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EXAMINATION OF THE ROLE OF DOPAMINE D3 RECEPTORS
IN BEHAVIOURAL SENSITIZATION TO ETHANOL
by
Sarah Jane Harrison, B.Sc., M.Sc., M.A.
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Graduate Department of Psychology
University of Toronto
© Copyright by Sarah Jane Harrison, 2008
Thesis Title
Examination of the Role of Dopamine D3 Receptors in Behavioural Sensitization to
Ethanol.
Doctor of Philosophy (2008) Graduate Department of Psychology
Sarah Jane Harrison, B.Sc., M.Sc., M.A. University of Toronto
Abstract
Dopamine D3 receptors (D3Rs) have been implicated in mediating behavioural
sensitization to various drugs of abuse, but their role in ethanol (EtOH) sensitization has
not been directly examined. Neil Richtand proposed a role for D3Rs in the modulation of
sensitization by acting as an inhibitor of D1/D2 receptor-mediated behaviours, and
several reports suggest D3Rs up-regulate in response to chronic drugs of abuse. In
separate experiments, we examined EtOH sensitization in D3R knockout (KO) as well as
in D1R and D2R KO mice. We also examined amphetamine sensitization in D3R KOs
compared to wild type mice. We challenged C57Bl/6 and DBA/2 mice with a D3R
agonist (PD128907) and antagonist (U99194A) to examine how acute and chronic D3R
activation and inactivation may affect the induction and expression of EtOH sensitization.
We investigated D1/D3R interactions in sensitized and control mice and examined
whether EtOH sensitization leads to changes in D3R binding using [125I]-7-OH-PIPAT
autoradiography.
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Results showed that D3R KOs, were resistant to EtOH but not amphetamine
sensitization. Chronic but not acute D3R blockade with U99194A inhibited the
induction, whereas acute D3R activation with PD128907 attenuated the expression of
EtOH sensitization. In our D1/D3R interaction study we observed that although
PD128907 attenuated D1 agonist-induced hyperactivity with SKF81297, this effect was
the same in sensitized and control animals, even though sensitized mice were more
responsive to PD128907 than controls. This enhanced response, which suggests a
functional up-regulation of D3Rs, was not accompanied by changes in D3R binding as
indicated by autoradiography, and could mean that functional changes in the D3R
associated with EtOH sensitization occur elsewhere than at the level of the membrane-
bound receptor.
Taken together, these results suggest a modulatory role for the D3R in EtOH but
not amphetamine sensitization, where D3R activation attenuates the expression and D3R
blockade prevents the induction of EtOH sensitization. These results are important
because a better understanding of the role of the D3R in EtOH sensitization may help not
only to identify some of the underlying neural mechanisms of sensitization, but also help
in the identification of treatment strategies for patients that may be susceptible to alcohol
abuse.
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Acknowledgements
I would first like to thank my supervisor Dr. José Nobrega for the support and
opportunities he has given over the years. I would like to thank him for his mentorship,
insight and counsel and most importantly for encouraging me to become an independent
research scientist.
I would also like to thank the members of my supervisory committee, Dr. John
Yeomans and Dr. Eve DeRosa, for their continued support over the years. I have enjoyed
working with them both as their respective teacher’s assistants and in turn I appreciate
their valuable input on my research and thesis. Thank you for your time and assistance.
A special thank you must be extended to the expert staff of the Animal and
Transgenic Facilities, to Ashlie Soko, Tiffany Antopolski and Katrina Deverell, for
breeding, caring for and genotyping the many mice used in my experiments. I am also
extremely grateful to Roger Raymond and Mustansir Diwan for their technical and moral
support both in and out of the laboratory.
Finally on a personal note, I would like to thank my family for being so
supportive throughout my many years of school. Thank you to my Mom, Dad, Michael,
Maureen and Siobhan, for the wonderful home, the great laughs, and for always listening
when I needed it. You are all, in your own special way, a unique role model to me.
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Table of Contents
ABSTRACT ....................................................................................................................... II ACKNOWLEDGEMENTS.................................................................................................... IV TABLE OF CONTENTS ....................................................................................................... V LIST OF FIGURES ............................................................................................................. IX LIST OF TABLES .............................................................................................................. XI LIST OF APPENDICES......................................................................................................XII
CHAPTER 1...................................................................................................................... 1 GENERAL INTRODUCTION ................................................................................................ 1
Behavioural Sensitization .......................................................................................... 1 Definition ............................................................................................................ 1 Characteristics of Behavioural Sensitization...................................................... 2 Induction and Expression of Behavioural Sensitization ..................................... 4 Sensitization in Humans...................................................................................... 6
Ethanol Sensitization ................................................................................................. 9 Features of Ethanol Sensitization ....................................................................... 9 Why Study Ethanol Sensitization? .................................................................... 13
CHAPTER 2.................................................................................................................... 14
Dopamine D3 Receptors .......................................................................................... 14 Distribution ........................................................................................................... 14 D3 Receptor Affinity ............................................................................................. 17 D3-Preferring Agonists and Antagonists.............................................................. 19 D3 Receptor-Mediated Behaviours....................................................................... 20 The Role of Dopamine D3 Receptors in Sensitization .......................................... 22
CHAPTER 3.................................................................................................................... 27 Differences in Mouse Strains .................................................................................. 27
C57Bl/6 vs. DBA/2 ................................................................................................ 27 Dopamine Receptor Mutant Mice ......................................................................... 31
Dopamine D1 Receptor Knockouts (D1 KOs) .................................................. 31 Dopamine D2 Receptor Knockouts (D2 KOs) .................................................. 33 Dopamine D3 Receptor Knockouts (D3 KOs) .................................................. 35
GENERAL OBJECTIVES ................................................................................................... 38
CHAPTER 4.................................................................................................................... 40 STUDY 1: EXAMINATION OF THE DEVELOPMENT AND EXPRESSION OF SENSITIZATION IN MICE LACKING THE D3 DOPAMINE RECEPTOR ................................................................ 40
Purpose ................................................................................................................. 40 Experiment 1A: Comparison of EtOH sensitization in C57 and DBA mice ......... 41
Materials & Method.............................................................................................. 41 Subjects ............................................................................................................. 41
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Drug .................................................................................................................. 42 Apparatus .......................................................................................................... 42 Ethanol Sensitization (Procedural Overview) .................................................. 43
