Sellings L, Vargas-Perez H, Ting-A-Kee R, Siu EC, Tyndale RF, van der Kooy
D (2010) Dopaminergic signaling mediates the motivational response underlying the
opponent process to chronic but not acute nicotine. Neuropsychopharmacol 35: 943-954.
46) Griffiths RR, Woodson PP (1988) Reinforcing properties of caffeine studies in humans and
laboratory animals. Pharmacol Biochem Be 29:419-427.
47) Grill HJ, Norgren R (1978) The taste reactivity test. I. Mimetic responses to gustatory stimuli
in neurologically normal rats. Brain Res 143:263-279.
48) Hasin DS, O’Brien CP, Auriacombe M, Borges G, Bucholz K, Budney A, Compton WM,
Crowley T, Ling W, Petry NM, Schuckit M, Grant BF (2013) DSM-5 criteria for substance
use disorders: recommendations and rationale. Am J Psychiat 170:834-851.
49) Hefco V, Yamada K, Hefco A, Hritcu L, Tiron A, Nabeshima T (2003) Role of the
mesotelencephalic dopamine system in learning and memory processes in the rat. Eur J
Pharmacol 475:55-60.
50) Heimer L, Zahm DS, Churchill L, Kalivas PW, Wohltmann C (1991) Specificity in the
projection patterns of accumbal core and shell in the rat. Neuroscience 41:89-125.
51) Higgins GA, Grzelak ME, Pond AJ, Cohen-Williams ME, Hodgson RA, Varty GB (2007)
The effect of caffeine to increase reaction time in the rat during a test of attention is mediated
through antagonism of adenosine A2A receptors. Behav Brain Res 185:32-42.
52) Hurd YL, Weiss F, Koob GF, And N-E, Ungerstedt U (1989) Cocaine reinforcement and
extracellular dopamine overflow in rat nucleus accumbens: an in vivo microdialysis study.
Brain Res 498:199-203.
53) Huston JP, de Sousa Silva MA, Topic B, Müller CP (2013) What’s conditioned in
conditioned place preference? Trends Pharmacol Sci 34:162-166.
95
54) Ikemoto S (2007) Dopamine reward circuitry: Two projection systems from the ventral
midbrain to the nucleus accumbens-olfactory tubercle complex. Brain Res Rev 56:27-78.
55) Ikemoto S, Panksepp J (1999) The role of nucleus accumbens dopamine in motivated
behavior: a unifying interpretation with special reference to reward-seeking. Brain Res Rev
31:6-41.
56) Istvan J, Matarazzo JD (1984) Tobacco, alcohol, and caffeine use: A review of their
interrelationships. Psychol Bull 95:301-326.
57) Ito R, Hayen A (2011) Opposing roles of nucleus accumbens core and shell dopamine in the
modulation of limbic information processing. J Neurosci 31:6001-6007.
58) Ito R, Robbins TW, Everitt BJ (2004) Differential control over cocaine-seeking behavior by
nucleus accumbens core and shell. Nat Neurosci 7:389-397.
59) Jhou TC, Fields HL, Baxter MG, Saper CB, Holland PC (2009a) The rostromedial tegmental
nucleus (RMTg), a GABAergic afferent to midbrain dopamine neurons, encodes aversive
stimuli and inhibits motor responses. Neuron 61:786-800.
60) Jhou TC, Geisler S, Marinelli M, DeGarmo BA, Zahm DS (2009b) The mesopontine
rostrotegmental nucleus: a structure targeted by the lateral habenula that projects to the
ventral tegmental area of Tsai and substantia nigra compacta. J Comp Neurol 513:566-596.
61) Jones SR, O'Dell SJ, Marshall JF, Wightman RM (1996) Functional and anatomical evidence
for different dopamine dynamics in the core and shell of the nucleus accumbens in the slices
of rat brain. Synapse 23:224-231.
