Download STUDY OF THE ROLE OF THE BED NUCLEUS OF

STUDY OF THE ROLE OF THE BED NUCLEUS OF
THE STRIA TERMINALIS D1- AND D2-LIKE
DOPAMINE RECEPTORS IN COCAINE SELFADMINISTERING RATS
by
Chia-Jung Cindy Chiang
A thesis submitted to the Centre for Neuroscience Studies
In conformity with the requirements for
the degree of Master of Science
Queen’s University
Kingston, Ontario, Canada
(August, 2010)
Copyright  Chia-Jung Cindy Chiang, 2010
Abstract
Previous studies have suggested the importance of the bed nucleus of the stria
terminalis (BST) in modulating drug seeking behaviors. With heavy reciprocal
projections from the BST to various dopamine rich areas (ventral tegmental area (VTA),
nucleus accumbens shell (NAc shell), retrorubral nucleus (RR), periaqueductal gray
region (PAG)), BST dopamine receptors are indeed an important part of the highly
integrated reward system and thus possess sensitivity to pharmacological rewards. In the
present study, the effects of bilateral intracranial injections of the D1- dopamine receptor
antagonist SCH-23390 (1.6, 3.2, and 6.4 µg total bilateral dose) , the D2- dopamine
receptor antagonist Sulpiride (3.2 µg total bilateral dose), and D2- dopamine receptor
agonist Quinpirole (3.0µg total bilateral dose), administered into the BST immediately
prior to self-administration sessions, were examined in both sucrose (180-minute
sessions) and cocaine (270-minute sessions) self-administering rats. Injections of SCH23390 (3.2 & 6.4 µg) to the cocaine group significantly reduced the final ratio of operant
lever responding (0.75 mg/kg/i.v. injection; progressive ratio schedule of reinforcement
(PR); timeout 20 sec); there was no effect of SCH-23390 in the sucrose group. Injections
of Sulpiride (3.2µg) to the cocaine group produced a significant attenuation in the final
ratio of operant lever pressing, with no effect in the sucrose group. Injections of
Quinpirole, however, resulted in no alternations in both the cocaine and sucrose selfadministration group. These results suggest that both D1 and D2 dopamine receptor
activation is occurring in the reinforcement of cocaine, with the possibility that D2
receptors are not as highly activated as the D1 receptors. This conclusion further
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implicates the complexity of the neurobiological factors involved in drug addiction and
the essential role of the BST in dopamine-facilitated reward reinforcement.
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Acknowledgements
I would like to thank my supervisor, Éric C. Dumont, for providing me with an
opportunity to acquire my MSc degree in such a fun, loving lab. He has helped to keep
me in check at times to ensure that I stay motivated and enlightened to finish my thesis
on time.
Most importantly, I would like to thank the people from the Dumont lab. Because
of them, the atmosphere in the lab has been a lot of fun. I particularly would like to
acknowledge Dasha Ianovskaia and Xenos Mason for providing me with useful
suggestions for experiments and my thesis. I also would like to thank Michal Krawczyk
for keeping things going smoothly in the lab.
Last but not least, I would like to thank my friends and family for providing me
with great support. Mom and Dad have always been there to encourage me. My friends,
Winni, Asin, Bing, and Eunice: thank you all.
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Table of Contents
Abstract ...................................................................................................................................................................... ii
Acknowledgments ............................................................................................................................................... iv
Table of Contents ...................................................................................................................................................v
List of Abbreviations ............................................................................................................................ vii
List of Figures .......................................................................................................................................... ix
List of Tables ........................................................................................................................................................... xi
Chapter 1: Introduction ..................................................................................................................................... 1
1.1 – What is drug addiction ............................................................................................................................1
1.1.1 -Definition .........................................................................................................................................1
1.1.2 - Animal model of drug addiction............................................................................................2
1.1.3 - Neural circuits of reward and motivation .........................................................................9
1.1.4 - Dopamine in motivated behaviours ................................................................................. 16
1.1.5 - Mechanisms of psychostimulants effects ....................................................................... 18
1.2 - The BST: an important component of the reward and motivation pathway and a
target for drugs of abuse ................................................................................................................................ 19
1.2.1 – Definition and anatomy ........................................................................................................ 19
1.2.2 – BST dopamine and motivated behaviour ...................................................................... 20
1.3 – Research aims and hypothesis ......................................................................................................... 22
1.3.1 – Research aims ........................................................................................................................... 22
1.3.2 - Hypothesis................................................................................................................................... 23
Chapter 2: Materials and Methods............................................................................................................. 24
2.1 - Animals........................................................................................................................................................ 24
2.2 - Drugs ............................................................................................................................................................ 24
2.3 - Apparatus ................................................................................................................................................... 25
2.4 - Cannulation surgery .............................................................................................................................. 26
2.5 - Intra-BST drug micro-injection ......................................................................................................... 29
2.6 – Behavioural procedure: operant conditioning-reinforcement paradigm ...................... 35
2.7 – Data analysis ............................................................................................................................................ 39
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2.8 - Histology / brain cannulation placements ................................................................................... 42
Chapter 3: Results ...............................................................................................................................................44
3.1 - Effect of SCH-23390 on self-administration ................................................................................ 44
3.2 - Effect of Sulpiride on self-administration ..................................................................................... 48
3.3 - Effect of Quinpirole on self-administration ................................................................................. 51
3.4 - Histology / Brain cannulation placements ................................................................................... 55
Chapter 4: Discussion .......................................................................................................................... 63
4.1 - Summary of current results ................................................................................................................ 63
4.2 - Selective D1 antagonist (SCH-23390) has no effect on sucrose self-administration,
but decreases cocaine self-administration.................................................................................................. 64
4.3 - D2-like antagonist (Sulpiride) has no effect on sucrose self-administration, but
decreases cocaine self-administration ..................................................................................................... 67
4.4 - D2-like agonist (Quinpirole) has no effect on sucrose- and cocaine selfadministration ........................................................................................................................................................ 70
4.5 - Implications of current finding ......................................................................................................... 71
4.6 - Conclusion.................................................................................................................................................. 74
References ............................................................................................................................................... 76
Appendix I: Behavioural traces showing drug pretreatment effects .................................85
Appendix II: Summary table for breakpoint, final ratio, and drug effects .................... 110
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List of Abbreviations
BP: Breaking Point
BST: Bed Nucleus of the StriaTerminalis
cAMP: Cyclic Adenosine Monophosphate
CeA: Central Nucleus of the Amygdala
CPP: Conditioned Place Preference
DR: Dorsal Raphe Nucleus
FR-1: Fixed Ratio-1 Schedule of Reinforcement
GA: Gauge
HCl: Hydrochloride
IP: Intra-Peritoneal
NAc: Nucleus Accumbens
PAG: Periaqueductal Gray Substance Region
PFA: Paraformaldehyde
PFC: Prefrontal Cortex
PR: Progressive Ratio Schedule of Reinforcement
Quinpirole: (D2-like DA receptor agonist) a.k.a. (-)-Quinpirole hydrochloride; Full
name: (-)-Quinpirole monohydrochloride, LY-171, 555, trans-(-)-(4aR)-4, 4a, 5, 6, 7, 8,
8a, 9-Octahydro-5-propyl-1H-pyrazolo[3,4-g]quinoline monohydrochloride; Linear
formula: C 13 H 21 N 3 ⋅HCl; Molecular weight: 255.79
RR: Retrorubral Nucleus
SCH-23390: (selective D1-like DA receptor antagonist) a.k.a. R (+)-SCH-23390
hydrochloride; Full name: R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5vii
tetrahydro-1H-3-benzazepine hydrochloride; Linear formula: C 17 H 18 CINO⋅HCl;
Molecular weight: 324.24
SNc: Substantia Nigra Pars Compacta
Sulpiride: (D2-like DA receptor antagonist) a.k.a. (S)-(-)-Sulpiride; Full name:
Levosulpiride, (S)-5-Aminosulfonyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2methoxybenzamide; Linear formula: C 15 H 23 N 3 O 4 S; Molecular weight: 341.43
VTA: Ventral Tegmental Area
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List of Figures
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Figure 12
Figure 13
Figure 14
Figure 15
Figure 16
Figure 17
Figure 18
Figure 19
Cocaine dose response curve ...............................................................................................8
Dose dependent effect of cocaine’s reinforcing efficacy ...........................................9
Extended amygdala and the drugs of abuse................................................................ 11
Schematic of dopamine projection from the ventral tegmental area (VTA) in
reward pathways ................................................................................................................... 12
Primary dopaminergic inputs into the extended amygdala ................................. 14
Reward circuitry involving the dopamine in the bed nucleus of the stria
terminalis (BST)...................................................................................................................... 15
Fixed ratio-1 (FR-1) operant response for sucrose self-administering rats . 36
Fixed ratio-1 (FR-1) operant response for cocaine self-administering rats .. 36
Operant response for sucrose self-administration under progressive ratio
(PR) schedule of reinforcement ....................................................................................... 37
Operant response for cocaine self-administration under progressive ratio
(PR) schedule of reinforcement ....................................................................................... 38
Bar graph showing the effects of vehicle and D1 selective dopamine receptor
antagonist, SCH-23390, on final ratio in sucrose and cocaine selfadministering rats.................................................................................................................. 45
Effects of vehicle and selective D1 dopamine receptor antagonist on sucrose
self-administration ................................................................................................................ 46
Effects of vehicle and selective D1 dopamine receptor antagonist on cocaine
self-administration ................................................................................................................ 47
Bar graph showing the effects of vehicle and D2-like dopamine receptor
antagonist, Sulpiride, on final ratio in sucrose and cocaine self-administering
rats................................................................................................................................................ 48
Effects of vehicle and D2-like dopamine receptor antagonist on sucrose selfadministration ......................................................................................................................... 49
Effects of vehicle and D2-like dopamine receptor antagonist on cocaine selfadministration ......................................................................................................................... 50
Bar graph showing the effects of vehicle and D2-like dopamine receptor
agonist, Quinpirole, on final ratio in sucrose and cocaine self-administering
rats................................................................................................................................................ 52
Effects of vehicle and D2-like dopamine receptor agonist on sucrose selfadministration ......................................................................................................................... 53
Effects of vehicle and D2-like dopamine receptor agonist on cocaine selfadministration ......................................................................................................................... 54
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Figure 20
Figure 21
Figure 22
Figure 23
Brain maps showing intra-cranial cannulation placements in sucrose selfadministering rats.................................................................................................................. 56
Brain maps showing intra-cranial cannulation placements in cocaine selfadministering rats.................................................................................................................. 59
Sucrose brain placement depicted through fluorescent beads at BST
injection site in actual rat brain under fluorescent microscopy ......................... 61
Cocaine brain placements depicted through fluorescent beads at BST
injection site in actual rat brain under transmitted microscopy and
fluorescent microscopy ....................................................................................................... 62
x
List of Tables
Table 1
Table 2
Table 3
Table 4
Table 5
Progressive ratio schedule of reinforcement table .....................................................6
Distribution of D1 and D2 receptors in the rat central nucleus of the
amygdala (CeA) and the bed nucleus of the stria terminalis (BST) .................. 15
Pseudorandom design of intra-BST drug injections ................................................ 32
Distribution of total intra-BST injections in sucrose- and cocaine- selfadministering rats .................................................................................................................. 33
Outline of intra-BST injections received by each rat .......................................... 34
xi
Chapter 1: Introduction
The bed nucleus of the stria terminalis (BST) is now considered an important
component of the reward and motivation circuit in the brain and a target for drugs of
abuse (Eiler et al., 2003; Walker et al., 2000; Epping-Jordan et al., 1998; Hasue and
Shammah-Lagnado, 2002). The BST contains several key components of the
dopaminergic system, including tyrosine hydroxylase positive terminals, dopamine and
cyclic adenosine monophosphate (cAMP) responsive Phosphoprotein of 32kDa (DARPP32), and dopamine receptors (Gustafson, 1990; Hasue and Shammah-Lagnado, 2002).
Given the key role of dopamine in motivated behaviours, we sought to determine whether
pharmacological manipulations of BST dopamine receptors would influence the
expression of naturally- or pharmacologically-driven operant behaviours in rats.
Specifically, the objective of the present study was to determine the role of BST D1- and
D2-like receptors in the expression of operant responding driven by either a natural
(sucrose) or a pharmacological (cocaine) reward combining the rat model of selfadministration with intra-cranial pharmacological manipulations. The general objective
of the study was to contribute to a better understanding of the neurobiological basis of
drug addiction, a necessary step towards better therapies for this neurological disease.
1.1 – What is Drug Addiction?
1.1.1 – Definition
Addiction is a widespread pathological condition characterized by drug-seeking
and taking, which is manifested as complex neural adaptations of learning the association
between the rewarding stimuli and other motivational/emotional cues. The Diagnostic
and Statistical Manual of Mental Disorders (American Psychiatric Association 2000)
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reports that the increase in energy and time drug users spend seeking access to drugs is a
salient feature of addictive behaviour.
1.1.2 – Animal Model of Drug Addiction
The use of drug self-administration in animal models has helped understanding of
certain elements of the process to drug addiction. Animal models of drug selfadministration have construct and predictive validity when it comes to its concordance
with the human form of addiction. Construct validity refers to the ability of the animal
model to provide explanatory power and to assess how various variables influence the
development of addiction. Predictive validity refers to the model’s ability to produce
valid predictions about the human form of addiction based on results from the animal
model (Koob and Le Moal, 2006). Another reason for using the animal model of drug
self-administration is that both animals and humans self-administer the same classes of
drugs. These drugs usually have very potent positive reinforcing properties such that
animals expend a tremendous amount of energy to perform many different tasks and
procedures to obtain them (i.e. pressing a lever multiple times to receive an intravenous
injection of drug, associating environmental cues with the reinforcing property of drugs
in conditioned place preference approaches) (Koob and Le Moal, 2006; Koob et al.,
1998). In summary, the animal model of self-administration is attractive because it
provides answers for a number of research questions: the abuse liability of reinforcing
compounds and their dose-response functions, patterns of drug intake, the effect of
response contingencies on drug intake, and the neural substrates of reinforcement (Arnold
and Roberts, 1997).