Results ................................................................................................................... 46 DBA/2 Mice....................................................................................................... 47 C57Bl/6 Mice .................................................................................................... 48
Discussion ............................................................................................................. 50 Experiment 1B: Comparison of EtOH sensitization in dopamine receptor knockouts.................................................................................................................. 52
Materials & Method.............................................................................................. 52 Subjects ............................................................................................................. 52 Genotyping ........................................................................................................ 53 Procedural Overview ........................................................................................ 53
Results ................................................................................................................... 54 Saline-treated Control Animals ........................................................................ 54 Experimental Animals (Sensitization in Knockout Mice) ................................. 57
Discussion ............................................................................................................. 60 Experiment 1C: Examination of amphetamine sensitization in D3 knockout mice................................................................................................................................... 67
Purpose ................................................................................................................. 67 Materials & Method.............................................................................................. 67 Results ................................................................................................................... 69 Discussion ............................................................................................................. 71
CHAPTER 5.................................................................................................................... 75 STUDY 2: EXAMINATION OF THE INDUCTION AND EXPRESSION OF ETHANOL SENSITIZATION FOLLOWING PHARMACOLOGICAL BLOCKADE WITH A D3R ANTAGONIST75
Purpose ................................................................................................................. 75 Experiment 2A: Effects of D3 antagonist on the expression of EtOH sensitization................................................................................................................................... 77
Materials & Method.............................................................................................. 77 Subjects ............................................................................................................. 77 Drug .................................................................................................................. 78 Procedural Overview ........................................................................................ 81
Results ................................................................................................................... 82 Discussion ............................................................................................................. 84
Experiment 2B: Effects of D3 antagonist on the induction of EtOH sensitization................................................................................................................................... 87
Materials & Method.............................................................................................. 87 Procedural Overview ........................................................................................ 87
Results ................................................................................................................... 87 C57Bl/6 Mice .................................................................................................... 87 DBA/2 Mice....................................................................................................... 89
Discussion ............................................................................................................. 90
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CHAPTER 6.................................................................................................................... 94
STUDY 3: EXAMINATION OF THE EXPRESSION OF ETHANOL SENSITIZATION FOLLOWING PHARMACOLOGICAL STIMULATION OF D3 RECEPTORS WITH AN AGONIST...................... 94
Experiment 3A: Effects of D3 agonist on expression of EtOH sensitization ........ 94 Purpose ................................................................................................................. 94 Materials & Method.............................................................................................. 95
Procedural Overview ........................................................................................ 95 Drug .................................................................................................................. 95
Results ................................................................................................................... 98 Discussion ........................................................................................................... 100
Experiment 3B: Effects of D3 agonist on induction of EtOH sensitization........ 102 Purpose ............................................................................................................... 102 Materials & Method............................................................................................ 102
Procedural Overview ...................................................................................... 102 Results ................................................................................................................. 103 Discussion ........................................................................................................... 106
CHAPTER 7.................................................................................................................. 109
STUDY 4: EXAMINATION OF D1/D3 RECEPTOR INTERACTIONS IN A PHARMACOLOGICAL CHALLENGE FOLLOWING SENSITIZATION TO ETHANOL................................................. 109
Purpose ............................................................................................................... 109 Materials & Method............................................................................................ 110
Procedural Overview ...................................................................................... 110 Drugs............................................................................................................... 113
Results ................................................................................................................. 114 Discussion ........................................................................................................... 116
CHAPTER 8.................................................................................................................. 118 STUDY 5: EXAMINATION OF CHANGES IN D3 RECEPTOR BINDING FOLLOWING SENSITIZATION TO ETHANOL IN THE MOUSE BRAIN....................................................... 118
Purpose ............................................................................................................... 118 Materials & Method............................................................................................ 119
Brain Sectioning.............................................................................................. 119 D3 Receptor Binding Autoradiography .......................................................... 119 Image Analysis ................................................................................................ 120
Results ................................................................................................................. 122 Discussion ........................................................................................................... 125
CHAPTER 9.................................................................................................................. 129
GENERAL DISCUSSION ................................................................................................. 129 D3 Knockout Mice .............................................................................................. 129 Pharmacological Intervention ............................................................................ 132 D1/D3 Interactions ............................................................................................. 139 Brain Analysis ..................................................................................................... 140