62) Juliano LM, Griffiths RR (2004) A critical review of caffeine withdrawal: empirical
validation of symptoms and signs, incidence, severity, and associated features.
Psychopharmacol 176:1-29.
96
63) Kalivas PW, Churchill L, Klitenick MA (1993) GABA and enkephalin projection from the
nucleus accumbens and ventral pallidum to the ventral tegmental area. Neuroscience
57:1047-1060.
64) Kaplan GB, Greenblatt DJ, Ehrenberg BL, Goddard JE, Cotreau MM, Harmatz JS, Shader
RI (1997) Dose-dependent pharmacokinetics and psychomotor effects of caffeine in humans.
J Clin Pharmacol 37:693-703.
65) Kaufling J, Waltisperger E, Bourdy R, Valera A, Veinante P, Freund-Mercier M-J, Barrot
Michel (2010) Pharmacological recruitment of the GABAergic tail of the ventral tegmental
area by acute drug exposure. Brit J Phamacol 161:1677-1691.
66) Keast RSJ, Swinburn BA, Sayompark D, Whitelock S, Riddell (2015) Caffeine increases
sugar-sweetened beverage consumption in a free-living population: a randomized controlled
trial. Brit J Nutr 113:366-371.
67) Kendler KS, Prescott CA (1999) Caffeine intake, tolerance, and withdrawal in women: a
population-based twin study. Am J Psychiat 156:223-228.
68) Kim JA, Pollak KA, Hjelmstad GO, Fields HL (2004) A single cocaine exposure enhances
both opioid reward and aversion through a ventral tegmental area-dependent mechanism. P
Natl Acad Sci USA 101:5664-5669.
69) Koob GF, Bloom FE (1988) Cellular and molecular mechanisms of drug dependence.
Science 242:715-723.
70) Koob GF, Le Moal (2001) Drug addiction, dysregulation of reward, and allostasis.
Neuropsychopharmacol 24:97-129.
71) Koob GF, Le Moal (2005) Plasticity of reward neurocircuitry and the ‘dark side’ of drug
addiction. Nature Neurosci 8:1442-1444.
97
72) Koob GF, Le Moal (2008a) Addiction and the brain antireward system. Annu Rev Psychol
59:29-53.
73) Koob GF, Le Moal (2008b) Neurobiological mechanisms for opponent motivational
processes in addiction. Philos Trans R Soc Lond B Biol Sci 363:3113-3123.
74) Koob GF, Stinus L, Le Moal M, Bloom FE (1989) Opponent process theory of motivation:
Neurobiological evidence from studies of opiate dependence. Neurosci Biobehav Rev
13:135-140.
75) Kourrich S, Calu DJ, Bonci A (2015) Intrinsic plasticity: An emerging player in addiction.
Nat Rev Neurosci 16:173-184.
76) Kuzmin A, Johansson B, Fredholm BB, Örgren, S (2000) Genetic evidence that cocaine and
caffeine stimulate locomotion in mice via different mechanisms. Life Sci 66:PL113-118.
77) Lammel S, Ion DI, Roeper J, Malenka RC (2011) Projection-specific modulation of
dopamine neuron synapses by aversive and rewarding stimuli. Neuron 70:855-862.
78) Lammel S, Hetzel A, Hackel O, Jones I, Liss B, Roeper J (2008) Unique properties of
mesoprefrontal neurons within a dual mesocorticolimbic dopamine system. Neuron 57:760-
773.
79) Lammel S, Lim BK, Ran C, Huang KW, Betley MJ, Tye K, Deisseroth K, Malenka RC
(2012) Input-specific control of reward and aversion in the ventral tegmental area. Nature
491:212-217.
80) Lao-Peregrin C, Ballesteros JJ, Fernandez M, Zamora-Moratalla A, Saavedra A, Lazaro MG,
Perez-Navarro E, Burks D, Martin ED (2016) Caffeine-mediated BDNF release regulates
long-term synaptic plasticity through activation of IRS2 signaling. Addict Biol 22:1706-
1718.