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Within the intravenous drug self-administration approach, several different
schedules of reinforcement exist. The self-administration approach is based on an
operant paradigm in which the reward is given and obtained in a response-contingent
manner. Thus, the animal earns the drug reward by meeting predetermined response
requirements (Skinner, 1938). Examples of schedules include the second-order schedule,
progressive-ratio (PR), and fixed ratio (FR) schedules of reinforcement. Second-order
schedule is where the completion of two successive schedules is required before a reward
is delivered. Therefore, the animal’s completion of the first schedule is followed by the
onset of the cue light in the chamber (i.e. FR10, where every 10 lever responses is
reinforced with the onset of light), which is contingent on the completion of the second
schedule (i.e. FR20, where every 20 light onsets is followed by the delivery of a single
reward). Thus, the animal gets the reward only after making 200 lever responses (Katz
and Goldberg, 1987). The PR schedule of reinforcement requires the animal to achieve
increasing number of lever responses for each subsequent reinforcer (Richardson and
Roberts, 1996). The FR schedule is where a reward is delivered after every nth response
(i.e. FR-1=every lever response is reinforced with a reward) (Richardson and Roberts,
1996). Using different schedules of reinforcement can provide important control
procedures for nonspecific motor and motivational actions such as increases in
exploratory activity and locomotion (Rowlett et al., 1996). Increasing the lever press
value requirement for obtaining a reinforcer, such as in a PR schedule, ensures that the
increase in behaviour is actually due to the animal maintaining the previous rate of drug
infusion to maintain the ‘high’ from the drug (Richardson and Roberts, 1996; Rowlett et
al., 1996). Another approach is to introduce a second, inactive lever into the
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experimental chamber so the selective increase in responding on the active lever can be
measured (Koob and Le Moal, 2006).
In the current study, animals were trained and tested under the FR-1 and PR
schedules of reinforcements. FR-1 schedule of reinforcement is where each lever
response is reinforced with a drug reward. Under this schedule, the rate of drug selfadministration is often the dependent measure for the drug’s reinforcing efficacy
(Richardson and Roberts, 1996; Arnold and Roberts, 1997). However, the rate of drug
self-administration in the FR-1 schedule is insensitive to changes in reinforcement
efficacy, especially with pharmacological manipulations of the reinforcing efficacy of
cocaine (Arnold and Roberts, 1997). Therefore, for the purpose of our experiment, the
FR-1 schedule was useful for training animals to associate lever pressing with drug
delivery. Once the rats attained stable pressing behaviour under the FR-1 schedule, they
graduate onto PR schedule (criteria outlined in ‘materials and methods’ section of thesis).
The PR schedule is useful for evaluating the reinforcing efficacy of a self-administered
drug (Richardson and Roberts, 1996; McGregor and Roberts, 1993; Roberts et al., 1989).
In the PR schedule, the response requirements for each successive reinforcer increases
systematically until the animal stops responding within a certain time window. This
point, defined as the breaking point (BP), is an indication of the maximum effort that an
animal is willing to expend to receive a certain number of drug infusion at a specific
concentration. Although the BP is defined as the point when the animal fails to attain a
drug infusion within a 60-minute period in most cases, this arbitrary rule is not applicable
in every case (Richardson and Roberts, 1996). Due to individual differences in pressing
patterns for each rat and differing levels of potency in neuropharmacological
4
manipulations, the criteria for defining BP vary substantially across studies. In some
cases, the BP criteria is set as the fixed amount of time for each subject to complete each
successive ratio, with variation from 12 minutes to 48 hours (Yanagita, 1973). (i.e. if the
fixed amount of time was set at 12 minutes, and the subject takes longer than 12 minutes
to achieve the response requirement for the second reward, then the second reward is
defined as the BP). In our study, however, we defined our BP as the point when the full
expression of operant behaviour has occurred during the experimental session (i.e. in
Sucrose self-administration group, the sessional length is set as 180 minutes; in Cocaine
self-administration group, the sessional length is set as 270 minutes). Thus, the BP for
sucrose- and cocaine- self-administering rats is the last reward received during their
sessional length time. Therefore, the BP would be defined as the final ratio completed
for the whole session length. The response ratio is the number of total lever presses
required for each reward, whereas the final ratio is the number of additional lever presses
between each successive reward (i.e. if the response ratio for the first reward is 1, and the
response ratio for the second reward is 3, then the final ratio between the first and the
second reward is 2. See table 1 for a table of final ratio and response ratio (total
pressing)). The PR schedule used in our study employed the PR series derived from the
equation: Response ratio = 5[𝑒𝑒 (𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖
𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛 ×0.2)
] − 5. Since the final ratio is
exponential in nature, a ln transformation was done on the final ratio so this value
conforms to the assumptions of parametric statistics (i.e. resulting in homogeneous
variance). Lastly, the most attractive reason for using PR schedule is that stable lever
pressing behaviour requires only 3 to 10 days to acquire. Once attained, stable behaviour
can be maintained for long periods of time, usually over 50 sessions (Richardson and
5
Roberts, 1996). The use of PR schedule of drug self-administration in animals provide
usable data because: the use of PR schedule is a valid and flexible method for measuring
drug reinforcement in humans, and stimulant drugs with abuse potential maintain
responding in humans under this schedule (Stoops, 2008).
Table 1. The progressive ratio schedule of reinforcement. Infusion is the
breakpoint (BP) determined from raw behavioural traces with lever presses. BP
is the last infusion before session ends. Each infusion value corresponds to a
final ratio value, which indicates the additional number of lever presses between
each successive infusion. Total pressing is the cumulative number of lever
presses. To use this table, match the number of infusions (BP) to its final ratio
value and calculate the effect of the drug (expressed as change in final ratio in
percentage) by dividing the final ratio of the injection day by the average final
ratio of the 3 pre-injection days.
Infusion
Final Ratio
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
1
2
4
6
9
12
15
20
25
32
40
50
62
77
95
118
145
178
219
268
328
402
492
603
737
6
Total Pressing
1
3
7
13
22
34
49
69
94
126
166
216
278
355
450
568
713
891
1110
1378
1706
2108
2600
3203
3940
For the current study, we chose to use the self-administration approach. This is
because the self-administration paradigm, combined with the use of the PR schedule of
reinforcement, more closely resembles the progressive and deteriorating human condition
of the drug addiction disorder. Human addicts often self-administer drugs intravenously,
and the degree of addiction slowly progresses with escalating dosage and time, up to a
certain point. More specifically, they self-administer cocaine to the extent of funds
available and within the limits of tolerable side effects (i.e. nervousness, dysphoria, and
depression) (Koob and Kreek, 2007; Liu et al., 2005). In addition, under the PR
schedule, it has been demonstrated that dose (Roberts et al., 1989, 2007), injection speed
(Liu et al., 2005), drug deprivation (Morgan et al., 2002), and circadian rhythm can all
interact to influence BP. Therefore, the animal model of self-administration can be used
to model the human condition very closely with some modifications in these factors.
The relationship between the stimulant effects of cocaine to dose can be best depicted on
an inverted U-shaped dose-response function (Figure 1) (Richardson and Roberts, 1996).
On the curve, the 1.5 mg/kg/injection cocaine dose at the tip of the U-shaped curve
suggests that this is the dose with the highest acute reinforcing efficacy. On the other
hand, the 3.0 mg/kg/injection dose is ‘over the top’, where it is starting to be on the
descending limb of the curve. Therefore, rats self-administering cocaine at this dose may
not find cocaine to be reinforcing. This is evident in an experiment showing that the
increases in cocaine’s reinforcing effects are dose-dependent. Breakpoints (BP)
maintained by 0.75 and 1.5 mg/kg/injection increase progressively over days, whereas
0.38 or 3.0 mg/kg/injection shows not much change (Figure 2) (Liu et al., 2005; Roberts
et al., 2007). Consequently, the rats in the current study self-administered a cocaine dose
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of 0.75 mg/kg/injection. This concentration is ideal because it is still on the ascending
limb of the curve, located approximately at about 25% away from the top dose (1.5
mg/kg/injection) (see Figure 1). With this dose, rats will still find cocaine relatively
reinforcing and their pressing behaviour can still increase or decrease with
pharmacological manipulations.
Figure 1. Cocaine dose-response curve showing the relationship between
dose of cocaine given (mg/kg/injection) and the final ratio completed on a
progressive ratio (PR) schedule. Graph depicts an inverted U-shape,
showing the maximal tolerable dose of cocaine that can be given to a rat
before the appearance of aversive effects at 1.5mg/kg/injection. To
facilitate the observation of the effects of intra-bed nucleus of stria
terminalis (BST) drugs given on cocaine self-administration behaviour, the
dosage used in the current experiment was set as 0.75mg/kg/injection.
(Adapted from Richardson and Roberts, 1996)
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Figure 2: Depiction of the dose-dependent effect of cocaine’s reinforcing
efficacy. Breakpoints maintained by 0.75 and 1.5mg/kg/injection increased
across days, showing that cocaine is reinforcing at these dosages (still on
the ascending limb of the cocaine dose-response curve). Cocaine dosages
that are either too low (0.38mg/kg/injection) or too high
(3.0mg/kg/injection) are not able to induce reinforcing feelings of cocaine
self-administration (so it shows stable response over days).
(Adapted from Roberts et al., 2007)
1.1.3 – Neural Circuits of Reward and Motivation
The reward circuitry involved in obtaining reinforcing stimuli (i.e. sex, cocaine,
food, water) requires the dopamine mesocorticolimbic system (Caine and Koob, 1994; Di
Chiara and Imperato, 1988; Thomas et al., 2008). This system comprises primarily of the
ventral tegmental area (VTA), the nucleus accumbens (NAc), the prefrontal cortex (PFC),
and associated limbic structures (i.e. hippocampus, amygdala) (Kauer and Malenka,
2007). The major site of dopamine neurons in the midbrain region is in the VTA. The
VTA is involved in the processing of rewarding and aversive properties of environmental
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stimuli. It provides information for the reward value of an action or stimulus to modify
future behaviour and addictive behaviours (Fields et al., 2007; Thomas et al., 2008;
Schultz, 1997; Wise, 1996). The NAc is the principal target of VTA dopamine neurons
and it mediates the rewarding properties of survival related natural stimuli (i.e. food and
sex) and drugs of abuse (Kelley and Berridge, 2002; Di Chiara and Imperato, 1988; Wise
and Rompre, 1989). Furthermore, the NAc contains two subregions with distinctive
functions, namely the core and the shell of the NAc. The NAc core is important for
instrumental learning and cue-induced reinstatement of drug-seeking behaviour (relapse),
and the NAc shell is involved with mediating the primary reinforcing effects of addictive
drugs (Beurrier, et al., 2001; Pennartz et al., 1994). The rewarding effect of drugs of
abuse is heavily dependent on the dopamine signaling in the NAc. Cocaine can be selfadministered into the rat NAc (McKinzie et al., 1999; Rodd-Henricks et al., 2002), and
reinstatement can be induced with injection of amphetamine into the NAc (Stewart and
Vezina, 1988).
The PFC correlates the overall motivational significance of the stimuli to
determine the intensity of behavioral responding (Goldstein and Volkow, 2002). Thus,
the cognitive processes required for goal-directed behaviours depend on the PFC function
(Goldstein and Volkow, 2002; Hyman, 2005). The amygdala, on the other hand, is
associated with fear conditioning and the processing of negative emotions. However,
since this structure is also involved in the processing of positive emotions and the
learning about the positive value of stimuli, the amygdala may act within the
mesocorticolimbic dopamine system to integrate the positive and negative value of an
environmental stimulus (i.e. drug of abuse, natural food reward, sex, stress).
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The dopaminergic connection between the mesolimbic dopamine system and the
extended amygdala is hypothesized to be responsible for the reinforcing effects of abused
drugs (Ettenberg et al., 1982; Koob 1999, 2003). The extended amygdala is made up of
several basal forebrain regions that share similar morphology, immunoreactivity, and
connectivity (Sun et al., 1991; Freedman and Cassel, 1994). The regions include the
BST, the central nucleus of the amygdala (CeA), and the shell of the NAc (Figure 3)
(Koob, 1999). The extended amygdala receives several afferents from limbic structures
(i.e. basolateral amygdala and hippocampus) and sends efferents to the VTA, and lateral
hypothalamus. Thus, the unique positioning and connectivity that the extended amygdala
has with other brain regions enables it to serve as an interface for emotional and motor
system (Koob, 2003). Consequentially, the extended amygdala works in close
association with the mesolimbic dopamine system for the generation of goal directed
motivational behaviour for rewards (see Figure 4).
Figure3: The involvement of each component of the extended amygdala in
drugs of abuse. Specifically, the bed nucleus of the stria terminalis (BST),
the shell of nucleus accumbens (NAc), and the central extended amygdala
(CeA) are all heavily associated with the reinforcing properties of cocaine.
They mediate these changes by regulating the level of dopamine
neurotransmission associated with cocaine addiction.
(Adapted from Koob, 1999)
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Figure 4: Schematic showing the dopaminergic projections from the
ventral tegmental area (VTA) to various areas of the brain implicated in
coordinating the reward and motivation neural circuit (i.e. basolateral
amygdala, prefrontal cortex (PFC), nucleus accumbens (NAc), and the
extended amygdala).
The BST, a key part of the extended amygdala, projects to several areas of the
brain (i.e. somatomotor, central autonomic control, neuroendocrine) and thus is
strategically placed in the brain to process afferent inputs and efferent outputs of
emotions, motivation and drugs of abuse (Koob, 1999; Dong et al., 2000, 2001; Dong and
Swanson, 2003, 2004).
The BST is highly interconnected with elements of the reward circuitry, the
mesolimbic dopamine system. The dorsal BST projects to the NAc shell and VTA (Dong
et al., 2001), therefore the BST also regulates the firing of dopaminergic cells within the
VTA (Georges and Aston-Jones, 2002). In addition, the BST also receives strong
12
dopaminergic inputs from the VTA, substantia nigra pars compacta, retrorubral area
(RR), and periaqueductal gray (PAG) through the medial forebrain bundle (Deutch et al.,
1988; Phelix et al., 1992; Freedman and Cassell, 1994; Hasue and Shammah-Lagnado,
2002; Meloni et al., 2006) (See Figure 5). In accordance to the fact that the BST is
heavily innervated by dopamine neurons, drugs of abuse such as opiates, ethanol,
amphetamine, nicotine, and cocaine have been shown to increase dialysate dopamine
levels in the BST, at an even higher level than in the NAc shell (Di Chiara and Imperato,
1988; Di Chiara et al., 1999; Carboni et al., 2000). The dopamine receptors in the BST
exist in a 3:2 ratio of D1: D2 receptors (Scibilia et al., 1992; Mengod et al., 1992) (See
Table 2), showing that there is a higher concentration of D1 receptors in the BST (See
Figure 6). Thus, the projections from and to the BST implicate the importance of this
structure in modulating the physiological and pathological reward-related behaviours
such as cocaine and food self-administration. (Aston-Jones and Harris, 2004; Dumont et
al., 2005).