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The D3 Receptor in Behavioural Sensitization to Ethanol: A Proposed Model of Receptor Induction................................................................................................. 141 Experimental Limitations and Future Directions ................................................ 150
REFERENCES ................................................................................................................ 156
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List of Figures
Figure 1: Parallel Distribution of D3 Receptor Protein and D3 mRNA Expression Observed in Mouse Brain Sections Following Different Assays……………………………14 Figure 2: Comparison of EtOH Sensitization in C57 vs. DBA Mice.………………………49 Figure 3: Comparison of Spontaneous Locomotor Activity in Saline-treated Dopamine Knockout Colonies Compared to Wild Type Mice...……………………………………….…55 Figure 4: Comparison of Locomotor Response to Chronic EtOH in Dopamine Knockout Colonies and Wild Type Mice…………………………………………………………………...58 Figure 5: Comparison of Amphetamine Sensitization in Wild Type and D3 KO Mice…..70 Figure 6: Dose-Response Curve for the D3 Receptor Antagonist U99194A……………...79 Figure 7: Time Course for Acute U99194A Across 15-minute Test Session……………....80 Figure 8: Expression of EtOH Sensitization in C57 and DBA Mice Following Acute Challenge with the D3 Antagonist U99194A……………………………………………….…83 Figure 9: Chronic Co-administration of D3 Antagonist U99194A with Chronic EtOH in C57 and DBA Mice……………………………………………………………………………….88 Figure 10: Dose-Response Curve for the D3 Receptor Agonist PD128907……………….96 Figure 11: Time Course for the D3 Agonist PD128907 Across 30-Minute Test Session..97 Figure 12: Acute PD128907 Challenge in Sensitized vs. Control DBA Mice…………..…99 Figure 13: Chronic Co-administration of the D3 Agonist PD128907 with EtOH Sensitization………………………………………………………………………………………104 Figure 14: Dose-Response Curve for the D1 Receptor Agonist SKF81297……………...111 Figure 15: Time Course for the D1 Agonist SKF81297 Across 90-minute Test Session.112 Figure 16: Comparison of EtOH Sensitized and Control Animals Following Administration of D1 and D3 Receptor Agonists……………………………………………115
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Figure 17: Sample Autoradiogram Images Obtained from [125I-]7-OH-PIPAT Receptor Binding Autoradiography Demonstrating D3 Receptor Protein Distribution in the Mouse Brain……………………………………………………………………………………………....123 Figure 18: The D3 Receptor in Behavioural Sensitization to Ethanol: A Proposed Model for Receptor Induction………………………………………………………………………….146
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List of Tables
Table 1: D3 Receptors Following Sensitization to Various Drugs of Abuse………………26
Table 2: Summary of Results from [125I]-7-OH-PIPAT Binding Autoradiography……..124
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List of Appendices
Appendix 1A: Raw Ambulatory Count Data for DBA mice during EtOH sensitization……………………………………………………………………………………....180 Appendix 1B: Raw Ambulatory Count Data for C57 mice during EtOH sensitization..181 Appendix 2A: PCR reaction components, cycling conditions and primer sequences for the dopamine D1 receptor knockout mice genotyping protocol from Jackson Laboratories…………………………………………………………………………………..…182 Appendix 2B: PCR reaction components, cycling conditions and primer sequences for the dopamine D2 receptor knockout mice genotyping protocol from Jackson Laboratories………………………………………………………………………………….....184 Appendix 2C: PCR reaction components, cycling conditions and primer sequences for the dopamine D3 receptor knockout mice genotyping protocol from Jackson Laboratories…………………………………………………………………………………...186
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CHAPTER 1
General Introduction
Behavioural Sensitization
Definition
Behavioural sensitization is the phenomenon that occurs when repeated,
intermittent drug administration leads to a progressive increase in a response to that drug
over time (Post, 1980; Post & Rose, 1976; Robinson, 1993; D. S. Segal & Mandell,
1974). A common physiological response to chronic drug administration is tolerance,
that is, drug effects generally become smaller with repeated usage, requiring more and
more drug to reach the same endpoint (Ramsay & Woods, 1997). When the body detects
drug-induced changes, it adapts to these changes by triggering reflexes that bring all
altered parameters back toward the pre-drug level. These adaptations form the
fundamental principle of homeostasis.
With respect to sensitization, the opposite response pattern occurs whereby the
body’s response increases with the same dose of drug, or requires less and less of the
drug each time to reach the same endpoint. This phenomenon therefore appears to be in
violation of homeostatic principles. Considered in this light then, behavioural
sensitization is quite a different phenomenon from tolerance, one that is, in fact, wasteful
and maladaptive (Woods & Ramsay, 2000).
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Characteristics of Behavioural Sensitization
In laboratory animals behavioural sensitization is characterized by increases in the
amount, speed and organization of locomotor activity (D. S. Segal & Mandell, 1974).
Behavioural sensitization can be readily measured in rodents by examining changes in
their induced locomotor activity in response to a drug. Locomotor activity is a reliable
and quantitative index to measure sensitization (increased responding) over the course of
drug treatment because it is easily measured in activity monitors and demonstrates clearly
that a change in behaviour has occurred (Eilam & Szechtman, 1989; Einat & Szechtman,
1993).
Although there are many variables that may determine whether tolerance or
sensitization occurs in response to repeated drug administration, one important factor in
determining the overall direction of response lies within the pattern of drug
administration. While chronic, continuous drug administration is often associated with
the development of tolerance, repeated intermittent drug administration is thought to be
more widely associated with the development of sensitization (Post, 1980). Furthermore
it has been repeatedly demonstrated that the behavioural sensitization effect (increased
response) observed following a course of repeated drug administration is considerably
enhanced when laboratory animals are challenged with a test dose of the drug following a
period of drug abstinence (Kalivas & Duffy, 1993; Paulson et al., 1991).
Repeated intermittent administration of many drugs of abuse lead to long-term
neuronal and molecular adaptations in the brain and behavioural sensitization is believed
to be an expression of these neuroadaptations (Pierce & Kalivas, 1997). Sensitization is
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not considered to be merely a result of an accumulation of drug within the system, but
manifests itself as an enduring behavioural change, persisting for some time following
termination of chronic drug administration (Robinson & Becker, 1986). For example,
sensitization to cocaine in rats has been shown to persist as long as 1-3 months after
termination of the drug (Henry & White, 1995; Shuster et al., 1977), while amphetamine
sensitization can last for at least one year in rats (Paulson et al., 1991). In humans,
pretreatment with dopaminergic drugs such as amphetamine can facilitate the later
acquisition of drug self-administration or drug-induced psychosis (Piazza et al., 1989;
Richtand et al., 2001; Sato, 1992; Vezina, 2004). These findings suggest that persistent
changes in neural mechanisms, particularly within the mesolimbic dopamine system,
underlie the manifestation of sensitization (Robinson, 1993; Wise & Leeb, 1993). It has
been postulated that these neural mechanisms become and remain increasingly sensitive
to the drug, and a relationship of this phenomenon to drug relapse after long periods of
abstinence has been proposed (Lessov & Phillips, 1998a, 1998b; Robinson, 1993;
Robinson & Berridge, 1993).
Various dopaminergic stimulants including cocaine, amphetamine, quinpirole and
L-DOPA have all been reported to induce behavioural sensitization (Angrist, 1983; Eilam
& Szechtman, 1989; Einat & Szechtman, 1993; Kalivas et al., 1993; Post & Contel,
1983; D. S. Segal et al., 1981; D. S. Segal & Mandell, 1974; D. S. Segal & Schuckit,
1983; Stewart & Badiani, 1993). Although the exact biochemical mechanisms
underlying behavioural sensitization are yet to be fully understood, some researchers
believe that these mechanisms are similar to those that underlie the development of
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behavioural pathologies associated with psychosis, drug abuse and relapse (Angrist,
1983; Kokkinidis & Anisman, 1980; Szechtman et al., 1998). A better understanding of
this phenomenon may be helpful in identifying treatment strategies for these disorders,
particularly in the realm of pharmacotherapy (Antelman, 1988).