98
81) Latini R, Bonati M, Castelli D, Garattini S (1978) Dose-dependent kinetics of caffeine in
rats. Toxicol Lett 2:267-270.
82) Laviolette SR, Nader K, van der Kooy D (2002) Motivational state determines the functional
role of the mesolimbic dopamine system in the mediation of opiate reward processes. Behav
Brain Res 129:17-29.
83) Laviolette SR, van der Kooy D (2003) Blockade of mesolimbic dopamine transmission
dramatically increases sensitivity to the rewarding effects of nicotine in the ventral tegmental
area. Mol Psychiatr 8:50-59.
84) Lawson CC, LeMasters GK, Wilson KA (2004) Changes in caffeine consumption as a signal
of pregnancy Reprod Toxicol 18:625-633.
85) Lazarus M, Shen HY, Cherasse Y, Qu WM, Huang ZL, Bass CE, Winsky-Sommerer R,
Semba K, Fredholme BB, Bolson D, Hayaishi O, Urade Y, Chen JF (2011) Arousal effect of
caffeine depends on adenosine A2A receptors in the shell of the nucleus accumbens. J
Neurosci 31:10067-10075.
86) Ljungberg T, Apicella P, Schultz W (1992) Responses of monkey dopamine neurons during
learning of behavioural reactions. J Neurophysiol 67:145-163.
87) Lorist MM, Snel J, Kok A, Mulder G (1996) Acute effects of caffeine on selective attention
and visual search processes. Psychophysiology 33:354-361.
88) Loughlin SE, Fallon JH (1984) Substantia nigra and ventral tegmental area projections to
cortex: Topography and collaterization. Neuroscience 11:425-435.
89) Lukas G, Brindle SD, Greengard P (1971) The route of absorption of intraperitoneally
administered compounds. J Pharmacol Exp Ther 178:562-566.
99
90) Lynch WJ, Carroll ME (1999) Sex differences in the acquisition of intravenously self-
administered cocaine and heroin in rats. Psychopharmacology 144:77-82.
91) Machado, A (1989) Operant conditioning of behavioural variability using a percentile
reinforcement schedule. J Exp Anal Behav 52:155-166.
92) Margolis EB, Lock H, Hjelmstad GO, Fields HL (2006) The ventral tegmental area revisited:
Is there an electrophysiological marker for dopaminergic neurons. J Physiol 577:907-924.
93) Marks V, Kelly JF (1973) Absorption of caffeine from tea, coffee, and coca cola. Lancet
1:827.
94) Matsui A, Williams JT (2011) Opioid-sensitive GABA inputs from rostromedial tegmental
nucleus synapse onto midbrain dopamine neurons. J Neurosci 31:17729-17735.
95) McPherson PS, Kim Y-K, Valdivia H, Knudson CM, Takekura H, Franzini-Armstron C,
Coronadot R, Campbell KP (1991) The brain ryanodine receptor: A caffeine-sensitive
calcium release channel. Neuron 7:17-25.
96) Millhouse OE (1969) A Golgi study of the descending medial forebrain bundle. Brain Res
15:341-363.
97) Mirenowicz, J, Schultz W (1996) Preferential activation of midbrain dopamine neurons by
appetitive rather than aversive stimuli. Nature 379:449-451.
98) Mucha RF, van der Kooy D, O’Shaughnessy M, Bucenieks P (1982) Drug reinforcement
studied by the use of place conditioning in rat. Brain Res 243:91-105.
99) Nader K, Bechara A, Roberts DCS, van der Kooy D (1994) Neuroleptics block high- but not
low-dose heroin place preferences: Further evidence for a two-system model of motivation.
Behav Neurosci 108:1128-1138.