13
RR
SNc
VTA DR
PAG
Figure 5: Bar graph showing the relative dopaminergic input into the
extended amygdala. (A8 = retrorubral nucleus (RR); A9 = substantia
nigra pars compacta (SNc); A10 = ventral tegmental area (VTA);
periaqueductal gray substance region (PAG); dorsal raphe nucleus
(DR)).
(Adapted from Hasue and Shammah-Lagnado, 2002)
14
Table 2. The distribution of D1 and D2 dopamine receptors in subregions of
the central nucleus of the amygdala (CeA) and the bed nucleus of the stria
terminalis (BST). The BST contains relatively higher densities of dopamine
receptors than the CeA. There are more D1 receptors than the D2 receptors in
the BST.
(Adapted from Scibilia et al., 1992)
Figure 6: Schematic showing the connection between the extended
amygdala circuit and the mesolimbic dopamine system in generating goaldirected motivational behaviours for attaining rewards. Specifically, the
bed nucleus of the stria terminalis (BNST) is innervated by dense
dopaminergic projections from several areas of the brain (i.e. ventral
tegmental area (VTA), retrorubral nucleus (RR), substantia nigra pars
compacta (SN), nucleus accumbens (NAcc), and the basolateral amygdala).
(Adapted from Koob and Le Moal, 2006)
15
1.1.4 – Dopamine in Motivated Behaviours
Dopamine, as a catecholamine neuromodulator in the mammalian brain, controls
a variety of functions including positive reinforcement of drugs, food intake, locomotor
activity and endocrine regulation (Pierce and Kumaresan, 2006). Dopamine receptors are
classically divided into five G protein-coupled receptor subtypes, namely the D1-like and
the D2-like receptor subtypes. The D1-like receptors consist of the D1 and the D5
dopamine receptors, and they generally couple to the G s protein to activate adenylyl
cyclase. The D2-like receptors consist of the D2, D3, and D4 dopamine receptors, and
they couple to the G i protein to inhibit adenylyl cyclase (Missale et al., 1998). With
chronic administration of opiates, cocaine and alcohol, there is an up-regulation of the
cAMP pathway in the NAc (Nestler and Aghajanian, 1997; Terwilliger et al., 1991; Ortiz
et al., 1995). Increased dopamine transmission in the NAc is critical for the maintenance
of the psychostimulant self-administration behaviour (Pierce and Kumaresan, 2006; Di
Chiara and Imperato, 1988). On the other hand, dopamine-depleting lesions in the NAc
decrease self-administration of cocaine (Caine and Koob, 1994; Gerrits and Van Ree,
1996; Di Chiara and Imperato, 1988). Drugs not abused by humans (i.e. atropine,
imipramine) do not modify synaptic dopamine concentrations (Di Chiara and Imperato,
1988).
In addition, during cocaine self-administration sessions, NAc dopamine levels
decline between cocaine infusions. This decrease may trigger responding for cocaine
self-administration in order to maintain extracellular dopamine above a certain threshold
level (Ranaldi, et al., 1999; Wise et al., 1995). Furthermore, self-administration studies
consistently show that systemically administered D1-like antagonists decrease the
16
reinforcing efficacy of cocaine (Bergman et al., 1990; Hubner and Moreton, 1991;
Winger, 1994) and D2-like antagonists reduce cocaine reinforcement (Bergman et al.,
1990; Caine et al., 2002). More specifically, bilateral intracerebral injections of the D1
receptor antagonist, SCH-23390, into the NAc attenuated the reinforcing effects of the
self-administered cocaine under both FR and PR schedule of reinforcement (Caine et al.,
1995; McGregor and Roberts, 1993). Thus, both the D1-like and the D2-like dopamine
receptors are essential for the reinforcing efficacy of psychostimulants.
Dopamine transmission in the brain is also responsible for the ability of food to
establish and maintain response habits and conditioned preferences. The auditory, visual,
tactile, olfactory and gustatory stimuli of foods activate the dopaminergic system in the
brain to energize feeding and reinforce food-seeking behaviour (Wise, 2006). Dopamine
neurons do not discriminate amongst various appetitive stimuli (i.e. between different
foods, liquids, sensory modalities) (Schultz, 1997; Romo and Schultz, 1990; Schultz and
Romo, 1990; Mirenowicz and Schultz, 1996). Pretreatment with dopamine antagonists
results in a dose-dependent decrease in how quickly the animals learn to lever-press for
food (Wise and Schwartz, 1981). Thus, sucrose operant-responding acts as a control for
the effect of dopamine on natural reinforcers in this study.
The dopamine inputs to the extended amygdala originate from three cell groups
labeled as A8 (retrorubral nucleus (RR)), A9 (substantia nigra pars compacta), and A10
(VTA). The BST and the CeA, the two major parts of the extended amygdala, are also
heavily innervated by the dopamine neurons. Thus, the extended amygdala is suggested
to be involved in the motor expression of drives and motivation through its projections to
17
the brainstem areas and the lateral hypothalamus (Di Chiara, 1995; Bjorklund and
Lindvall, 1984; Hasue and Shammah-Lagnado, 2002).
1.1.5 – Mechanisms of Psychostimulants Effects
Psychostimulant drugs, such as cocaine, are drugs of high-abuse potential. They
can be ingested via several routes of administration: intranasally, intravenously, and
smoked. When administered, cocaine produces stimulant effects such as sustained
performance, heightened arousal, alertness, and motor activity (‘stereotyped behaviour’,
characterized by increasing rate of repetitive behaviour), accompanied by feelings of
intense euphoria (Koob and Le Moal, 2006). They bind to monoamine transporters
located on monoaminergic nerve terminals. Cocaine inhibits all three monoamine
transporters: dopamine, serotonin, and norepinephrine; thereby, potentiating the
monoaminergic transmission. The relative monoamine transporter affinity of cocaine is
serotonin > dopamine > norepinephrine, where the interaction of cocaine with dopamine
best predicts the reinforcing potency of cocaine (Pierce and Kumaresan, 2006; Ritz and
Kuhar, 1989). The prevention of the reuptake of dopamine by the monoamine transporter
inhibitors results in the accumulation of dopamine levels within the synaptic cleft of
monoamine synapses.
The use of cocaine as a positive reinforcer in our experiment is beneficial because
the pharmacological manipulation of cocaine reinforcement can be observed more easily
due to its fast kinetics. Cocaine has a short half-life of about 40 minutes and thus it can
be excreted much faster (Javid et al., 1983). In addition, cocaine has a more robust
reinforcing effect than other drugs. Animals learn to self-administer cocaine more
readily than any other drug and they work harder for it. This is shown through an
18
experiment done by Yanagita (1975), where the reinforcing potency of cocaine was
compared with other drugs, under the PR schedule of reinforcement. In the BST, cocaine
dose dependently increases the extracellular dopamine (Carboni et al., 2000), suggesting
that the BST is indeed sensitive to the dopamine stimulant actions of cocaine.
1.2 – The BST: An Important Component of the Reward and Motivation Pathway and A
Target for Drugs of Abuse
1.2.1 – Definition & Anatomy
The BST, as an important part of the reward motivational pathway, lies next to the
anterior commissure in the basal forebrain to make up the rostral part of the extended
amygdala (Fudge and Haber, 2001; McDonald et al., 1999). It is a complex forebrain
structure that is composed of heterogeneous cell groups (Ju and Swanson, 1989). These
different subnuclei are contained within the anterior and the posterior divisions of the
BST (Ju and Swanson, 1989). The anterior division consists of the anterolateral
(consisting of oval, juxtacapsular, rhomboid, and fusiform nuclei) (Larriva-Sahd, 2004),
anterodorsal, and anteroventral (consisting of dorsomedial, dorsolateral, magnocellular,
and ventral nuclei) cell groups (Ju and Swanson, 1989). The posterior division, however,
lies caudal to the primary bundles of the dorsal and commissural bundles of the stria
terminalis and contains six recognizable components: the principal nucleus,
interfascicular nucleus, transverse nucleus, premedullary nucleus, dorsal nucleus, and
cell-sparse zone (Ju and Swanson, 1989). Each of the BST subnuclei, with its
heterogeneous cytoarchitectural nature and projections to various parts of the brain, is
responsible for coordinating different combinations of vital functions.
19
Specifically, with the dopaminergic inputs that the BST receives from the VTA,
substantial nigra pars compacta, RR and periaqueductal gray (Hasue and ShammahLagnado, 2002), the BST may thus be critical for the reward and motivation behaviours.
1.2.2 – BST Dopamine and Motivated Behaviour
Several studies have demonstrated the importance of both the D1- and the D2-like
dopamine receptors in modulating the reinforcing efficacy of both the natural food and
cocaine, with most of the research done via systemic, intra-NAc injections, intra-VTA
injections, intra-amygdala injections, intra-hippocampus injections, intra-PFC injections,
and intra-striatum injections (Weissenborn et al., 1996; Khroyan et al., 2003; Bergman et
al., 1990; Kuo, 2002; Bari and Pierce, 2005; Terry and Katz, 1992; Caine et al., 1995;
Johnson and Kenny, 2010; McGregor and Roberts, 1993, 1995; Ranaldi and Wise, 2001;
Berglind et al., 2006; Andrzejewski et al., 2006). In most of these studies, both the D1
and D2 antagonists decreased the reinforcing value of cocaine (Koob et al., 1987;
Egilmez et al., 1995; Caine and Koob, 1994; Hubner and Moreton, 1991). Studies with
knock-out mutant mice suggest that D1 receptors are essential (Caine et al., 2007) and D2
receptors might be less involved in cocaine self-administration than the D1 receptors
(Caine et al., 2002). However, studies using dopamine agonists gave less conclusive
results. Depending on the dose of the cocaine self-administered and the dosage of the
agonists given, the agonists can either produce a decrease or an increase in the rewarding
effects of the drugs (Weissenborn, et al., 1996; Katz et al., 2006; Caine et al., 1999).
Up to now, only a few studies demonstrate the potential significance of BST
dopamine in the regulation of rewarding behaviours. As mentioned above, the BST, the
CeA, and the NAc shell are homologous in nature and share a role in the acquisition and
20
expression of emotions and of appetitive behaviour (de Olmos and Heimer, 1999;
Dumont et al., 2005). Due to the dense dopamine innervations in the BST, it is suggested
that cocaine dose dependently increases the extracellular dopamine in the BST. This
implicates the fact that the BST, similar to the shell of the NAc, is particularly sensitive
to dopamine stimulant actions of drugs of abuse (Carboni et al., 2000). In addition, it is
suggested that the development of addiction may coincide with alternations of
dopaminergic activity following chronic exposure to substances of abuse. D1 and D2
receptor densities within the extended amygdala (i.e. in the NAc core, shell, and
amygdala) increase with both continuous alcohol intake and deprivation of alcohol (Sari
et al., 2006). Furthermore, the D1 receptors in the BST are particularly essential for
modulating alcohol-motivated behaviours. Specifically, SCH-23390 dose-dependently
reduces both alcohol- and sucrose-motivated responding; however, the D2 antagonist
only elicits small decreases in sucrose responding (Eiler II et al., 2003). D1 dopamine
receptor in the BST may be important in cocaine self-administration. Specifically, intraBST injections of SCH-23390 significantly increase the rate of cocaine selfadministration under the FR-5 schedule of reinforcement, implicating partial attenuation
of the reinforcing effects of cocaine (Epping-Jordan et al., 1998). However, the use of
the rate of lever pressing under the FR-5 schedule as the only dependent measure is
equivocal. This is because the rate of lever responding is not sensitive to changes in
reinforcement efficacy, especially with pharmacological manipulations of the reinforcing
efficacy of cocaine (Arnold and Roberts, 1997). In addition, in the Epping-Jordon et al.
(1998) study, the use of only cocaine infusions as the sole reinforcer and SCH-23390 as
the sole pharmacological manipulation agent may be intrinsically flawed. Since cocaine
21
is a highly abused drug, the decrease of the reinforcing efficacy of cocaine seen in rats
treated with SCH-23390 is predictable. Thus, testing the effect of SCH-23390 in a
different kind of reinforcer (i.e. food, sucrose) will provide a more complete picture of
the effect of SCH-23300 on BST dopamine receptors. With this in mind, we will use the
PR schedule of reinforcement to train and test our rats to self-administer either sucrose
pellets or cocaine infusions. With the increase in response requirement for each
successive reinforcer, it can be assured that our measure of BP and final ratio is truly a
reflection of the reinforcing efficacy of either sucrose or cocaine rewards, not due to the
animals’ random lever pressing behaviour (Richardson and Roberts, 1996; Koob and Le
Moal, 2006). In addition, the stable lever pressing response under the PR schedule and
the sensitivity of the PR schedule to various parameters (i.e. cocaine dose, dopamine
antagonists, injection speed) will enable us to quantify the rats’ behaviour as a reflection
of the effect of our pharmacological manipulations (Roberts et al., 1989, 2007; Liu et al.,
2005). Furthermore, as there are also D2 receptors in the BST, we will also test the
contribution of the BST D2 receptors to the reinforcing efficacy of both cocaine and
sucrose rewards. Therefore, in this study, we will be focusing on elucidating the
potential dopamine transmission mechanisms in the BST through the D1 and D2
receptors. This will be demonstrated via rats that self-administer cocaine infusions and
sucrose pellets.
1.3 – Research Aims and Hypothesis
1.3.1 – Research Aims
Since previous research showed that intra-BST injection of the selective D1receptor antagonist SCH-23390 decreased the reinforcing efficacy of cocaine in self-
22
administering rats under the FR schedule of reinforcement (Epping-Jordan et al., 1998;
Eiler II et al., 2003), and electrophysiological data (Krawczyk et al., 2010) demonstrated
an upregulation of BST D1 receptors and a downregulation of D2 receptors in rats with
chronic cocaine treatment, this behavioral study was designed to determine the relative
contributions of the BST D1- and D2 receptors to the expression of operant responding
driven by either a natural (sucrose) or pharmacological (cocaine) reward (Krawczyk et
al., 2010). Given that the BST contains both the D1-like and D2-like dopamine
receptors, rats will receive intra-BST microinjections of the selective D1 antagonist SCH23390, the D2-like antagonist sulpiride, and D2-like agonist quinpirole. All of the selfadministration training and experiments will be performed under the PR schedule of
reinforcement.
1.3.2 - Hypothesis
Based on the fact that rats with chronic cocaine treatment exhibited an
upregulation of BST D1 receptors and a downregulation of BST D2 receptors (Krawczyk
et al., 2010), and because the BST is critical in natural and pharmacological reward selfadministration in rats due to its heavy dopaminergic inputs from neighboring structures
and its expression of dopamine receptors, we hypothesize that pharmacological
manipulation of the BST D1 and D2 receptors influences the reinforcing efficacy of
pharmacological (cocaine) rewards under the PR schedule of reinforcement.