Induction and Expression of Behavioural Sensitization
The process of behavioural sensitization can be separated into two distinct
components, both temporally and anatomically, termed ‘induction’ (also called
‘development’, ‘acquisition’ or ‘initiation’) and ‘expression’ (Kalivas & Stewart, 1991;
Robinson & Becker, 1986). The induction or initiation of behavioural sensitization is
operationally defined as the transient sequence of cellular and molecular events
precipitated by chronic drug administration that leads to enduring changes in neural
function responsible for behavioural augmentation. Expression is defined as the enduring
neural alterations arising from the initiation process that directly mediate the augmented
behavioural response (Pierce & Kalivas, 1997).
Induction of sensitization is primarily mediated by the ventral tegmental area
(VTA) (Bjijou et al., 2002; Kalivas & Stewart, 1991; Pierce & Kalivas, 1997; Vezina,
1996) whereas expression of sensitization is believed to result primarily from changes in
the nucleus accumbens (NAcc) (Cador et al., 1995; Kalivas et al., 1993; Pierce &
Kalivas, 1997) as well as other terminal projection sites of the reward pathway including
the amygdala and prefrontal cortex (Bjijou et al., 2002; Pierce & Kalivas, 1997).
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Locomotor sensitization is produced by systemic or local infusions of various
drugs of abuse including amphetamine, morphine and cocaine into the VTA, but not by
infusions of these same drugs into a number of dopamine neuron terminal fields
including the NAcc (Cador et al., 1995; Dougherty & Ellinwood, 1981; Kalivas &
Weber, 1988; Perugini & Vezina, 1994; Vezina, 2004; Vezina & Stewart, 1990).
Instead, local infusions of drugs of abuse such as cocaine and amphetamine to the NAcc
lead to locomotor excitation in a manner similar to acute drug administration, but chronic
administration of these drugs does not produce an augmented response over time (Cador
et al., 1995). These findings indicate that the NAcc is important for the expression, but
not the induction of behavioural sensitization (Vezina, 2004).
Dopamine D1 receptors in the VTA have been most widely cited as playing a
necessary role in the induction of behavioural sensitization (Bjijou et al., 1996; Cador et
al., 1995; Kalivas et al., 1993; Kalivas & Stewart, 1991; Pierce & Kalivas, 1997; Vezina,
1996). Blockade of D1 receptors in the VTA with the D1 selective antagonist SCH23390
has been shown to block the development of sensitization to systemic or locally infused
amphetamine; however, this effect was not observed with D2 receptor antagonists
sulpiride, spiperone or eticlopride, indicating that D1, but not D2 receptors are necessary
for the induction of behavioural sensitization (Vezina, 1996).
There are many neurochemical changes that mediate the expression of
sensitization, most notable are the long-term changes that occur in the NAcc (Wolf,
1998). Some of these changes include persistent increases in D1 receptor
electrophysiological responsiveness (Henry & White, 1991, 1995; Higashi et al., 1989;
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Xu et al., 1997), upregulation of cAMP signal transduction (Nestler et al., 1990;
Terwilliger et al., 1991) an enhancement of the increase in extracellular dopamine
elicited by stimulants (Kalivas & Duffy, 1990; Robinson et al., 1988), and an increase in
response to D2 and D3 agonists such as quinpirole (De Vries et al., 2002). There are
many other persistent neurochemical changes that are associated with the expression of
sensitization, many of which may be beyond the scope of this introduction. However, the
above-mentioned changes indicate that long-term alterations in the mesolimbic dopamine
system and increased responsiveness of D1, D2 and D3 receptors in the NAcc play a key
role in mediating the expression of behavioural sensitization.
Sensitization in Humans
Although locomotor sensitization to many drugs of abuse can be readily induced
in laboratory animals, evidence that sensitization occurs in humans has been more
difficult to demonstrate (Robinson & Berridge, 2001). Until recently, the only evidence
that repeated exposure to psychostimulants could produce sensitization in humans came
from studies of cocaine and amphetamine psychosis where pre-exposure to
psychostimulants could exacerbate subsequent psychotomimetic effects of these drugs
(Post & Contel, 1983; Sato, 1992; D. S. Segal & Schuckit, 1983).
More recently however, several reports have attempted to examine direct
evidence for behavioural sensitization to stimulant drugs in humans, which have
produced inconsistent results. Variability in these results may reflect differences in drug
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dose (5-30 mg, p.o), dosing regimen (2-5 doses; inter-dose intervals of 1 day-12 months),
subject population (healthy controls vs. substance dependent), and dependent measures
(Leyton, 2007).
In several of the earlier human studies, many investigators chose, as their primary
dependent measure, changes in subject-rated ‘highs’ or euphoria levels; however
emerging evidence suggests that increased dopamine levels may be more closely related
to motivational salience of the reward, rather than to the pleasurable or hedonic nature of
the reward (Berridge & Robinson, 1998; Blackburn et al., 1992; Leyton et al., 2005;
Robinson & Berridge, 1993, 2000, 2001). Furthermore in stimulant abusers, there is a
reported tolerance to the euphorigenic effects of the drug despite increases in drug-
seeking behaviour (Volkow et al., 1997; Volkow et al., 1999). Thus using subject-rated
reports of euphoria alone may not be a useful dependent measurement with which to
study sensitization in humans. More useful data has been gathered when dependent
measures have been expanded to include, self-reported mood and energy levels, drug
choice, eye-blink rate, and clinician-rated scales for manic symptoms such as increased
speech and overall physiological vigor (Leyton, 2007).
To date, seven studies have examined the behavioural effects of repeated
amphetamine administration in humans in the laboratory. In two studies, in which 5-10
mg, p.o., of amphetamine was administered, no evidence of behavioural sensitization was
observed (Johanson & Uhlenhuth, 1981; T. H. Kelly et al., 1991). However, in studies
that used higher doses (20-30 mg, p.o.), increases in self-reported mood and energy levels
(Boileau et al., 2006; Strakowski & Sax, 1998; Strakowski et al., 1996), as well as
7
clinician-rated indices of vigor and energy (Strakowski & Sax, 1998; Strakowski et al.,
1996) were reported in addition to potentiated eye-blink responses (Boileau et al., 2006;
Strakowski & Sax, 1998; Strakowski et al., 1996). These changes in behaviour were not
associated with increases in self-reported ‘drug-liking’, which either did not change
(Strakowski & Sax, 1998) or decreased (Strakowski et al., 2001) with repeated
amphetamine administration.