100
100) Nader K, van der Kooy D (1997) Deprivation state switched the neurobiological substrates
mediating opiate reward in the ventral tegmental area. J Neurosci 17: 383-390.
101) Nair-Roberts RG, Chatelain-Badie SD, Benson E, White-Cooper H, Bolam JP, Ungless MA
(2008) Stereological estimates of dopaminergic, GABAergic and glutamatergic neurons in
the ventral tegmental area, substantia nigra and retrorubral field in the rat. Neuroscience
152:1024-1031.
102) Namburi P, Beyeler A, Yorozu S, Calhoon GG, Halbert SA, Wichmann R, Holden SS,
Mertens KL, Anahtar M, Felix-Ortiz AC, Wickersham IR, Gray JM, and Tye KM (2015) A
circuit mechanism for differentiating positive and negative associations. Nature 520:675-
678.
103) Nauta WJH, Smith GP, Faull RLM, Domesick VB (1978) Efferent connections and nigral
afferents of the nucleus accumbens septi in the rat. Neuroscience 3:385-401.
104) Nehlig A (1999) Are we dependent upon coffee and caffeine? A review on human and animal
data. Neurosci Biobehav Rev 23:563-576.
105) Nehlig A, Debry G (1994) Caffeine and sports activity: A review. Int J Sports Med 15:215-
223.
106) Nestler EJ (1992) Molecular mechanisms of drug addiction. J Neurosci 12:2439-2450.
107) Nestler EJ (2005) Is there a common molecular pathway for addiction? Nature Neurosci
8:1445-1449.
108) Noschang CG, Pettenuzzo LF, von Pozzer Toigo E, Andreazza AC, Krolow R, Fachin A,
Avila MC, Arcego D, Crema LM, Diehl LA, Goncalvez CA, Vendite D, Dalmaz C (2009)
Sex-specific differences on caffeine consumption and chronic stress-induced anxiety-like
behavior and DNA breaks in the hippocampus. Pharamcol Biochem Be 94:63-69.
101
109) Oades RD, Halliday GM (1987) Ventral tegmental (A10) system: Neurobiology. 1. Anatomy
and connectivity. Brain Res Rev 12:117-165.
110) Ogawa N, Ueki H (2007) Clinical importance of caffeine dependence and abuse. Psychiat
Clin Neuros 61:263-268.
111) Olds J, Milner P (1954) Positive reinforcement produced by electrical stimulation of septal
area and other regions of rat brain. J Comp Physiol Psych 47:419-427.
112) Olmstead MC, Munn EM, Franklin KBJ, Wise RA (1998) Effects of pedunculopontine
tegmental nucleus lesions on responding for intravenous heroin under different schedules of
reinforcement. J Neurosci 18:5035-5044.
113) Olson VG, Nestler EJ (2007) Topographical organization of GABAergic neurons within the
ventral tegmental area of the rat. Synapse 61:87-95.
114) Olson VG, Zabetian CP, Bolanos CA, Edwards S, Barrot M, Eisch AJ, Hughes T, Self DW,
Neve RL, Nestler EJ (2005) Regulation of drug reward by cAMP response element-binding
protein: Evidence for two functionally distinct subregions of the ventral tegmental area. J
Neurosci 25:5553-5562.
115) Parker LA, Hills K, Jensen K (1984) Behavioral CRs elicited by a lithium- or an
amphetamine-paired contextual test chamber. Anim Learn Behav 12:307-315.
116) Patkina NA, Zvartau EE (1998) Caffeine place conditioning in rats: comparison with cocaine
and ethanol. Eur Neuropsychopharmacol 8:287-291.
117) Pavlov IP (1927) Conditioned reflexes: An investigation of the physiological activity of the
cerebral cortex. London: Oxford University Press.
118) Paxinos G, Watson C (2005) The rat brain in stereotaxic coordinates, Ed 5. San Diego:
Academic Press.