23
Chapter 2: Materials and Methods
2.1 - Animals
Twenty-seven male Long-Evans rats (Charles River Laboratories, St. Constant,
Quebec, Canada) weighing between 225 -250 g upon arrival were housed in pairs and
were kept on a 12-hour reversed light-dark cycle (09:00 hour lights off –21:00 hour lights
on) in a temperature-controlled animal holding room (temperature: ~22°C; humidity:
~34%). They were handled every day for 3 days before cannulation surgeries. After the
surgery, rats were moved into the experimental room, and they had 3 days of postoperational recovery period before the start of the experiment. All behavioral testing was
conducted during the dark active phase, where water and food were provided ad libitum.
Cages and bedding were changed once a week. Treatment and housing of the rats were
in accordance to the guidelines of the Canadian Council on Animal Care (CCAC) and all
conducted experiments were approved by the Queen’s University Animal Care
Committee (UACC). Some rats (n = 5) were not included in the data analysis because of
complications after cannulation surgeries. The total number of rats used in the final data
analysis was 22.
2.2 - Drugs
The three drugs used in the present study were: SCH-23390 [R(+)-7-Chloro-8hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (R (+)SCH-23390 hydrochloride; Sigma-Aldrich, Oakville, Ontario, Canada) in a total bilateral
dose of either 1.6, 3.2, or 6.4 µg/µL]; Sulpiride [ Levosulpiride, (S)-5-Aminosulfonyl-N[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxybenzamide ((S)-(-)-Sulpiride; Sigma-Aldrich,
Oakville, Ontario, Canada) in a total bilateral dose of 3.2 µg/µL], and Quinpirole [(-)24
Quinpirole monohydrochloride, LY-171, 555, trans-(-)-(4aR)-4, 4a, 5, 6, 7, 8, 8a, 9Octahydro-5-propyl-1H-pyrazolo[3,4-g]quinoline monohydrochloride ((-)-Quinpirole
hydrochloride; Sigma-Aldrich, Oakville, Ontario, Canada) in a total bilateral dose of 3.0
µg/µL. All of the drugs prepared for intra-BST microinjections were dissolved and
diluted in sterile saline. Intra-BST saline microinjections were also given to serve as a
vehicle control. All the drugs and saline solutions were stored in 20µL aliquots at -20°C
until experiment day.
Cocaine-hydrochloride (HCl) (MediscaPharmaceutique, Saint-Laurent, QC) was
dissolved in sterile saline (0.9% NaCl isotonic saline) in a concentration of half-strength
(0.75 mg/kg/injection) and adjusted to the rats’ physiological pH of 7.2 – 7.6.
2.3 - Apparatus
Behavioral data were collected using six operant conditioning chambers
(MedAssociates, St-Albans, VT) with access to food and water ad libitum. Each chamber
was equipped with a Plexiglass front and back panel (W(30cm) x H(30cm) x L(30cm)), a
cue light, and an operant retractable lever mounted 5 cm above the floor. With each
depression on the operant lever, it would administer either one 45mg sucrose pellet (Test
Diet, Richmond, IN) for the sucrose self-administration group, or a 0.12 mL infusion of
2.5mg/mL (equivalent to 0.75 mg/kg/injection) cocaine-HCl for the cocaine selfadministration group. Following each reward, the lever was retracted for 20 seconds
(time-out period of 20 sec), followed by the simultaneous activation of the cue light.
After the time-out period of 20 seconds, the operant chamber went dark again, with the
re-appearance of the operant lever. Intravenous infusions of cocaine were delivered by a
Razel infusion pump (MedAssociates) via polyethylene-50 tubing (PE-50) (HRS Plastics
25
One) enclosed within a metal spring tether that was reaching towards a swivel to allow
free movement of the animal, and the intravenous catheter port linking onto the dorsal
region of the rat. Raw behavioral data traces for each experimental session were
recorded using the MedPCIV software (MedAssociates) for later analysis.
2.4 - Cannulation Surgery
Upon arrival, rats were allowed a minimum of 3 days to acclimatize to the new
surroundings before the cannulation surgery. Bilateral intracranial guide cannula
implantation surgery was performed to allow direct infusion of the drug solution into the
oval region of the dorsal BST. In addition, intravenous cannulation surgery was done to
allow intravenous infusion of cocaine for the cocaine self-administration group of rats.
When rats were ready for surgery, they were weighed and anesthetized with
ketamine-xylazine (75mg/kg: 10mg/kg, intra-peritoneal (IP) injection). Stock ketamine
hydrochloride injection (100mg/mL) (Bioniche Animal Health, Belleville, ON, Canada).
Stock xylazine (20mg/mL) sterile injectable (Bayer HealthCare Animal Health Division,
Toronto, ON, Canada) via intraperitoneal injection in the animal holding room and then
transported in their home cages into the surgery room. The use of injectable induction
agents facilitated stereotaxic placement (TSE Systems) into the ear bars, and decreased
peripheral bleeding to allow for the maintenance of surgical site clearance.
Sucrose self-administration rats received intracranial surgery. After the rats have
been anesthetically induced, 2% xylocane jelly (Mississauga, ON, Canada) was applied
to their ears, then their heads were shaved and fixated into the stereotaxic instrument and
secured with nose-tooth bar and non-rupture ear bars where they received 1.75%
isoflurane gas (from 99% isoflurane inhalation anaesthetic, distributed by Benson,
26
Markham, ON, Canada) mixed with oxygen in a vaporizer system (Forane vaporizer
system, Ohio Medical Products, Madison, WIS). The oxygen (Praxair, Mississauga, ON,
Canada) was delivered inside the chamber at 5.0 L/min for the entire duration of the
surgery to maintain anaesthesia. Throughout the whole duration of the surgery, the rat’s
respiratory rate was closely monitored so the level of isoflurane gas may be adjusted to
ensure the rat stayed under constant stable anaesthesia. Tear gel (Novartis
Pharmaceuticals, Mississauga, ON, Canada) was applied to the eyes and reapplied during
surgery to prevent rat’s eyes from drying out. Then, an injection of Anafen (5mg/mL,
subcutaneous; Merial Canada, Quebec, Canada) was given, followed by an injection of
10mL of saline (0.9% NaCl, subcutaneous; Baxter Corporation, Mississauga, ON,
Canada). Before making the incision for the intracranial surgery, 4% Germi-stat
(Germiphene Corporation, Brantford, ON, Canada), 70% isopropyl alcohol (HealthCare
Plus Jedmon Products, LTD, Toronto, ON, Canada), and iodine (1% free iodine, Rougier
Pharma, Canada) was applied twice in this sequence to sanitize the surgical site and
prevent potential infections during surgery. A vertical incision was made from between
the eyes to slightly behind the ears. Then, the incision site was cleaned up so Bregma
could be identified. Two holes were drilled through which two single guide cannulae
(6.5mm in length, Plastics One Inc, HRS Scientific, Roanoke, VA) were bilaterally
implanted into the oval region of the dorsal BST (-0.6 A.P. ± 1.9 M.L. -6.5 D.V.,
measured relative to Bregma). [**Sucrose self-administration rats with Rat IDs that are
before L143 were implanted with a shorter set of bilateral guide cannuale: 5.8mm in
length, Plastics One Inc, HRS Scientific, Roanoke, VA] Four additional holes were
drilled around the first two where anchoring screws (0.080 x 0.125”, Plastics One Inc,
27
HRS Scientific, Roanoke, VA) were inserted, and dental acrylic cement was filled around
the screws and guide cannulae to hold the head attachment securely in place. To protect
from pathogens and ensure patency, an autoclaved dummy cannula (30GA stylette,
0.014in-0.35mm, with 0 protrusion) was inserted into the guide cannulae and covered
with a sterile dust cap. Once the dental acrylic had hardened, the head incision was
sutured up, the anaesthetic gas was turned off and oxygen (1.0 L/min) was given for 5
minutes. An injection of 10mL of saline (0.9% NaCl, subcutaneous; Baxter Corporation,
Mississauga, ON, Canada) was given to supplement for fluid loss during surgery. For the
whole duration of the surgery, the rats were kept warm while they were under anaesthesia
on the stereotaxic apparatus with a heating pad beneath them (medium heat). For
analgesia following surgery, Anafen (5mg/mL, subcutaneous; Merial Canada, Quebec,
Canada) was given for three days after the surgery, during which they were kept in the
experimental room for recovery. After three days of recovery, the rats started their
experiments.
Cocaine self-administration rats received intravenous cannulation surgery right
after the completion of the intracranial cannulation surgery. The jugular cannula
implantation surgery started with the transport of the rat from the stereotaxic apparatus to
the intravenous surgery station. Rats were kept under anaesthesia by continuing to
receive 1.75% isoflurane gas (from 99% isoflurane inhalation anaesthetic, distributed by
Benson, Markham, ON, Canada) mixed with oxygen in a vaporizer system (Forane
vaporizer system, Ohio Medical Products, Madison, WIS). The oxygen was delivered
inside the chamber at 5.0 L/min for the entire duration of the surgery. The right jugular
vein was isolated so a silastic cannula (cannula made up of: Polypropylene mesh 500
28
micron (28mm in diameter), Silicone tubing (Outer diameter: 0.63mm x Inner diameter:
0.30mm x Wall: 0.17mm), silicon bead that is 90mm away from main catheter, stainless
steel tubing: (Outer diameter: 0.71mm x Inner diameter: 0.406mm), CamCaths, Ely, UK)
could be inserted 32mm into the vein, towards the right atrium. The cannula was
connected to a port secured onto the dorsal side of the rat via a hernia mesh sutured to the
muscle wall using 3-0 silk thread (Ethicon, Somerville, NJ, USA). After the mesh was
sutured in place, the rat’s back was sutured so that his skin is tightly lying against the
dorsal port. In addition, the neck incision from the insertion of the silastic cannula was
sutured up using both internal suture and a normal suture to ensure the threads stayed in
place. After the surgery, anaesthetic gas was turned off and oxygen (1.0 L/min, Praxair,
Mississauga, ON, Canada) was given for 5 minutes or longer to allow rats to recover
from surgery faster. Then, they received an injection of 10mL of saline injection (0.9%
NaCl, subcutaneous; Baxter Corporation, Mississauga, ON, Canada). Afterwards, they
were returned to the experimental room, where they received three days of postoperational Anafen injections (5mg/mL, subcutaneous; Merial Canada, Quebec, Canada)
for analgesia. After surgery, intravenous cannulae were flushed with heparin (70U in
0.2mL sterile saline) twice a day throughout the rats’ whole experimental career to
prevent clotting and conserve patency.
2.5 - Intra-BST Drug Micro-Injection
Rats received micro-injections based on pre-established criteria. Firstly, rats were
trained on the progressive ratio (PR) schedule of reinforcement on the operant paradigm
for equal or more than 10 days before they could receive their first micro-injection.
Secondly, the candidate must reach a stable BP over a period of one to three consecutive
29
days (total rewards received at BP ±3, based on a criterion of ≤8% deviation from the LN
final ratio average) (see data analysis section of the thesis for explanation and formulae
used to calculate the LN final ratio average deviation). In this experiment, a BP was
defined as the last infusion received before the end of the session, at which the full
expression of operant responding has occurred (i.e. in Sucrose self-administration group,
the session length is set as 180 minutes; in Cocaine self-administration group, the session
length is set as 270 minutes). Therefore, the BP is the final ratio completed for the whole
session length. This is used as a measure of the maximum motivational effort that the rat
is willing to exert to attain the reward. Final ratio is the number of presses in the last bout
of pressing required to achieve BP. If all the criteria were met, the rat would receive
intracranial injections into the BST.
Micro-injections occurred immediately prior to self-administration experiments
and were delivered over a 90-second interval. For the microinjection, a pair of 10µL
microsyringes (Microliter #1701; Hamilton, Reno, Nevada, USA), cleaned and flushed
with 70% ethanol (diluted from 100% reagent alcohol, Fisher Scientific, NJ, USA) and
distilled water, were mounted on a Razel micro-infusion pump via a 1RPM motor. An
injector set (28GA, 6.5mm with 1.2mm protrusion=7.7mm total length, HRS Scientific,
Plastics One, Roanoke, VA), was 1.2mm longer than the bilateral guide cannula.
[**Sucrose self-administration rats with Rat IDs that are before L143 used shorter
injector sets: 21GA, 5.8mm with 0.3mm protrusion=6.1mm total length, HRS Scientific,
Plastics One, Roanoke, VA] It was inserted into polyethylene tubing PE50 (thin
walled,0.023x0.041 HRS Scientific, Plastics One, Roanoke, VA). The tubing and
injector were flushed and filled with sterile saline (0.9% NaCl saline, Baxter Corporation,
30
Mississauga, ON, Canada) before they were attached to the microsyringes. A small
volume (about 5µL) of sterile saline (0.9% NaCl saline, Baxter Corporation, Mississauga,
ON, Canada) was drawn back into the injector and tubing, followed by 1µL of air to
ensure the integrity of the saline and drug. Lastly, 1µL of drug was drawn up into the
injectors, ready for intra-BST micro-injection.
Before each injection, a test run of the Razel micro-infusion pump was performed
to make sure that the drug solution was coming out of the injector set. Then, the injectors
were inserted into the bilateral guide cannulae on the head of each rat, and 0.5 µL of the
diluted drug (or saline vehicle) was injected into the BST on each side of the brain over
60 seconds. Injectors were left in the guide cannulae for an additional 30 seconds, and
the injectors were retracted slowly to minimize the amount of residual drug due to suction
force. The stylets were put back in the guide cannulae and the rat was immediately
placed inside an operant chamber to start the self-administrative experiment. The
procedure was repeated for all the rats that met the pre-established criteria for microinjections. A different set of injectors was used for different drugs and rats to prevent
cross-contamination across rats.
Experimental design for the order of intra-BST micro-injected drugs:
At the initial stages of our experiment, we started out with a pseudorandom design
for the order of the intra-BST drugs given to each animal. This is a double blind
procedure, where both the animal and the experimenter were blind to the drugs given, so
potential experimental bias and manipulation could be minimized. Under this design,
each animal received a maximum of 5 intra-BST drug injections. Among these 5
injections, the animal received 2 injections of the target drug at the same concentration, 1
31
injection of the same drug at a different concentration, and 2 injections of saline. Each
injection was coded by a letter and a number, and the order of the injections was mixed
randomly (see Table 3 for an example of the pseudorandom design of drug injections).
Under this design, each drug injection group, represented by a letter, only contains one
kind of the drug at 2 different concentrations. The ordering of the injections was
designed to counterbalance any potential carry-over effects from previous injections and
to provide good experimental control.