The effects of repeated administration of other psychostimulants such as cocaine
have not been examined in drug-naïve subjects. Studies of sensitization in cocaine-
dependent individuals have not demonstrated any increases in subjective effects or
physiological responses (Gorelick & Rothman, 1997; Nagoshi et al., 1992; Rothman et
al., 1994). However it is entirely possible that cocaine-dependent individuals might be
maximally sensitized to additional administration of a familiar drug. Given the risk of
inducing addictive behaviours in drug-naïve-subjects, the ethics of studying sensitization
in human subjects is clearly limiting. Nonetheless, from the few previously described
studies examining changes in response to amphetamine in drug-naïve subjects, the overall
pattern of responses suggests that behavioural sensitization to amphetamine (when
administered at moderately high doses such as 20-30 mg), can occur in as little as 2-3
administrations, and can last up to at least one year in humans (Boileau et al., 2006;
Leyton, 2007). Taken together, there is increasing evidence to support the theory that
behavioural sensitization to psychostimulants can occur in humans (Sax & Strakowski,
2001).
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Ethanol Sensitization
Features of Ethanol Sensitization
A widely held view regarding the neurochemical basis of substance abuse, is that
most, if not all drugs of abuse ultimately lead to increases in mesolimbic dopamine
release in the NAcc, which is a critical hallmark for the development of addictive
behaviours (Di Chiara & Imperato, 1988; Robinson & Berridge, 1993; Wise, 2002).
Some drugs of abuse have more direct effects of accumbal dopamine release than others.
For example, drugs that are classified as psychostimulant drugs, such as cocaine and
amphetamine, block the dopamine re-uptake transporter, thus elevating the levels of
dopamine in the synapse. These drugs act directly on the NAcc, and for the purposes of
this thesis, will be referred to as direct dopaminergic agents. Other drugs of abuse lead to
elevation of accumbal dopamine in a less direct manner. Some drugs of abuse such as
nicotine can excite the VTA, whereas others such as morphine and ethanol can disinhibit
the VTA. Excitation of the dopaminergic cell bodies in the VTA leads to the activation
of the mesolimbic pathway, thus ultimately increasing dopamine release in the NAcc
(Wise, 2002; Wise & Leeb, 1993). For the purposes of this thesis these drugs that act to
increase the mesolimbic pathway via the VTA will be referred to as indirect
dopaminergic agents.
Although behavioural sensitization has been widely studied with the use of direct
psychomotor stimulants, such as amphetamine and cocaine it has also been observed to
occur with chronic administration of other indirect dopaminergic drugs of abuse such as
morphine, phencyclidine, MDMA, nicotine and ethanol (Itzhak & Martin, 1999; Masur &
9
Boerngen, 1980; Robinson & Berridge, 1993, 2000; Stinus et al., 1985). Despite the fact
that the number of studies on ethanol sensitization research remains small in comparison
to studies on ethanol tolerance, there is a growing literature that suggests that repeated
ethanol administration does indeed produce psychomotor sensitization, particularly in
mice (Crabbe et al., 1982; Cunningham & Noble, 1992; Lessov & Phillips, 1998a; Masur
et al., 1986).
Although it has been difficult to study the psychomotor activating effects of drugs
that also have motor depressant effects, such as morphine and ethanol, previous studies
have obviated this problem by isolating features of these models that focus on the
stimulant aspect of the drug effects. For example in studies of ethanol sensitization in
mice, stimulant effects are observed within the first 0-15 minutes of drug administration,
after which point, the sedative effects are observed to dominate the behavioural profile
(Crabbe et al., 1982; Lessov & Phillips, 1998a; Masur & Boerngen, 1980). In fact
Tamara Phillips’ group has shown that the locomotor stimulating effects of EtOH are
immediate and peak within 5 minutes of intraperitoneal administration (Lessov &
Phillips, 1998a; Phillips et al., 1991; Phillips et al., 1995). Therefore, by understanding
the biphasic time course of ethanol administration, examination of locomotor activity
within the first 15 minutes of administration makes it possible to isolate the stimulant
effects of this drug from the subsequent sedative effects.
Ethanol’s stimulant effects in mice are also highly dose-dependent. Masur and
Boerngen demonstrated that in comparison to saline-treated animals, mice show no
change in response to acute low doses of 1.0-1.5 g/kg, an immediate excitatory response
10
to acute injections of ethanol at doses from 2.0-2.5 g/kg, and an immediate sedative effect
to higher doses of 3.5-4.0 g/kg (Masur & Boerngen, 1980). With chronic exposure to
ethanol, the initial excitatory doses produced a progressive increase in response to the
drug over time. This sensitized response remained high and showed no signs of
developing tolerance even after 5 months of chronic treatment (Masur et al., 1986). On
the other hand, mice that were chronically treated with ethanol at initially depressive
doses demonstrated a tolerance to the sedative effects by day 15, and by day 30 a small
but significant increase in locomotor activity was observed and remained even after 45
days of treatment. These data suggest that even at doses where initial sedative effects
take place, once tolerance to the depressive effects have taken place, an increase in
response to the stimulant effects of alcohol can emerge and remain stable within the
behavioural profile (Crabbe et al., 1982; Masur & Boerngen, 1980; Tabakoff &
Kiianmaa, 1982).