102
119) Pechlivanova D, Tchekalarova J, Nikolov R, Yakimova K (2010) Dose-dependent effects of
caffeine on behavior and thermoregulation in a chronic unpredictable stress model of
depression in rats. Behav Brain Res 209:205-211.
120) Persad LAB (2011) Energy drinks and the neurophysiological impact of caffeine. Front
Neurosci 5:1-8.
121) Pettit ML, DeBarr KA (2011) Perceived stress, energy drink consumption, and academi
performance among college students. J Am Coll Health 59:335-341.
122) Phillipson OT, Griffiths AC (1985) The topographic order of inputs to nucleus accumbens
in the rat. Neuroscience 16:275-296.
123) Picciotto MR, Zoli M, Rimondini R, Lena C, Marubio LM, Pich EM, Fuxe K, Changeux J-
P (1998) Acetylcholine receptors containing the β2 subunit are involved in the reinforcing
properties of nicotine. Nature 391:173-177.
124) Poller WC, Madai VI, Bernaud R, Laube G, Veh RW (2013) A glutamatergic projection
from the lateral hypothalamus targets VTA-projecting neurons in the lateral habenula of the
rat. Brain Res 1507:45-60.
125) Pontieri FE, Tanda G, Di Chiara G (1995) Intravenous cocaine, morphine, and amphetamine
preferentially increase extracellular dopamine in the "shell" as compared to the "core" of the
rat nucleus accumbens. P Natl A Sci USA 92:12304-12308.
126) Reavill C, Jenner P, Kumar R, Stolerman IP (1988) High affinity binding of [3H] (-)-nicotine
to rat brain membranes and its inhibition by analogues of nicotine. Neuropharmacology
27:235-241.
127) Richards G, Smith A (2015) Caffeine consumption and self-assessed stress, anxiety, and
depression in secondary school children. J Psychopharmacol 29:1236-1247.
103
128) Roberts DCS, Koob GF (1982) Disruption of cocaine self-administration following 6-
hydroxydopamine lesions of the ventral tegmental area in rats. Pharmacol Biochem Be
17:901-904.
129) Robinson TE, Berridge KC (1993) The neural basis of drug-craving: an incentive-
sensitization theory of addiction. Brain Res Rev 18:247-291.
130) Rodriguez-Lopez C, Clasca F, Prensa L (2017) The mesoaccumbens pathway: a retrograde
labeling and single-cell axon tracing analysis in the mouse. Front Neuroanat 11:1-15.
131) Russo SJ, Festa ED, Fabian SJ, Gazi FM, Kraish M, Jenab S, Quinones-Jenab V (2003)
Gonadal hormones differentially modulate cocaine-induced conditioned place preference in
male and female rats. Neuroscience 120:523-533.
132) Rygula R, Abumaria Nm Flugge G, Fuchs E, Ruther E, Havemann-Reinecke U (2005)
Anhedonia and motivational deficits in rats: Impact of chronic social stress. Behav Brain Res
162:127-134.
133) Sanchez-Catalan MJ, Faivre F, Yalcin I, Muller MA, Massotte D, Majchrzak M, Barrot M
(2017) Response of the tail of the ventral tegmental area to aversive stimuli.
Neuropsychopharmacol 42:638-648.
134) Sanchez-Catalan MJ, Kaufling J, Georges F, Veinante P, Barrot M (2014) The antero-
posterior heterogeneity of the ventral tegmental area. Neuroscience 282:198-216.
135) Schultz W (2016) Dopamine reward prediction-error signaling: a two-component response.
Nat Rev Neurosci 17:183-195.
136) Schultz W, Apicella P, Ljungberg T (1993) Responses of monkey dopamine neurons to
reward and conditioned stimuli during successive steps of leaning a delayed response task. J
Neurosci 13:900-913.
104
137) Sellings LHL, Baharnouri G, McQuade LE, Clarke PBS (2008) Rewarding and aversive
effects of nicotine are segregated within the nucleus accumbens. Eur J Neurosci 28:342-352.