Table 3. Pseudorandom design of drug injections. Each drug injection group
is designated by a letter and contains 5 injections. Of these 5 injections: 2
injections are vehicle saline, the other 2 injections are the drug at one
concentration, and 1 injection is the same drug at a different concentration. The
order of these injections is randomized to avoid experimenter bias.
Group A (SCH-23390)
A1. SCH 1.6 μg/μL
A2. Saline (Vehicle)
A3. SCH 3.2 μg/μL
A4. Saline (Vehicle)
A5. SCH 1.6 μg/μL
From the middle towards the end stages of our experiments, as we realized that
the order of the injections did not affect the behaviour of the animals, and experimenters
were not biased by knowing what the drugs were, we maximized the use of our rats by
giving them many injections until they were too old for more experiments. Therefore,
each rat could receive more than one kind of drugs. The injections from various rats
were pooled into individual drug treatment groups and the resulting dependent measures
32
were analyzed using one-way ANOVA (see Table 4 and Table 5 for the distribution of
injection N numbers for each animal and drug treatment group).
Table 4. Distribution of total intra-BST injections in sucrose- and cocaineself-administering rats. For both groups, the same saline treatment data was
used to compare to other drug treatment groups (SCH-23390, Sulpiride,
Quinpirole). Numerical data from all our drugs of interest were pooled from
various rats.
Drug
# of injections in each group (N numbers)
Sucrose self-admin
Cocaine self-admin
Saline (vehicle)
12
11
SCH 1.6μg/μL
4
5
SCH 3.2μg/μL
6
4
SCH 6.4μg/μL
4
5
Sulpiride 3.2μg/μL
5
6
Quinpirole 3.0μg/μL
4
7
33
Table 5. An outline of the intra-BST injections received by each rat (in
terms of the kinds of drugs received, the concentrations of the drugs, and the
number of injections received). The number in each cell represents the
number of injections given in each condition.
Rat ID
Saline
SCH
SCH
SCH
Sulpiride
Quinpirole
1.6μg/μL
3.2μg/μL
6.4μg/μL
3.2μg/μL
3.0μg/μL
Sucrose Rats (number in each cell represents the number of injections given in each
condition)
2
1
2
L111
L112
2
L113
1
L126
1
L135
1
1
L138
2
1
L169
2
L170
1
2
1
1
2
2
2
1
1
L194
1
L197
1
1
2
L198
1
L196
Total N
12
4
6
4
5
4
Cocaine Rats (number in each cell represents the number of injections given in each
condition)
1
2
1
L159
L160
1
L162
1
1
2
1
34
1
1
L176
1
L188
L189
1
1
L191
2
1
L207
3
1
L213
1
1
L215
1
Total N
11
2
1
1
1
1
2
1
1
2
1
5
4
5
6
7
2.6 - Behavioural Procedure: Operant Conditioning-Reinforcement Paradigm
Operant Training
All experiments and training were run between 9:00 hour to 21:00 hour in the
dark phase (active) of the reversed light/dark cycle. For both the cocaine and sucrose
self-administration groups, rats were trained on a fixed-ratio-1 (FR-1, one lever press
gives one reward) schedule until they achieved a controlled, titrating pattern of pressing
(40 lever presses for 40 rewards for sucrose (see Figure 7) and 25 lever presses for 25
infusions for cocaine (see Figure 8), where infusions were self-administered at equal time
intervals for 3 consecutive days. The rats then graduated to the progressive atio training
(PR) for both sucrose (see Figure 9) and cocaine (see Figure 10) self-administration
groups.
35
Figure 7: Example behavioural trace showing the acquisition of operant
lever pressing response under the fixed ratio-1 (FR-1) schedule of
reinforcement in sucrose self-administering rats. Rats demonstrate
consistent pressing behaviour (40 presses for 3 consecutive days) in a
controlled manner. Actual lever presses (operant response) are
demonstrated by vertical increments, and the receipt of each reward
(45mg sucrose pellet) is represented by tick marks. On the FR-1
schedule, each operant response is paired with one reward.
Figure 8: Example behavioural trace showing the acquisition of
operant lever pressing response under the fixed ratio-1 (FR-1) schedule
of reinforcement in cocaine self-administering rats. Rats demonstrate
consistent pressing behaviour (25 presses for 3 consecutive days) in a
controlled and titrating manner. Actual lever presses (operant
response) are demonstrated by vertical increments, and the receipt of
each reward (0.12mL of 0.75mg/kg/injection of cocaine) is represented
by tick marks.
36
Figure 9: Example behavioural trace showing the expression of operant
lever pressing behaviour under the progressive ratio (PR) schedule of
reinforcement for the sucrose self-administration group. The behaviour of
each rat is graphed as cumulative number of lever presses against time in
minutes. The receipt of each sucrose reward (45mg/pellet), is represented
by tick marks. Typically, rats on the PR schedule are trained for at least 10
days until stable operant responding is achieved so they can receive
intracranial microinjections of our drugs of interest. Under the PR
reinforcement schedule, each reward after the first requires a progressively
greater number of operant responses. On each experimental day, the data
collected for quantification of behaviour is breakpoint (BP), which is the last
reward received before session ends (in the sucrose self-administering rats,
the whole session lasts for 180 minutes). The ln (final ratio) scale on the
right corresponds to the total number of lever presses on the left. From the
total number of lever presses, the number of infusions (breakpoint) is
derived and final ratio (the number of additional lever presses between each
reinforcer) is determined from Table 1. The ln (final ratio) value is used to
calculate the ≤8% deviation from the LN final ratio average for the 1 to 3
consecutive days before the actual injection day. This formula is outlined in
detail in the method section. Drug effect is calculated as the change in final
ratio in percentage. This is calculated by dividing the final ratio of the
injection day by the average of the 3 pre-injection day final ratio.
37
Figure 10: Example behavioural trace showing the expression of operant
lever pressing behaviour under the progressive ratio (PR) schedule of
reinforcement for the cocaine self-administration group. The behaviour of
each rat is graphed as cumulative number of lever presses against time in
minutes. The receipt of each cocaine infusion (0.75mg/kg/infusion) is
represented by tick marks. Typically, rats on the PR schedule are trained
for at least 10 days until stable operant responding is achieved so they can
receive intracranial microinjections of our drugs of interest. Under the PR
reinforcement schedule, each reward after the first requires a progressively
greater number of operant responses. On each experimental day, the data
collected for quantification of behaviour is breakpoint (BP), which is the
last reward received before session ends (in the cocaine self-administering
rats, the whole session lasts for 270 minutes). The ln (final ratio) scale on
the right corresponds to the total number of lever presses on the left. From
the total number of lever presses, the number of infusions (breakpoint) is
derived and final ratio (the number of additional lever presses between each
reinforcer) is determined from Table 1. The ln (final ratio) value is used to
calculate the ≤8% deviation from the LN final ratio average for the 1 to 3
consecutive days before the actual injection day. This formula is outlined in
detail in the method section. Drug effect is calculated as the change in final
ratio in percentage. This is calculated by dividing the final ratio of the
injection day by the average of the 3 pre-injection day final ratio.
38
Experimental Testing (Intra-BST Micro-Injection of Drugs)
For each experimental session, a maximum number of 2 rats were micro-injected
(if there were that many rats ready at the time). After the rats received intra-BST
microinjected drugs, they were put into the experimental chambers and the selfadministrative sessions were initiated. The rats were observed through the transparent
plexiglass of the chambers and any irregularities in their behaviours due to potential
effects of the drugs were recorded. For the sucrose self-administration group, both the
FR-1 and PR sessions were 180 minutes in length. The cocaine self-administration group
lasted 270 minutes in length for both the FR-1 and PR sessions. MedPCIV software
(MedAssociates, St-Albans, VT) was used to record the sessional behaviour data for
analysis (i.e. number of lever presses, BP).
2.7 - Data Analysis
In total, 22 rats were used in the final data analysis. All of these rats were used
for intra-BST drug micro-injections and histology was done to ensure proper cannulae
placement.
Behavioural changes (i.e. traces) recorded by MedPCIV were compared within
the two test groups: sucrose self-administration and cocaine self-administration (example
behavioural traces used to calculate the effects of dopamine drugs administered are
shown in Figures 9 and 10). On each behavioural trace, each tick represents the receipt
of one reward, either in the form of a single sucrose pellet or a single cocaine infusion.
The number of lever presses on the left vertical axis of the behavioural trace corresponds
to the total pressing column in Table 1, which is calculated using the response ratio
39
formula for the PR schedule series (Response ratio = 5[𝑒𝑒 (𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖
𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛 ×0.2)
] − 5).
Each value of total pressing corresponds to an infusion number, which is taken as the BP
in our experiment since we defined our BP as the last infusion reward received before
experiment session ends. If the animal’s total pressing value is less than the specified
total pressing value outlined in Table 1, then the BP will be one less (i.e. if the animal
achieved total pressing of 10, the BP for this animal would be 3, not 4, because 10 is less
than the specified total pressing value of 13 on the table, which corresponds to a BP of 4).
Each BP value then corresponds to a final ratio value (Table 1), which is then ln
transformed to ensure it conforms to the assumptions of parametric statistics. This is
because the final ratio is derived from a natural ln equation, so values at the higher end of
the scale will have a larger variance than values at the low end. Consequentially, the
ln(final ratio) scale on the right side of the behavioural trace (Figure 9 and 10) was used
in the calculation for deciding if an animal could receive an intra-BST drug injection (our
criteria was that the candidate must reach a stable BP over a period of one to three
consecutive days, with total rewards received at BP ±3, based on a criterion of ≤8%
deviation from the LN final ratio average). The formula used to determine if there was a
≤8% deviation from the LN final ratio average is outlined as follows:
|((ln[final ratio Day1 ])-(ln[final ratio Day2 ]))|
|(ln[final ratio Day1 ])|
+ |((ln[final ratio Day2 ])-(ln[final ratio Day3 ]))|
x100
|(ln[final ratio Day2 ])|
2
Once the intra-BST micro-injection was given, we determined the effect of our
dopaminergic drugs of interest. The change in final ratio relative to 3 pre-injection day
40
average baseline was measured for each drug treatment group: Vehicle (sterile saline),
SCH-23390 (1.6, 3.2, or 6.4 µg/µL), Sulpiride (3.2 µg/µL), and Quinpirole (3.0 µg/µL).
For each injection, the final ratio of the intra-BST injection day was compared with the
average final ratio of the 3 pre-injection days and expressed as a change in percentage.
The formula used in the calculation of the drug effect is outlined as follows:
final ratio injection day
Average (final ratio preinjection day1to3 )
- 1
x 100
The reason for not doing a ln(final ratio) transformation for the calculation of the drug
effect using the above formula is because the numerical values generated by this formula
is more reflective of the actual drug effect on the behaviour of the animals. After the
drug effects were calculated, the numerical results from each animal were pooled into
separate drug treatment groups. So, within one drug treatment group, the injections could
come from a various drugs and some animals were used for more than one kind of drug
injection (see Table 4 and Table 5 for clarification). All the data were checked for
normality and equality of variances and then all the values were compared by using the
one-way ANOVA test and post hoc comparisons using Dunnett’s method for comparison
with control to test for statistical significance between the saline treatment group and the
individual drug treatment groups. Because of the way that the intra-BST drug injection
data were collected, the drug effect values obtained were not independent measures.
Therefore, we performed one-way ANOVA for our statistical analysis
.
41
2.8 - Histology / Brain Cannulation Placements
Once an experiment was completed, the brains of the rats were examined in order
to determine whether the bilateral guide cannulae were placed within the oval BST. To
prepare fluorescent microscope slices, rats were given intra-BST microinjections of
1.0µL (0.5µL in each side of brain) of micro-fluorescent beads (FluoSpheres carboxylate
modified microspheres; 0.04µm, red-orange fluorescent; actual size: 0.036µm; invitrogen
Molecular Probes, Oregon, USA) using the same procedure as previous intra-BST microinjections. To prepare for brain extraction, rats were anaesthetized with sodium
pentobarbital (60mg/ml; from Euthanyl stock solution of 240mg/ml; Bimeda-MTC
Animal Health Inc, Cambridge, ON) until they reached surgical plane for perfusion (each
animal started at 0.6mL, and were gradually given additional incremental amounts of
0.2mL, depending on their weight). A horizontal incision was made across the rat’s
belly, with the underlying muscle tissue cut open to expose the interior organs. The
sternum was clamped down to expose the diaphragm. Then, the diaphragm was cut open
to expose the heart. To attain a better surgical view of the heart, the rib cage was cut
open even further up from both the left and right sides. The right atrium was clipped to
release the potential pressure that might build up during perfusion. Then, the tip of a 16G
needle (Becton Dickinson, Franklin Lakes, NJ, USA) was inserted into the apex of the
heart (at the left ventricle) for saline and 4% paraformaldehyde (PFA) solution to be
pumped through. After this, the rats were then perfused transcardially with 250 mL of
saline or until the liquid dripping down from the rat’s body was transparent in
appearance. This was then followed by pumping 500 mL of 4% PFA solution
42
transcardially into the rat. Perfusion was only considered successful if the rat’s body was
solid hard by the end of the 4% PFA solution.
Then, the brains were extracted and placed in the 4% PFA solution for postfixation overnight (in 4°C refrigerator). At this stage, a successfully extracted brain
would appear white in color, with no blood vessels showing on the cortex. To ensure
cryoprotection, the brains were then transferred to a 30% sucrose-PFA solution for at
least 2-3 days (in 4°C refrigerator) to ensure complete infusion. Then, using a cryotome
(Leica SM2000R, Germany), the brain was partitioned into 50µm slices taken from the
dorsolateral BST. The slices were then mounted and viewed under a fluorescing
microscope (Leica Mikroskopie and Systeme GmbH Wetzlar, Germany) via the
projection of green fluorescent light (ebq100, Germany) to confirm the placement of the
cannulae guides.
43
Chapter 3: Results
Due to individual variations in behavioral responses and for general statistical
analysis considerations, first saline injections for cocaine rats L191, L159, L215, and
L213 were excluded. In addition, the first SCH-23390 (1.6µg/µL) injection for cocaine
rat L188, and the first Sulpiride (3.2 µg/µL) injection for cocaine rat L162 were excluded
because the resulting drug effects were more than 2 standard deviations away from the
mean for those injections and treatment groups. For the calculation of the change in
baseline final ratio or BP (to show the effect of the drug), the average of the final ratio
from 1 to 3 days before the injection date was used. For more detailed behavioural traces
and data of all the rats with each individual drug treatment, see appendix I, and II.