The behavioural profile that was initially observed by Masur and Boerngen has
also been replicated in other laboratories, using differing injection regimes and time
courses as well as differing routes of administration, (oral vs. intraperitoneal)
demonstrating that this pattern is very strong in mice and not easily disrupted by inter-
laboratory differences (Crabbe et al., 1982; Tabakoff & Kiianmaa, 1982). In rats, initial
excitatory responses from ethanol did not appear at any acute dose and even though rats
demonstrate a tolerance to initial sedative effects of chronic ethanol, an unmasking of a
sensitizing response did not appear at any time (Masur et al., 1986). The authors suggest
that since this behaviour is so apparent in mice but not in rats, the tolerating and
11
sensitizing responses to ethanol may be mediated by at least partially divergent
mechanisms that may be under genetic control and manifesting themselves differently in
various species (Masur et al., 1986). Since the Masur study reported this apparent
resistance to ethanol sensitization in rats, the vast majority of studies examining the
stimulant effects of chronic ethanol in rodents are performed in mice. To state
conclusively that all rats are resistant to EtOH sensitization would be premature in the
absence of a large-scale comparison of various rat strains using different sensitization
protocols. However since a study of this nature has not been performed, it is difficult to
say with any certainty whether rats are capable of demonstrating sensitization to ethanol.
The effects of ethanol sensitization in mice have been found to persist for at least
1 month after termination of the drug, suggesting that there are long-term
neuroadaptations that may result from chronic ethanol administration (Lessov & Phillips,
1998a). Furthermore, behavioural sensitization to ethanol has been suggested to
contribute to uncontrolled alcohol drinking and seems to enhance voluntary ethanol
consumption in mice (Camarini & Hodge, 2004; Hunt & Lands, 1992; Lessov & Phillips,
1998a). In humans, sons of alcoholics who are at increased risk for the development of
alcoholism show sensitization to a number of physiological responses to alcohol
including skin conductance, finger pulse amplitude and finger temperature whereas sons
of non-alcoholics show tolerance to these same measures (Newlin & Thomson, 1991,
1999). It has also been suggested that the differences in tendency to develop sensitization
to ethanol may represent a predictor for future alcoholism (Brodie, 2002; Schmidt et al.,
2000).
12
Why Study Ethanol Sensitization?
The reasons for using ethanol in the present studies are twofold. First, ethanol is
the most extensively abused drug in society, and yet despite the vast literature that
surrounds ethanol, the studies on ethanol-induced sensitization are sparse (Lessov et al.,
2001). Most studies of ethanol focus on the sedative effects of alcohol and tolerance to
alcohol. In light of the potential role of sensitization in drug abuse, it is worthwhile to
investigate the differences in tendency to develop ethanol sensitization as a potential
predictor for alcoholism. A close examination of this ethanol sensitization model may
help to identify neural mechanisms that may be common to all drugs of abuse and
therefore may give insight into common mechanisms of addiction, as well as identifying
features of this model that may be unique to ethanol sensitization itself.
Second, the study of behavioural sensitization with ethanol is of particular interest
for studying dopaminergic mechanisms. Most studies thus far have examined changes in
the dopaminergic system while looking at sensitization induced by dopaminergic agents,
which entails the risk of circularity. By investigating whether ethanol can induce changes
in the dopaminergic system, analyses of brain mechanisms of behavioural sensitization
may be widened to include agents that do not directly act on dopamine receptors
themselves.
13
CHAPTER 2
Dopamine D3 Receptors
Distribution
Pierre Sokoloff and his colleagues first cloned and characterized the D3 dopamine
receptor sub-type in 1990 (Sokoloff et al., 1990). Based on amino acid sequence and
gene organization, the D3 receptor has been classified as a member of the D2-like family
of dopamine receptors that possesses a 7-transmembrane domain structure and is coupled
to inhibitory G-proteins (Schwartz et al., 1995). The D3 receptor shares a 75% sequence
homology with the D2 receptor in the transmembrane domains, with greatest differences
existing in the third intracytoplasmic loop, the domain that is known for coupling to G
proteins (Sokoloff et al., 1990).
In contrast to the D2 dopamine receptor subtype which demonstrates a wide
distribution throughout the CNS, the D3 receptor protein is less abundant and expressed
in a much more restricted manner than the D2 receptor protein. The overall number of
D3 receptors in the whole brain is estimated to be lower than that of the D2 receptor
protein by approximately 2 orders of magnitude, which is also in the same range as that
of corresponding mRNA (Bouthenet et al., 1991; Levesque et al., 1992; Sokoloff et al.,
1990). The D3 dopamine receptor distribution is primarily restricted to limbic brain
areas, with highest densities found in the islands of Calleja, olfactory tubercle, nucleus
accumbens (NAcc) as well as lobules 9 and 10 of the cerebellum (i.e., archicerebellum)
(Levesque et al., 1992). In the nucleus accumbens, D3 receptors have been detected in
the rostral pole and ventromedial portion of the shell, but are absent in the core and septal
14
pole (cone) of the shell, both of which are areas known to be rich in D2 receptors (Diaz et
al., 1994). Other brain areas have been reported to express low to moderate amounts of
D3 receptors as well, including the ventral pallidum (VP), lateral substantia nigra pars
compacta (lSNc), medial mammillary nucleus (MM), and the dentate gyrus of the
hippocampus (Bouthenet et al., 1991; Diaz et al., 2000; Stanwood et al., 2000a).
The distribution of the D3 dopamine receptor protein is approximately parallel to
that of the D3 receptor mRNA (Figure 1), suggesting that the receptor protein and mRNA
are highly co-localized (Bouthenet et al., 1991; Shafer & Levant, 1998). And although
most D3 mRNA expressing cells also express D2 mRNA in humans (Gurevich & Joyce,
1999), D2 and D3 receptors in the rodent brain appear to have predominantly
complementary rather than overlapping patterns of expression (Bouthenet et al., 1991).
Therefore, in rodent brains, high densities of D2 receptors are found in brain areas such
as the caudate-putamen, pituitary and the core region of the NAcc, with very low to no
densities of D3 receptors found in these regions. This distribution is in contrast to high
densities of D3 receptors found in the islands of Calleja, olfactory tubercle and NAcc
shell in the rodent brain, where low densities of D2 receptors have been observed
(Bouthenet et al., 1991; Sokoloff et al., 1990).
In contrast to the more complementary D2/D3 brain distribution, D1 and D3
receptors are often found to be co-expressed on the same neurons. More specifically, in
the granule cells of the islands of Calleja, a class of substance P-containing neurons
receiving dopaminergic input from the mesencephalon shows a large degree of D1/D3
receptor coexistence (Schwartz et al., 1998a). Additionally the D3 receptor expressing
15
Figure 1:
A: D3 receptor binding autoradiogramusing [3H]-7-OH-DPAT.