138) Sellings LHL, Clarke PBS (2003) Segregation of amphetamine reward and locomotor
stimulation between nucleus accumbens medial shell and core. J Neurosci 23:6295-6303.
139) Silverman K, Evans SM, Strain EC, Griffiths RR (1992) Withdrawal syndrome after the
double-blind cessation of caffeine consumption. New Engl J Med 327:1109-1114.
140) Skinner BF (1950) Are theories of learning necessary? Psychol Rev 57:193-216.
141) Smellie FW, Davis CW, Daly JW, Wells JN (1979) Alkylxanthines: Inhibition of adenosine-
elicited accumulation of cyclic AMP in brain slices and of brain phosphodiesterase activity.
Life Sci 24:2475-2481.
142) Smith AP (2009) Effects of caffeine in chewing gum on mood and attention. Hum
Psychopharmacol Clin Exp 24:239-247.
143) Solinas M, Ferré S, You ZB, Karcz-Kubicha M, Popoli P, Goldberg SR (2002) Caffeine
induces dopamine and glutamate release in the shell of the nucleus accumbens. J Neurosci
22:6321-6324.
144) Solomon RL, Corbit JD (1973) An opponent-process theory of motivation: II. Cigarette
addiction. J Abnorm Psychol 81:158-171.
145) Solomon RL, Corbit JD (1974) An opponent-process theory of motivation: I. Temporal
dynamics of affect. Psychol Rev 81:119-145.
146) Steffensen SC, Svingos AL, Pickel VM, Henriksen SJ (1998) Electrophysiological
characterization of GABAergic neurons in the ventral tegmental area. J Neurosci 18:8003-
8015.
105
147) Stuber GD, Evans SB, Higgins MS, Pu Y, Figlewicz DP (2002) Food restriction modulates
amphetamine-conditioned place preference and nucleus accumbens dopamine release in the
rat. Synapse 46:83-90.
148) Sturgess JE, Ting-A-Kee RA, Podbielski D, Sellings LHL, Chen JF, van der Kooy D (2010)
Adenosine A1 and A2A receptors are not upstream of caffeine's dopamine D2 receptor-
dependent aversive effects and dopamine-independent rewarding effects. Eur J Neurosci
32:143-154.
149) Swanson LW (1982) The projections of the ventral tegmental area and adjacent regions: A
combined fluorescent retrograde tracer and immunofluorescence study in the rat. Brain Res
Bull 9:321-353.
150) Tao S, Abdel-Rahman AA (1993) Neuronal and cardiovascular responses to adenosine
microinjection into the nucleus tractus solitarius. Brain Res Bull 32:407-417.
151) Ting-A-Kee R, Dockstader C, Heinmiller A, Grieder T, van der Kooy D (2009) GABAA
receptors mediate the opposing roles of dopamine and the tegmental pedunculopontine
nucleus in the motivational effects of ethanol. Eur J Neurosci 29:1235-1244.
152) Ting-A-Kee R, Vargas-Perez H, Bufalino M-R, Bahi A, Dreyer J-L, Tyndale RF, van der
Kooy D (2013) Infusion of brain-derived neurotrophic factor into the ventral tegmental area
switches the substrates mediating ethanol motivation. Eur J Neurosci 37:996-1003.
153) Thorndike EL (1898) Some experiments on animal intelligence. Science 7:818-824.
154) Tuerke KJ, Winters BD, Parker LA (2012) Ondansetron interferes with unconditioned lying-
on belly and acquisition of conditioned gaping induced by LiCl as models of nausea-induced
behaviors in rats. Physiol Behav 105:856-860.
106
155) van der Kooy D, Mucha RF, O’Shaughnessy M, Bucenieks P (1982) Reinforcing effects of
brain microinjections of morphine revealed by conditioned place preference. Brain Res
243:107-117.