3.1 - Effect of SCH-23390 on Self-Administration
In the sucrose self-administration group, the change in final ratio pressing was
calculated for intracranial injections of vehicle (sterile saline = -12.11±7.34%, n = 12),
and the D1 selective antagonist SCH-23390 (1.6µg/µL = -26.71±12.13%, n = 4;
3.2µg/µL = -17.22±8.53%, n = 6; 6.4µg/µL = 21.61±14.45%, n = 4). Oneway ANOVA
test revealed no statistical significance between the drug treatment groups, F (3, 21) =
2.57, p = 0.081. (see Figure 11). Example behavioral traces for the effect of saline and
SCH-23390 in sucrose self-administering rats were shown in Figure 12.
In the cocaine self-administration group, the change in final ratio pressing was
calculated for intracranial injections of vehicle (sterile saline = 2.64±8.86%, n = 11) and
the D1 selective antagonist SCH-23390 (1.6µg/µL = -14.41±18.99%, n = 5; 3.2µg/µL =
-34.90±5.81%, n = 4; 6.4µg/µL = -34.34±5.48%, n = 5) (Figure 11). Oneway ANOVA
test (using Dunnett’s method for comparison with control) revealed that there was
44
statistical significance in cocaine rats treated with SCH-23390 at concentrations of
3.2µg/µL and 6.4µg/µL when compared to the saline treatment group (SCH-23390
3.2µg/µL: p = 0.024; SCH-23390 6.4µg/µL: p = 0.028) (see Figure 11). Example
behavioral traces for the effect of saline and SCH-23390 in cocaine self-administering
rats were shown in Figure 13.
Effect of SCH-23390 (D1 antagonist) on
Reinforcement
Delta final ratio pressing (%)
40.00
30.00
20.00
N=5
N=11
N=4
N=5
10.00
0.00
-10.00
-20.00
-30.00
-40.00
N=12
N=4
N=6
saline
1.6 μg/μL
3.2 μg/μL
6.4 μg/μL
Cocaine self-admin
2.64
-14.41
-34.90
-34.34
Sucrose self-admin
-12.11
-26.71
-17.22
21.61
-50.00
Cocaine self-admin
*
N=4
Sucrose self-admin
Figure 11: Bar graph showing the effects of vehicle (saline) and SCH-23390 (at
total bilateral dosages of 1.6, 3.2, and 6.4 µg/µL) on the breakpoint (percentage
change in final ratio) of rats self-administering sucrose and cocaine under the
progressive ratio (PR) schedule of reinforcement. Mean (±SEM) changes in
final ratio pressing in percentage (%) were averaged across all rats in each drug
treatment group, with N numbers outlined on the graph for each treatment group.
The effect of the drug is calculated by comparing the final ratio on the day of the
injection with the average of the 3 day pre-injection final ratio. Asterisk (*)
indicates that responding for SCH-23390 at concentrations of 3.2 µg/µL, and 6.4
µg/µL in the cocaine self-administering rats was significantly different from
responding under the saline vehicle treatment ( p < 0.05) by post hoc
comparisons using Dunnett’s method for comparison with control.
45
Figure 12: The effects of intra-BST micro-injections on sucrose self-administration
under the progressive ratio (PR) schedule of reinforcement. These graphs illustrate
the change (black dashed line) from the 3-day pre-injection operant response
baseline (red lines) by 1µL (0.5µL per side) injections of either vehicle saline (top
graph) or the selective D1- dopamine receptor antagonist, SCH-23390, at a total
bilateral dosage of 3.2µg/µL (bottom graph). Each reward received is represented
by ticks on the graph and breakpoint (BP) is defined as the last reward received
before the session ends (180 minutes). BP is used to determine the final ratio for
calculation of drug effects expressed as percentage change in final ratio pressing
(see methods section for explanation and formulae).
46
Figure 13: The effects of intra-BST micro-injections on cocaine selfadministration under the progressive ratio (PR) schedule of reinforcement.
These graphs illustrate the change (black dashed line) from the 3-day preinjection operant response baseline (red lines) by 1µL (0.5µL per side)
injections of either vehicle saline (top graph) or the selective D1-dopamine
receptor antagonist, SCH-23390, at a total bilateral dosage of 6.4µg/µL
(bottom graph). Each reward received is represented by ticks on the graph
and breakpoint (BP) is defined as the last reward received before the
session ends (270 minutes). BP is used to determine the final ratio for
calculation of drug effects expressed as percentage change in final ratio
pressing (see methods section for explanation and formulae).
47
3.2 - Effect of Sulpiride on Self-Administration
In the sucrose self-administration group, the change in final ratio pressing was
calculated for intracranial injections of vehicle (sterile saline = -12.11±7.34%, n = 12)
and the D2-like antagonist Sulpiride (3.2µg/µL = 2.74±16.57%, n = 5). Oneway
ANOVA test revealed no statistical significance between the drug treatment groups, F (2,
18) = 2.52, p = 0.11 (see Figure 14). Example behavioral traces for the effect of saline
and Sulpiride in sucrose self-administering rats were shown in Figure 15.
Effect of Sulpiride (D2 antagonist) on
Reinforcement
Delta final ratio pressing (%)
30.00
N=11
N=6
20.00
10.00
0.00
-10.00
-20.00
N=5
N=12
-30.00
-40.00
*
-50.00
Saline
3.2 μg/μL
Cocaine self-admin
2.64
-31.00
Sucrose self-admin
-12.11
2.74
Cocaine self-admin
Sucrose self-admin
Figure 14: Bar graph showing the effects of vehicle (saline) and Sulpiride (at total
bilateral dosage of 3.2µg/µL) on the breakpoint (percentage change in final ratio) of
rats self-administering sucrose and cocaine under the progressive ratio (PR)
schedule of reinforcement. Mean (±SEM) changes in final ratio pressing in
percentage (%) were averaged across all rats in each treatment group, with N
numbers outlined on the graph for each treatment group. The effect of the drug is
calculated by comparing the final ratio on the day of the injection with the average
of the 3 day pre-injection final ratio. Asterisk (*) indicates that responding for
Sulpiride at the concentration of 3.2 µg/µL in the cocaine self-administering rats
was significantly different from responding under the saline vehicle treatment ( p <
0.05) by post hoc comparisons using Dunnett’s method for comparison with control.
48
Figure 15: The effects of intra-BST micro-injections on sucrose selfadministration under the progressive ratio (PR) schedule of reinforcement.
These graphs illustrate the change (black dashed line) from 3-day pre-injection
operant response baseline (red lines) by 1µL (0.5µL per side) injections of
either vehicle saline (top graph) or the D2-like dopamine receptor antagonist,
Sulpiride, at a total bilateral dosage of 3.2µg/µL (bottom graph). Each reward
received is represented by ticks on the graph and breakpoint (BP) is defined as
the last reward received before the session ends (180 minutes). BP is used to
determine the final ratio for calculation of drug effects expressed as
percentage change in final ratio pressing (see methods section for explanation
and formulae).
49
Figure 16: The effects of intra-BST micro-injections on cocaine selfadministration under the progressive ratio (PR) schedule of reinforcement.
These graphs illustrate the change (black dashed line) from the 3-day preinjection operant response baseline (red lines) by 1µL (0.5µL per side) injections
of either vehicle saline (top graph) or the D2-like dopamine receptor antagonist,
Sulpiride, at a total bilateral dosage of 3.2µg/µL (bottom graph). Each reward
received is represented by ticks on the graph and breakpoint (BP) is defined as
the last reward received before the session ends (270 minutes). BP is used to
determine the final ratio for calculation of drug effects expressed as percentage
change in final ratio pressing (see methods section for explanation and
formulae).
50
In the cocaine self-administration group, the change in final ratio pressing was
calculated for intracranial injections of vehicle (sterile saline = 2.64±8.86%, n = 11) and
the D2-like antagonist Sulpiride (3.2µg/µL = -31.00±6.20%, n = 6) (Figure 14). Oneway
ANOVA test (using Dunnett’s method for comparison with control) revealed that there
was statistical significance in cocaine rats treated with Sulpiride 3.2µg/µL when
compared to the saline treatment group (Sulpiride 3.2µg/µL: p = 0.0203) (see Figure 14).
Example behavioral traces for the effect of saline and Sulpiride in cocaine selfadministering rats were shown in Figure 16.
3.3 - Effect of Quinpirole on Self-Administration
In the sucrose self-administration group, the change in final ratio pressing was
calculated for intracranial injections of vehicle (sterile saline = -12.11±7.34%, n = 12)
and the D2-like agonist Quinpirole (3.0µg/µL = -5.69±12.08%, n = 4) (Figure 17).
Oneway ANOVA test revealed no statistical significance between the drug treatment
groups F(1, 14) = 0.20, p = 0.67 (see Figure 17). Example behavioral traces for the
effect of saline and Quinpirole in sucrose self-administering rats were shown in Figure
18.
In the cocaine self-administration group, the change in final ratio pressing was
calculated for intracranial injections of vehicle (sterile saline = 2.64±8.86%, n = 11) and
the D2-like agonist Quinpirole (3.0µg/µL = -20.20±11.61%, n = 7) (Figure 17). Oneway
ANOVA test revealed no statistical significance between the drug treatment groups F(1,
16) = 2.50, p = 0.13 (see Figure 17). Example behavioral traces for the effect of saline
and Quinpirole in cocaine self-administering rats were shown in Figure 19.
51
Effect of Quinpirole (D2 agonist) on
Reinforcement
60.00
Delta final ratio pressing (%)
50.00
40.00
30.00
20.00
N=11
N=7
10.00
0.00
-10.00
-20.00
-30.00
-40.00
N=12
N=4
Saline
3.0 μg/μL
Cocaine self-admin
2.64
-20.20
Sucrose self-admin
-12.11
-5.69
Cocaine self-admin
Sucrose self-admin
Figure 17: Bar graph showing the effects of vehicle (saline) and Quinpirole (at
total bilateral dosage of 3.0 µg/µL) on the breakpoint (percentage change in final
ratio) of rats self-administering sucrose and cocaine under the progressive ratio
(PR) schedule of reinforcement. Mean (±SEM) changes in final ratio pressing in
percentage (%) were averaged across all rats in each treatment group, with N
numbers outlined on the graph for each treatment group. The effect of the drug is
calculated by comparing the final ratio on the day of the injection with the average
of the 3 day pre-injection final ratio. There were no significant differences
between the saline and the Quinpirole treatment groups.
52
Figure 18: The effects of intra-BST micro-injections on sucrose selfadministration under the progressive ratio (PR) schedule of reinforcement.
These graphs illustrate the change (black dashed line) from 3-day preinjection operant response baseline (red lines) by 1µL (0.5µL per side)
injections of either vehicle saline (top graph) or the D2-like dopamine
receptor agonist, Quinpirole, at a total bilateral dosage of 3.0µg/µL (bottom
graph). Each reward received is represented by ticks on the graph and
breakpoint (BP) is defined as the last reward received before the session
ends (180 minutes). BP is used to determine the final ratio for calculation
of drug effects expressed as percentage change in final ratio pressing (see
methods section for explanation and formulae).
53
Figure 19: The effects of intra-BST micro-injections on cocaine selfadministration under the progressive ratio (PR) schedule of reinforcement.
These graphs illustrates the change (black dashed line) from the 3-day preinjection operant response baseline (red lines) by 1µL (0.5µL per side)
injections of either vehicle saline (top graph) or the D2-like dopamine
receptor agonist, Quinpirole, at a total bilateral dosage of 3.0µg/µL
(bottom graph). Each reward received is represented by ticks on the graph
and breakpoint (BP) is defined as the last reward received before the
session ends (270 minutes). BP is used to determine the final ratio for
calculation of drug effects expressed as percentage change in final ratio
pressing (see methods section for explanation and formulae).
54
3.4 - Histology / Brain Cannulation Placements
The 2 groups (sucrose and cocaine self-administration) included a total of 22 rats.
Of these, 12 rats were sucrose rats and 10 rats were cocaine rats. In the sucrose selfadministration group, 4 rats had traces of fluorescent beads in the ventricle, suggesting
inaccurate cannulation placements. However, this only occurred with a few rats in the
very initial stage of our experiment. Our cannulation placements were much more
accurate later on. The majority of our intra-cranial cannulations were directly in the BST,
with different rats showing its optimal placement in the BST at somewhat different brain
levels. In the cocaine self-administration group, 1 rat had traces of fluorescent beads in
the ventricle. However, the rest of the rats in this group all had injection tracts and sites
directly in the BST. The cannulae placements for each rat in each group are shown in
Figure 20 (a)~(g) (sucrose rats) and Figure 21 (a)~(d) (cocaine rats), and photographs of
coronal sections showing typical BST cannulae placement are presented in Figure 22
(sucrose rats) and Figure 23 (cocaine rats).
55
Figure 20. These brain maps show the intra-cranial cannulation placements in sucrose
self-administering rats at different brain levels. Parts (a) ~ (g) depict various rat brain
levels, marked with anterior-posterior coordinates from Bregma. Cannulae placements in
the bed nucleus of the stria terminalis (BST) are marked by thicker lines along the
ventricle and ovals in the BST region. Each rat has a representative brain level for which
its optimal injection site is located, which is marked on the brain maps with the rat’s ID.
The localization of the intra-BST injection is outlined by the existence of the fluorescent
beads. The IDs of the rats are labelled on the arrows pointing to the lines along the
ventricle and in the ovals in the BST region. Although only one side of the brain is
marked, all of these are bilateral brain placements. The right side of the brain map
facilitates identification of the anatomy of the brain, especially the BST.
(Adapted from L. W. Swanson Brain Maps, 3rd Edition)
a) Level 15 (AP = +0.45 from Bregma)
b) Level 16(AP = +0.10 from Bregma)
56
c) Level 18(AP = -0.11 from Bregma)
d) Level 19(AP = -0.26 from Bregma)
e) Level 20(AP = -0.46 from Bregma)
57
f) Level 21(AP = -0.51 from Bregma)
g) Level 23(AP = -1.08 from Bregma)
58
Figure 21. These brain maps show the intra-cranial cannulation placements in
cocaine self-administering rats at different brain levels. Parts (a) ~ (d) depict
various rat brain levels, marked with anterior-posterior coordinates from Bregma.
Cannulae placements in the bed nucleus of the stria terminalis (BST) are marked
by thicker lines along the ventricle and ovals in the BST region. Each rat has a
representative brain level for which its optimal injection site is located, which is
marked on the brain maps with the rat’s ID beside it. The localization of the intraBST injection is outlined by the existence of the fluorescent beads. The IDs of the
rats are labelled on the arrows pointing to the lines along the ventricle and in the
ovals in the BST region. Although only one side of the brain is marked, all of
these are bilateral brain placements. The right side of the brain map facilitates
identification of the anatomy of the brain, especially the BST.