B: D3 mRNA expression using in situ hybridization.
ICj Olf ICj
NAcc-Sh
CtxCtx
CPu CPu
NAcc-Sh
Parallel distribution of D3 receptor protein and D3 mRNA expression observed in mousebrain sections following different assays: Panel A represents D3 receptor protein distributionin limbic regions as visualized by using [3H]-7-OH-DPAT autoradiography. Panel B showsD3 receptor mRNA distribution as visualized by using [35S]-UTP in situ hybridization. Bothof these assays were performed in our laboratory where these images were generated (S. J.Harrison, 2004). Although the autoradiogram of the D3 receptor binding lacked visualresolution, it remained clear that binding specificity was well established and parallels the D3receptor mRNA distribution. Regions with the highest binding signals include the islands ofCalleja (ICj), olfactory tubercle (Olf ) and shell region of the nucleus accumbens (NAcc-Sh).Very low to no signal could be detected in the caudate-putamen (CPu) nuclei or the cortex(Ctx), which were indistinguishable from background. Additional autoradiogram images using[125I]-7-OH-PIPAT autoradiography can be seen on page 123 (Figure 17).
16
neurons in the shell portion of the NAcc are mainly GABAergic medium spiny neurons
that co-express the D1 receptor, as well as dynorphin and substance P mRNA (Ridray et
al., 1998). Given the close proximity of these two receptor types, researchers have been
interested in the interactions between these two receptors and the functional relevance of
this co-expression.
D3 Receptor Affinity
Of the 5 sub-types of dopamine receptors, the D3 receptor appears to have the
highest overall affinity for endogenous dopamine, which binds at nanomolar levels
(Levesque et al., 1992; Sokoloff et al., 1992b). Another major property of the native
receptor is the low modulatory effect of guanyl nucleotides on its binding. In the
presence of guanyl nucleotides, when D2 and D3 receptors exist in the low affinity state,
D3 receptors have been found to have ~70-fold higher affinity for endogenous dopamine
than D2 receptors, (Richfield et al., 1989; Richtand et al., 2000; Sokoloff et al., 1992b).
In the absence of guanyl nucleotides, D2 and D3 receptors exist in the high affinity state,
where dopamine now has ~20-fold higher preference for D3 receptors than D2 receptors.
Where the D2 receptor exhibits approximately a 100-fold higher affinity for agonists in
the high affinity state compared to the low affinity state, the D3 receptor exhibits only
about a 5-10-fold increase in affinity between the high and low states (Levesque et al.,
1992; Shafer & Levant, 1998). Although the difference in affinity is not as great when
both the D2 and D3 receptors exist in the high affinity state, it has been proposed that
17
since D3 receptors in the rodent brain are insensitive to guanyl nucleotides that D3
receptors, like D1 receptors, may exist predominantly in the low affinity state in vivo
(Richfield et al., 1989; Richtand et al., 2001; Shafer & Levant, 1998).
In the low affinity state, the D3 receptor has the highest affinity for endogenous
dopamine Ki = 30 nM (Sokoloff et al., 1992b), which is close to basal extracellular
concentrations (3-5 nM)(Kalivas & Duffy, 1993; Parsons & Justice, 1992) and synaptic
dopamine concentrations (50 nM) (Ross, 1991). In contrast, D1 and D2 receptor affinity
for dopamine are much lower: D1 Ki = 2300 nM and D2 Ki = 2000 nM (Sokoloff et al.,
1992a). Following stimulant drug administration, dopamine concentrations are elevated
for prolonged periods with average concentrations of 750 nM (Zetterstrom et al., 1983).
At that concentration, calculated low-affinity state receptor occupancy is 96% for the D3
receptor, compared to 25% for D1 and 27% for D2 receptor (Richtand, 2006). Given the
limited number and distribution of D3 receptors in the brain as compared to D1 and D2
receptors, these reports indicate that endogenous dopamine will bind to and saturate D3
receptors long before saturation of D1 and D2 receptors has been reached. The
functional consequences of this discrepancy are not yet certain.
18
D3-Preferring Agonists and Antagonists
Due to the high homology of the D3 dopamine receptor to the D2 dopamine
receptor, the pharmacological profile of the D3 receptor is generally similar to that of the
D2 receptor (Sokoloff et al., 1990). To date, there are no known agonists that are purely
selective for D3 receptors over D2 receptors (Burris et al., 1995). Several putatively D3-
selective ligands have been identified and employed for binding studies in the brain;
however the degree of D2/D3 selectivity has been variable and appears to be highly
dependent on the in vitro assay conditions used (Flietstra & Levant, 1998).
Beth Levant and her colleagues have established a rank order of potency of
dopamine agonists for the D3 receptor. The results of their studies indicate that (+)-trans-
3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3b]-1,4-oxasin-9-ol
(PD128907) and R-(+)-7-hydroxy-2-(N,N-di-n-propylamino)tetralin (7-OH-DPAT) have
the highest known affinity for the D3 receptor, followed by quinpirole and dopamine
(Bancroft et al., 1998). Dopamine agonists exhibited the following rank order potency in
competition with [3H]-PD128907: PD128907 (Ki = 0.53 ± 0.11 nmol/L) ≈ 7-OH-DPAT
(Ki = 0.42 ± 0.06 nmol/L) ≈ quinpirole (Ki = 0.74 ± 0.07) > dopamine (Ki = 20 ± 2.0)
(Bancroft et al., 1998). Comparatively, dopamine agonists exhibited the following rank
order potency in competition with [3H]-7-OH-DPAT: PD128907 (Ki = 0.73 ± 0.17
nmol/L) ≥ 7-OH-DPAT (Ki = 1.1 ± 0.38 nmol/L) ≈ quinpirole (Ki = 2.0 ± 0.16 nmol/L) ≥
dopamine (Ki = 5.5 ± 1.0 nmol/L) (Bancroft et al., 1998). Therefore the binding profile
19
of 7-OH-DPAT and PD128907 share relatively equivalent rank order potencies
(correlation coefficient=0.707) (Bancroft et al., 1998).