156) van der Poel A, Rodenburg G, Dijkstra M, Stoele M, van de Mheen D (2009) Trends,
motivations and settings of recreational cocaine use by adolescents and young adults in the
Netherlands. Int J Drug Policy 20:143-151.
157) Vautrin S, Pelloux Y, Costentin J (2005) Preference for caffeine appears earlier in non-
anxious than in anxious mice. Neurosci Lett 386:94-98.
158) Veening JG, Swanson LW, Cowan WM, Nieuwenhuys R, Geeraedts LMG (1982) The
medial forebrain bundle of the rat. II. An autoradiographic study of the topography of the
major descending and ascending components. J Comp Neurol 206:82-108.
159) Ventura R, Cabib S, Alcaro A, Orsini C, Puglisi-Allegra S (2003) Norepinephrine in the
prefrontal cortex is critical for amphetamine-induced reward and mesoaccumbens dopamine
release. J Neurosci 23:1879-1885.
160) Vezina P, Stewart J (1989) The effect of dopamine receptor blockade on the development of
sensitization to the locomotor activating effects of amphetamine and morphine. Brain Res
499:108-120.
161) Volkow ND, Wang GJ, Logan J, Alexoff D, Fowler JS, Thanos PK, Wong C, Casado V,
Ferre S, Tomasi D (2015) Caffeine increases striatal dopamine D2/D3 receptor availability in
the human brain. Transl Psychiat 5:e549-554.
162) Watanabe H, Uramoto H (1986) Caffeine mimics dopamine receptor agonists without
stimulation of dopamine receptors. Neuropharmacology 25:577-581.
107
163) Walsh SL, Stoops WW, Moody DE, Lin SN, Bigelow GE (2009) Repeated dosing with oral
cocaine in humans: Assessment of direct effects, withdrawal and pharmacokinetics. Exp Clin
Psychopharmacol 17:205-216.
164) Westerink BHC, Kwint HF, deVries JB (1996) The pharmacology of mesolimbic dopamine
neurons: a dual-probe microdialysis study in the ventral tegmental area and nucleus
accumbens of the rat brain. J Neurosci 16:2605-2611.
165) Wise RA (1987) The role of reward pathways in the development of drug dependence.
Pharmacol Therapeut 35: 227-263.
166) Wise RA, Morales M (2010) A ventral tegmental CRF-glutamate-dopamine interaction in
addiction. Brain Res 1314:38-43.
167) World Health Organization (1992) The ICD-10 classification of mental and behavioural
disorders: clinical descriptions and diagnostic guidelines. Geneva: World Health
Organization.
168) Xie H, McHugo GJ, Fox MB, Drake RE (2005) Substance abuse relapse in a ten-year
prospective follow-up of clients with mental and substance use disorders. Psychiat Serv
56:1282-1287.
169) Yamaguchi T, Sheen W, Morales M (2007) Glutamatergic neurons are present in the rat
ventral tegmental area. Eur J Neurosci 25:106-118.
170) Young AMJ, Joseph MH, Gray JA (1993) Latent inhibition of conditioned dopamine release
in rat nucleus accumbens. Neuroscience 54:5-9.
171) Yukl G, Wexley KN, Seymore JD (1972) Effectiveness of pay incentives under variable ratio
and continuous reinforcement schedules. J Appl Psychol 56:19-23.
108
172) Zahm DS (1999) Functional-anatomical implications of the nucleus accumbens core and
shell subterritories. Ann NY Acad Sci 877:113-128.
173) Zahm DS, Heimer L (1990) Two transpallidal pathways originating in the rat nucleus
accumbens. J Comp Neurol 302:437-446.
174) Zahm DS, Heimer L (1993) Specificity in the efferent projections of the nucleus accumbens
in the rat: Comparison of the rostral pole projection patterns with those of the core and shell.
J Comp Neurol 372: 220-232.