(Adapted from L. W. Swanson Brain Maps, 3rd Edition)
a) Level 15 (AP = +0.45 from Bregma)
b) Level 19(AP = -0.26 from Bregma)
59
c) Level 20(AP = -0.46 from Bregma)
d) Level 21(AP = -0.51 from Bregma)
60
Level 18-19 (between AP = -0.11 ~ -0.26 from Bregma)
Figure 22. Picture showing actual rat brain under fluorescent microscopy
for sucrose self-administering rats. The shiny beads outline the injection
site and tract along the BST region. Specifically, the injection site was
located at brain level 18-19 (between AP = -0.11 ~ -0.26 from Bregma).
61
Ventricle
BST
aco
a) Level 20(AP = -0.46 from Bregma). Fluorescent beads and injection tract at BST injection
site on both sides of the brain.
Ventricle
b) Level 20(AP = -0.46 from Bregma). No fluorescent beads lining the ventricle on either side
of the brain.
Figure 23. Pictures showing actual rat brain under both transmitted and fluorescent
microscopy for cocaine self-administering rats. In picture (a), the photo on the left
shows the rat brain with beads under transmitted microscope; the photo on the right
depicts the same picture under fluorescent microscope. The shiny beads outline the
injection site and tract along the bed nucleus of the stria terminalis (BST) region.
Specifically, the injection site was located at around rat brain level 20 (AP = -0.46
from Bregma). In picture (b), the photo shows that there are no beads lining the
ventricle.
Abbreviations for this figure are as follows: aco, anterior commissure; BST, bed
nucleus of the stria terminalis; ventricle, lateral ventricle.
62
Chapter 4: Discussion
4.1 - Summary of Current Results
In this study, our results suggested the significance of dopaminergic response to
cocaine rewards in the BST. Dopaminergic drugs administered directly into this structure
manipulated the expression of addictive behaviours related to pharmacological (i.e.
cocaine), but not natural (i.e. sucrose) rewards. Delivery of dopamine antagonists into
the BST decreased motivated behaviours: both the D1 selective antagonist SCH-23390
(3.2 & 6.4 µg/µl) and the D2-like antagonist Sulpiride (3.2 µg/µl) significantly reduced
operant behaviour in animals self-administering cocaine. However, none of these
dopamine agents had any significant effect on sucrose self-administration. The D2-like
agonist, Quinpirole (3.0 µg/µl) also failed to induce any changes in operant responding.
This general finding is in partial concordance with previous studies demonstrating
changes in the reinforcing properties of both food and drugs resulting from blockade of
dopamine receptors in other brain structures: the VTA (Shibata et al., 2009), the
amygdala (Berglind et al., 2006; McGregor et al., 1994; McGregor and Roberts, 1993),
and the NAc (McGregor and Roberts, 1993; Hodge, et al., 1997; Anderson et al., 2003;
Anderson et al., 2006; Schmidt and Pierce, 2006). Since relatively few studies have
examined the contribution of the BST dopamine receptors in the reinforcing properties of
sucrose and pharmacological rewards (Epping-Jordan et al., 1998; Walker et al., 2000;
Eiler II et al., 2003), our results will provide further confirmation of the sensitivity of the
BST dopamine receptors on reinforcement, especially in cocaine. Furthermore, the
results will confirm that the BST is well positioned for the role of addiction. The BST’s
connection to the mesolimbic dopamine system, its heavy innervations to and from
63
midbrain dopamine releasing areas (i.e. VTA, PAG, RR, SNc), and its expression of D1and D2- dopamine receptors (Freedman and Cassell, 1994; Hasue and ShammahLagnado, 2002; McDonald et al., 1999; Scibilia et al., 1992) all serve to facilitate rewardelicited dopamine related modulation at the BST (Carboni et al., 2000).
4.2 - Selective D1 Antagonist (SCH-23390) Had No Effect on Sucrose SelfAdministration, But Decreased Cocaine Self-Administration
In our experiment, rats pre-treated with SCH-23390 before their selfadministration sessions produced very different results. In the sucrose self-administration
group, treatment of SCH-23390 at various concentrations (1.6, 3.2, & 6.4 µg/µl)
produced no significant effects compared to the vehicle (saline) treatment group.
Although saline and SCH-23390 treatment at 1.6 µg/µl and 3.2 µg/µl generated slight
decreases in responding compared to baseline, which was somewhat consistent with
previous research (Eiler II et al., 2003), the slight increase in responding at 6.4 µg/µl of
SCH-23390 made the role of the BST in modulating natural reinforcement more obscure.
However, the fact that there were still decreases in sucrose maintained operant
responding indicated that BST dopamine receptors maybe still be partially blocked or
manipulated by the D1 dopamine receptor antagonist following chronic sucrose intake.
Since food reward foraging is imperative for survival and reproduction, brain systems
have evolved pathways to direct and motivate this behaviour. Studies have shown that
dopaminergic projection from the VTA to the NAc is not only essential for modulating
goal-directed behaviour, but the changes in the firing rate of NAc neurons also encode
information related to the operant response for food reward (Wise et al., 1978; Carelli,
2002; Roitman et al., 2004). In our case, the BST projects moderately to other brain
64
areas involved in locomotor or exploratory behaviour control, reward prediction and
ingestive behaviour control (Dong and Swanson, 2003). Therefore, it is not surprising
that dopamine released in the BST can have a modifying effect on food seeking. The fact
that there was no influence on operant responding maintained by food suggests that the
decrease of cocaine seeking by intra-BST SCH-23390 injection was not due to general
motor impairment (Stairs et al., 2010; Anderson et al., 2003). Other studies showing an
effect of sucrose responding under SCH-23390 (Eiler II et al., 2003; Barrett et al., 2004)
may be inconsistent with our results due to different schedules of reinforcement
employed (i.e. they used FR schedules with differing time-out periods) and different
methods of food / sucrose delivery (i.e. sucrose solution was self-administered instead of
sucrose pellets) under the self-administration paradigm. The use of a range of sucrose
solution concentrations may be maintained by different rates of responding which will
subject the results to more variability in interpretation.
In the cocaine self-administration group, treatment of saline produced no change
in responding, however, treatment of SCH-23390 at concentrations of 3.2 µg/µl and 6.4
µg/µl yielded statistically significant attenuations in responding. This finding is in full
concordance with the study conducted by Epping-Jordan et al (1998), which showed
decreases in the reinforcing efficacy of cocaine (0.75mg/kg/intravenous injection)
following intra-BST injection of SCH-23390 at concentrations of 3.2 µg/µl and 6.4 µg/µl.
However, all experimental trials in this study were carried out under the FR-5 schedule of
reinforcement with timeout periods of 20 seconds, while the PR schedule of
reinforcement was used in our study. With the FR schedule, the dependent variable is the
rate of self-administration. Since the rate of drug intake is inversely proportional to unit
65
injection dose, Yokel and Wise (1975) have proposed that the increase in rate of drug
self-administration serves as a compensatory response to reflect the decrease in drug
reinforcing efficacy; conversely, the decrease in rate reflect an increase in reinforcing
efficacy (Richardson and Roberts, 1996; Yokel and Wise, 1975). On the other hand, with
the PR schedule, the dependent variable is the breaking point, which is defined as the
final ratio completed by an animal where responding ceases as the animal reaches the
maximum number of infusions for the effort it is willing to put in for the drug reward.
Therefore, a decrease in breaking point, as observed in our study, is indicative of
attenuation in the ability of the drug to maintain patterns of self-administration
(Richardson and Roberts, 1996). The effect of SCH-23390 was calculated as percentage
change in baseline final ratio by comparing the final ratio of the drug treatment day with
the average final ratio of three-day before drug treatment. The fact that we included the
whole 270-minute session data in our analysis for final ratio and breaking point ensured
the full potency of our drug of interest (i.e. both the psychostimulant effect of cocaine
and the dopamine receptor antagonist and agonists) was scrutinized for their effect on the
motivational aspect of drug seeking. In the Epping-Jordan et al. study, results were
expressed as both percentage change in rate of cocaine self-administration for the first
20-minute of the experiment and the whole 3-hour experimental session. With both of
these times, there were significant increases in the rate of cocaine self-administration
under the FR schedule. In our study, SCH-23390 decreased both the cumulative number
of lever presses and the breaking point under the PR schedule. The orderly interreinforcer time shown on the trace (Figure 13) suggests that the animals were able to
maintain a controlled, titrated self-administering behaviour without inducing a
66
cumulative dosing effect. Since SCH-23390 treatment yielded similar inter-reinforcer
time compared to the 3 day pre-injection baseline, it is suggestive that SCH-23390 does
not generate a general impairment of the motor functioning in the animals. In addition,
the fact that the breaking point from the SCH-23390 treatment day occurred so much
earlier than the non-drug treatment days, where the breaking point spans the whole
duration of the experimental session, implicates that this is due to blockade of the D1
receptor in the BST. Other studies also showed that SCH-23390 injected into the BST
dose-dependently decreased alcohol motivated responding under FR4 operant schedule,
both in responding rates and cumulative responding (Eiler II et al., 2003). Although the
suppression of responding signifies an increase in reinforcing efficacy of cocaine with
SCH-23390 treatment in the BST, this phenomenon may not be completely pertinent to
our results due to the researchers’ use of FR operant schedule, the training of rats to selfadminister increasing dosages of ethanol, and the difference in the dosage of SCH-23390
infused into the BST via a different injection rate. Overall, our results are supported by
studies conducted under the PR schedule of cocaine reinforcement with injection of SCH23390 into the NAc (McGregor and Roberts, 1993), VTA (Ranaldi and Wise, 2001), and
subcutaneously (Ward et al., 1996).
4.3 - D2-like Antagonist (Sulpiride) Has No Effect on Sucrose Self-Administration, But
Decreases Cocaine Self-Administration
Pretreatment of rats with Sulpiride did not affect operant responding in the
sucrose self-administration group when compared to the pattern of responding in the
saline treated group. This is in agreement with previous studies (Caine et al., 2002; Eiler
II et al., 2003; Bari and Pierce, 2005), where it was shown that D2 receptors were not as
67
important in modulating natural reinforcer motivated behaviours. Again, this no effect of
natural reinforcer with the D2 receptor antagonist treatment serves as a very good
experimental control for the D2 receptors to be not involved in motor function in our
case.
For the cocaine self-administration group, only Sulpiride at 3.2 µg/µl produced
significant attenuations in operant responding. This is not surprising according to studies
of D2 receptor knock-out mice showing that they were still able to self-administer
cocaine at higher rates than heterozygous and wild type mice when high doses of cocaine
on the descending limb of the dose response curve is available. The effect of increased
response rates with pretreatment of D2 antagonist (eticlopride) and the effect of D2
agonist (quinelorane) as a positive reinforcer for cocaine was only present in wild type
mice. Taken together, these results suggest that the D2 receptor is not necessary for
cocaine self-administration (Caine et al., 2002). In our study, however, the use of
Sulpiride as our D2-receptor antagonist provided us with some significance of D2
receptor in the cocaine reinforcement because Sulpiride is about 100 times more selective
for D2 receptors as for D3 receptors and about 400 times more selective for D2 receptors
than for D4 receptors (Vallone et al., 2000). The specificity of Sulpiride generated
reliable decreases in both the number of cumulative decreases in lever press and breaking
point under PR schedule. In addition, the specificity of Sulpiride as a hydrophilic
compound for D2 receptors is especially apparent when administered at lower dosages,
whereas higher dosages of Sulpiride may result in non-specific binding for both D2 and
D3 receptors (Arnt, 1985). Although the BST projects heavily to areas of the brain
involved in generating locomotor (foraging) behaviour for reward acquisition (Dong and
68
Swanson, 2004; Dong et al., 2000; Dong and Swanson, 2003; Dong et al., 2001), and
studies have suggested that the D2-like receptor antagonist are prone to produce a greater
effect on motor impairment function than the D1-like antagonist (Fowler and Liou, 1994;
Neisewander et al., 1995), our results showed no such complication. The fact that the
actual behavioral traces (Figure 16) showed similar pressing pattern and decreases in time
to reach final breaking point on the Sulpiride injection day compared to the 3 day
baseline suggests that the abrupt cessation of responding on the Sulpiride treatment day
was purely due to attenuations in motivation. Furthermore, by looking at our baseline
traces with no Sulpiride treatment (Figure 16) the responding also plateaued in the latter
half of the session. Since there was no antagonist given, the cessation of responding here
must be a reflection of the maximal effort the animal was willing to put in. The trace on
the Sulpiride injection day did not show a uniform decrease or cessation of responding,
instead, there was still gradual increase in responding in the initial stage of the session
followed by a gradual tapering off of responding, all achieved at lower cumulative lever
presses. Overall, our results suggest that the significance of the D2 dopamine receptor in
mediating reward (i.e. sucrose or cocaine) rely greatly on its localization in the brain.
Since the D2 dopamine receptor has been shown to modulate the reinstatement of cocaine
seeking in the NAc shell by Sulpiride (Anderson et al., 2006; Schmidt and Pierce, 2006),
and in the basolateral amygdala (Berglind et al., 2006), our results in the BST may well
serve to be the first experiment to show that the D2-like dopamine receptor in the BST
plays a role in mediating cocaine seeking behaviour in rats.
69
4.4 - D2-like Agonist (Quinpirole) Has No Effect on Sucrose- And Cocaine SelfAdministration
Pre-treatment of the sucrose self-administration group with the D2-like agonist,
Quinpirole, had no effect on both the breaking point and the cumulative number of lever
presses. As shown on the behavioral trace (Figure 18), responding on the Quinpirole
injection day persisted for the whole duration of the drug taking session and the general
pattern of self-administration was identical to the 3-day baseline traces. A comparison
between the saline treated group with the Quinpirole treated group revealed no statistical
difference and significance. As for the cocaine self-administration group, Quinpirole
resulted in decreases in lever pressing and breaking point. However, no statistical
significance was found between the saline and Quinpirole treated groups. A possible
explanation for this effect could be the dosage of Quinpirole used was not enough to
modulate the psychostimulant effect elicited by the amount of cocaine ingested during the
experiment. As it was shown previously that Quinpirole (0.3 to 3.0 µg) administered into
the NAc increased locomotor activity (Dreher and Jackson, 1989), reinstated cocaine
self-administration behaviour (Khroyan et al., 2000), and reduced electrically evoked
dopamine release in slice preparation studies (Yamada et al., 1994), we would have
expected to see Quinpirole to alter the cocaine-maintained responding by changing the
cocaine-evoked dopamine release in the BST. However, our results could have occurred
due to Quinpirole’s D3-preferring nature as a D2/D3 receptor agonist (Vallone et al.,
2000). Therefore, it is probable that Quinpirole could produce a biphasic effect of
cocaine-reinforced responding through its dose-related differential action at the various
dopamine receptor subtypes. At the low dose (up to 3.0 µg- ) of Quinpirole, D3 receptors
70
might have been more involved, whereas at the higher doses (3.0 to 10 µg) , the D2
receptors might have been activated as well. At the concentration used in our study (3.0
µg/µl), however, both the D2 and D3 receptors could have been involved to produce the
response seen in both of our sucrose and cocaine rats.
4.5 - Implications of Current Finding
In this study, the use of different pharmacological agents and schedules of
reinforcement served to facilitate our understanding of the relative contributions of
dopamine receptor subtypes to different reinforcing behaviours within the BST. It should
be noted that our approach to combine FR-1 and PR schedules of reinforcement was well
chosen. Firstly, FR-1 schedule enabled the animal to rapidly acquire robust associations
of the contextual cues in the operant chamber (i.e. cue light, time-out period, lever
retraction / protrusion) with the acquisition of a reward. Following this, under the PR
schedule, the effect on the reinforcing properties of the rewards after the administration
of various dopaminergic agents was quantified. Since the BP under PR schedule was
sensitive to various pharmacological (i.e. dose, injection speed, pretreatment of
dopaminergic agents intracranially) and non-pharmacological manipulations (i.e.
environmental cues, food restriction) (Stoops, 2008; Arnold and Roberts, 1997), this
schedule is ideal for studying and evaluating the reinforcing effects of rewarding
compounds. The FR and PR schedule serve to measure different aspects of dopamine
receptor functioning within the central nervous system (McGregor and Roberts, 1993).
Specifically, the changes in the rate of drug self-administration under the FR schedule are
not as sensitive or predictive to alternations in drug reinforcement efficacy as assessed by
the PR schedule (Arnold and Roberts, 1997; McGregor et al., 1994). In addition,
71
depending on the method and the area of manipulation, the inverse relationship between
rate and BP does not always hold true. In addition, under our experimental condition,
cocaine served as a far more potent reinforcer than sucrose. Consequently, the
reinforcing efficacy of cocaine was assessed under PR schedule. This is because it was
shown previously that highly preferred stimuli (i.e. cocaine) were usually the more
effective reinforcers under high schedule requirements, whereas both the highly and the
less preferred stimuli (i.e. sucrose) were equally effective under low schedule
requirements (Penrod et al., 2008). Another parameter that served to eliminate the
possibility that the effects obtained from the current study were partially due to
impairment in motor functioning was inter-reinforcer time (i.e. inter-reinforcer time
refers to the time in between each successive reward). Despite studies showing the
contribution of dopamine modulators on motor functioning (Neisewander et al., 1995), by
looking at the time between each reward (represented by ticks on our behavioral traces),
it is apparent that in some cases, this time interval was shortened by the dopamine agent
administered. If motor deficits occurred, we would have seen a lengthening of this time
interval. Lastly, by looking at the 3-day post injection traces, we can conclude that there
was no cumulative carrying over effects of the dopamine agents that could potentially
influence self-administration responding after the treatment. Since the post-treatment
traces showed rebounds of the responding back to baseline on the first day after injection
day (i.e. pressing pattern before treatment), the effects of the dopamine agents lasted for
only one day.
Conditioned place preference (CPP), another approach that is commonly used to
evaluate the reinforcing properties of drugs, scrutinizes very different aspects of drug
72
addiction. It appears that the CPP specifically examines the association between
environmental cues and the additive properties of the drug. With sufficient training, it
was shown that dopamine agonists were able to modulate the length of time spent in
environments paired with either aversive or preferred stimuli (Hoffman and Beninger,
1988; Graham et al., 2007; Hoffman et al., 1988). However, for the purpose of this
study, it is more logical and beneficial to use the self-administration paradigm. This is
because in order for us to elucidate the contribution of the BST dopamine receptor in the
reinforcing efficacy of the sucrose and cocaine reinforcer, we need to keep track of the
alternations of the extracellular dopamine concentration before and after dopamine
receptor modulation. In the self-administration approach, studies have shown that
dopamine concentrations in the NAc change at different points of the drug taking
behaviour. For both sucrose and cocaine self-administration, there is an increase in
dopamine concentration before the lever presses, which coincided with the initiation of
drug seeking behaviours. After the lever presses when sucrose was delivered and
consumed, there is no further increase in dopamine (Roitman et al., 2004). For cocaine,
there are still increases in dopamine after the lever presses with the concurrent
presentation of cocaine related cues (Phillips et al., 2003). On the other hand, however,
under the CPP approach, there is no discrete timing of dopaminergic signals during
contextual conditioning and expression of conditioned behaviour (Graham et al., 2007).
Therefore, the animal model of self-administration under PR schedule of reinforcement
enabled us to elucidate the potential dopaminergic contribution and mechanisms within
the BST through quantified behavioral responses.
73
Since the BST is surrounded by dopamine rich areas both laterally (globus
pallidus/caudate putamen) and cranially (NAc shell), we produced brain cannulation
brain slices to support the fact that the effects seen in our study was not due to dopamine
diffusion from adjacent areas, but was due to changes in the BST dopamine. Figure 20
and figure 22 show the BST cannulation placements for sucrose self-administering rats,
and figure 21 and figure 23 show the BST brain placements for the cocaine selfadministering rats. Although there were variations in the accuracy of the placement of
the cannula injectors into the BST region, the overall results showed that the approximate
localization of the injection was in the BST and there was minimal diffusion into the
lateral ventricle.
4.6 - Conclusion
In summary, the major findings in this study are: 1) cocaine operant responding
decreased with blockade of both D1- and D2- dopamine receptors, 2) sucrose operant
responding did not show significant change with either blockade or activation of
dopamine receptors. Our results have partially confirmed our hypothesis that
pharmacological manipulation of dopamine receptors influences the reinforcing efficacy
of natural (sucrose) or pharmacological (cocaine) rewards. This implies that
manipulation of the dopamine receptors in the BST contributes to the associative pairing
of pharmacological rewards to operant behaviour. This observation is also supported by
other work from the Dumont lab. Behaviorally, bilateral administration of the BP897 (a
selective D3 partial agonist) into the BST produced a significant reduction in the final
ratio of operant response in cocaine rats and no effect in sucrose rats. In addition,
electrophysiological data in our lab have shown that rats with chronic cocaine treatment
74
exhibited an upregulation of D1 receptor, accompanied by a change from D2-mediated
pre-synaptic decrease (in normal rats) to post-synaptic D1 mediated increase in GABAergic neurotransmission. Taken together, it was implicated that chronic cocaine
treatment was probably ensued by the BST undergoing changes in synaptic strength
(Dumont et al., 2005; Weitlauf et al., 2004). This is due to dopamine’s critical
contribution in increasing the strength of neuronal connections at the synaptic level
within the reward pathways of the brain in performing reward related behaviours (Hyman
et al., 2006; Dong et al., 2004; Winder et al., 2002; Dumont et al., 2005). Collectively,
these results highlighted the importance of BST dopamine receptor mediation in the
maintenance of cocaine addiction. Specifically, the effect of both D1 and D2 receptor
antagonism on cocaine reinforcement seemed to have affected self-administration
behaviour throughout the whole session. With the incomplete inhibition of behaviours
shown in this study, the BST may very well be just a part of the complex reward system
with various selective and non-selective antagonists and agonists working with several
reinforcing agents. However, with the BST’s strategic placement in the brain, this
research serves to generate knowledge that will serve as key for developing treatments
for drug addiction.
75
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Appendix: I
(Behavioural traces showing the effects of dopamine antagonists and agonist
pretreatment in all the rats)
**Dashed black trace = behavioural effect for drug treatment day
**Red regular trace = lever pressing for days before actual drug treatment day**
Sucrose Rats (Operant control):
L111:
85
March 10 (SCH1.6): only has one day preinjection because there was decreased
trend of breakpoint to injection day (March
7-March 8). This is to minimize drug effect
due to instability of behavior in this case.
86
L112:
Exclude Feb 10 baseline trace
to remove effect of upward
trend of breakpoint and make
drug effect more realistic.
87
Exclude March 12 baseline trace
to remove downward trend of
breakpoint and make drug effect
more realistic.
L113:
88
L126:
Exclude May 2 baseline
trace to make time to reach
breakpoint more uniform
with other days.
L135:
89
March 29 baseline line trace missing,
but data for that day was recorded
and calculated accordingly.
L138:
90
91
L169:
Exclude June 13 baseline trace
to eliminate downward trend
of breakpoint to injection date
and make drug effect more
realistic.
92
August 30 baseline line trace
missing, but numerical data for that
day was still recorded and calculated
accordingly.
L170:
93
94
L194:
L196:
L197:
Aug 30 baseline line trace
missing, but numerical data
for that day was still
recorded and calculated
accordingly.
95
L198:
Aug 30 baseline line trace
missing, but numerical data for
that day was still recorded and
calculated accordingly.
96
Cocaine Rats:
L159:
Exclude June 24 baseline trace
to minimize effect of downward
trend of breakpoint to make
drug effect more realistic.
97
L160:
98
L162:
99
L176:
Aug 28 baseline line trace
missing, but numerical data
for that day was still
recorded and calculated
accordingly.
100
L188:
101
L189:
102
103
L191:
104
105
106
L207:
107
L213:
108
L215:
109
Appendix: II
Summary table for breakpoint, final ratio, and drug effect in all the rats.
Sucrose Rats (Operant control):
Rat ID #
Drug
L111
SCH3.2µg/µl
Saline
Saline
SCH3.2µg/µl
SCH1.6µg/µl
Breakpoint
Final Ratio
15
14
14
13
14
13
13
13
14
15
14
13
14
12
13
13
13
12
95
77
77
62
77
62
62
62
77
95
77
62
77
50
62
62
62
50
Average
breakpoint
on non-drug
days for each
rat
13.1
L112
Sulpiride 3.2
µg/µl
Sulpiride 3.2
µg/µl
Saline
14
15
14
14
77
95
77
77
16
17
19
118
145
219
16
15
16
15
118
95
118
95
110
Saline
15
14
14
95
77
77
14.5
L113
Saline
SCH1.6µg/µl
11
11
12
12
12
11
12
11
40
40
50
50
50
40
50
40
13
11
10
11
9
9
9
62
40
32
40
25
25
25
12
12
12
10
10
13
11
11
50
50
50
32
32
62
40
40
11.8
L126
Saline
Sulpiride
3.2µg/µl
10.6
L135
Saline
SCH1.6µg/µl
9.8
L138
SCH3.2µg/µl
Saline
Saline
14
14
14
12
14
15
13
14
13
13
11
12
14
12
77
77
77
50
77
95
62
77
62
62
40
50
77
50
111
SCH3.2µg/µl
SCH1.6µg/µl
12
14
13
13
9
50
77
62
62
25
18
16
16
178
118
118
19
19
19
17
14
16
15
17
16
18
18
16
219
219
219
145
77
118
95
145
118
178
178
118
13
14
15
14
62
77
95
77
13.2
L169
Sulpiride
3.2µg/µl
Saline
Saline
Sulpiride
3.2µg/µl
Quinpirole
3.0µg/µl
14.4
L170
SCH3.2µg/µl
Saline
SCH3.2µg/µl
14
14
15
15
12
13
14
11
14
16
16
13
15
13
13
77
77
95
95
50
62
77
40
77
118
118
62
95
62
62
112
SCH6.4µg/µl
13
62
14.3
L194
SCH6.4µg/µl
15
15
15
17
95
95
95
145
12.1
L196
SCH6.4µg/µl
13
13
14
14
62
62
77
77
11.8
L197
Quinpirole
3.0µg/µl
SCH6.4µg/µl
11
11
11
12
40
40
40
50
10
11
11
12
32
40
40
50
10.2
L198
Quinpirole
3.0µg/µl
Quinpirole
3.0µg/µl
10
9
10
8
32
25
32
20
14
13
14
13
77
62
77
62
11.4
113
Cocaine Rats:
Rat ID #
Drug
L159
Sulpiride
3.2µg/µl
Sulpiride
3.2µg/µl
Saline
Quinpirole
3.0µg/µl
Breakpoint
Final Ratio
12
13
15
11
50
62
95
40
20
19
17
268
219
145
18
16
16
15
16
16
15
14
178
118
118
95
118
118
95
77
Average
breakpoint
on non-drug
days for each
rat
16.0
L160
SCH3.2µg/µl
Saline
SCH3.2µg/µl
SCH1.6µg/µl
17
17
17
16
19
16
17
19
17
17
16
14
16
16
16
13
145
145
145
118
219
118
145
219
145
145
118
77
118
118
118
62
15.0
L162
15
15
95
95
114
Quinpirole
3.0µg/µl
SCH6.4µg/µl
Saline
16
15
118
95
16
17
17
15
14
13
14
15
118
145
145
95
77
62
77
95
15.2
L176
Quinpirole
3.0µg/µl
9
9
11
7
25
25
40
15
17
18
18
15
13
16
16
13
15
15
14
13
12
11
12
10
145
178
178
95
62
118
118
62
95
95
77
62
50
40
50
32
11
10
12
8
40
32
50
20
18
19
18
178
219
178
9.25
L188
SCH3.2µg/µl
SCH6.4µg/µl
SCH6.4µg/µl
Sulpiride
3.2µg/µl
Quinpirole
3.0µg/µl
12.1
L189
115
Sulpiride
3.2µg/µl
Saline
Quinpirole
3.0µg/µl
Sulpiride
3.2µg/µl
SCH1.6µg/µl
16
118
18
17
18
18
16
14
16
17
178
145
178
178
118
77
118
145
15
16
13
13
95
118
62
62
16
15
16
12
118
95
118
50
15.6
L191
SCH3.2µg/µl
Saline
SCH1.6µg/µl
Quinpirole
3.0µg/µl
Sulpiride
3.2µg/µl
17
17
17
15
16
18
17
17
14
13
15
16
14
16
14
14
145
145
145
95
118
178
145
145
77
62
95
118
77
118
77
77
13
15
14
14
62
95
77
77
15
95
116
Saline
Quinpirole
3.0µg/µl
SCH6.4µg/µl
15
15
14
13
11
13
11
95
95
77
62
40
62
40
16
14
15
14
118
77
95
77
16
13
14
11
16
14
15
15
13
13
12
15
11
13
11
11
13
13
14
13
118
62
77
40
118
77
95
95
62
62
50
95
40
62
40
40
62
62
77
62
13.3
L207
SCH6.4µg/µl
SCH1.6µg/µl
Saline
Saline
Saline
12.8
L213
SCH1.6µg/µl
Saline
14
14
14
13
13
11
11
11
77
77
77
62
62
40
40
40
19
18
219
178
12.4
L215
117
Saline
20
18
268
178
18.1
118