Although PD128907 has a slightly higher affinity for the D3 receptor over the D2
receptor in vivo than 7-OH-DPAT, it has been demonstrated that in vitro, the D2/D3
selectivity of these compounds is highly dependent on the assay conditions, particularly
the buffer composition (Burris et al., 1995; Levesque et al., 1992). Specifically, the
greatest D2/D3 selectivity with 7-OH-DPAT can be obtained in the absence of Mg2+ and
in the presence of EDTA, since the high affinity state of D2 receptors is not favoured in
the absence of Mg2+ (Bancroft et al., 1998; Sibley & Creese, 1983). Other D3-preferring
agonists include quinelorane, pramipexole and 7-trans-OH-PIPAT (Levant et al., 1995).
Several antagonists possessing modest selectivity for the D3 receptor over the D2
receptor have been identified and include l-nafadotride, (+)-S-14297, U-99194A, and
GR103691 (Audinot et al., 1998; Sautel et al., 1995).
D3 Receptor-Mediated Behaviours
Although the D3 receptor has been implicated in several behaviours including
agonist-induced yawning (Collins et al., 2005), penile erection and ejaculatory behaviour
(Ahlenius & Larsson, 1995; Ferrari & Giuliani, 1995), the receptor is most widely cited
in the modulation of locomotor activity. In contrast to the D2 dopamine receptor
subtype, where stimulation is thought to increase locomotor activity, stimulation of the
D3 receptor is believed to inhibit locomotor activity (Daly & Waddington, 1993; Waters
20
et al., 1993). Moreover, administration of D3-preferring agonists such as 7-OH-DPAT
and PD128907 produces a dose-dependent biphasic effect on locomotor activity in rats
and mice, where locomotion is inhibited at low doses and stimulated at higher doses
(Daly & Waddington, 1993; Pugsley et al., 1995; M. S. Starr & Starr, 1995). The
inhibitory effects have been attributed to the action of these drugs at the D3 receptor,
whereas the stimulatory effects have been attributed to the actions of higher doses of the
drug at D2 receptors (Ahlenius & Salmi, 1994; Daly & Waddington, 1993; M. S. Starr &
Starr, 1995; Svensson et al., 1994). Support for this interpretation was demonstrated by
the finding that inhibitory effects of 7-OH-DPAT were produced at doses of the drug that
produced significant D3 receptor occupancy but did not produce significant occupancy of
D2 receptors in vivo (Levant et al., 1996).
On the other hand, the D3-preferring antagonist nafadotride produced biphasic
effects on locomotor activity, stimulating locomotor activity at low doses and inhibiting
locomotion at high doses (Sautel et al., 1995). Again, the doses of nafadotride used to
stimulate locomotion produced significant D3 and negligible D2 occupancy in vivo,
whereas those doses used to inhibit locomotion produced significant D2 occupancy
(Levant & Vansell, 1997).
It has been demonstrated that mutant mice lacking the D3 receptor exhibit
enhanced behavioural sensitivity to injections of drugs that have simultaneous actions on
both D1 and D2 receptors such as cocaine and amphetamine, suggesting that one function
of the D3 receptor is to modulate behaviours by inhibiting the cooperative effects of
postsynaptic D1 and D2 receptors at the systems level (Xu et al., 1997). In addition,
21
mice that demonstrated D1 agonist-induced hyperactivity with the administration of the
D1-selective agonist SKF81297 experienced an attenuation of this hyperactivity when
they were subsequently injected with the D3-preferring agonist, PD128907 (Mori et al.,
1997). Taken together these findings indicate the probable involvement of the D3
receptor in the modulation of locomotor activity in a manner opposite to that of the
D1/D2 dopamine receptor stimulation (Depoortere, 1999; Stanwood et al., 2000b).
The Role of Dopamine D3 Receptors in Sensitization
Considerable evidence implicates dopaminergic mechanisms in the development
and expression of sensitization (Kalivas & Stewart, 1991; Muller & Seeman, 1979; Post
& Contel, 1983; Robinson & Berridge, 1993; Wise & Bozarth, 1987). While the role of
dopamine D1 and D2 receptors has received most of the attention, it has been recently
hypothesized that the dopamine D3 receptor may play a role in the expression of
sensitization (Guillin et al., 2001; Le Foll et al., 2003; Richtand et al., 2000; Richtand et
al., 2003; Richtand et al., 2001). Because the distribution of D3 receptors is largely
restricted to the limbic system, especially the nucleus accumbens, a terminal projection
site of mesolimbic dopamine neurons, D3 receptors have also been suggested to play a
role in mediating reward-related behaviours, (Caine & Koob, 1993; Chaperon & Thiebot,
1996) as well as being involved in the mechanisms of drug dependence and addiction
(Heidbreder et al., 2005; Le Foll et al., 2005b).
22
As mentioned previously, activation of D3 and D1/D2 dopamine receptor
subtypes has opposing functional consequences on behaviour. This has been most clearly
characterized in rodent locomotion where considerable evidence supports the widely held
view that D1/D2 activation increases locomotor activity, while D3 receptor stimulation
inhibits locomotion (Depoortere, 1999; Xu et al., 1997). Thus the behavioural action of
the dopamine D3 receptor is believed to act as a ‘brake’ on D1/D2 mediated behaviours
(Glickstein & Schmauss, 2001; Richtand et al., 2001).
Neil Richtand suggests that behavioural sensitization may be the result of an
imbalance of D3 receptors relative to D1/D2 receptors. If this is the case, then his
hypothesis may even be extended to suggest that an imbalance of D3 receptors may serve
as a marker for a genetic predisposition towards drug abuse. Such an imbalance may also
potentially account for a reason why some individuals are more likely to become
susceptible to drug-seeking behaviour, while others are more resistant. Richtand suggests
that those individuals that express sensitization may therefore express lower numbers of
D3 receptors or experience a down-regulation of D3 receptors that accompanies
sensitization (Richtand et al., 2001). This reduction in D3 receptors then, would lessen
the effective inhibitory brake on D1/D2 mediated hyperactivity leading to a greater
susceptibility for sensitization. As a result, Richtand’s hypothesis is that there is a
decrease in postsynaptic D3 receptor density, or in D3 receptor function that accompanies
sensitization in animals that express behavioural sensitization.
Several studies have examined D3 receptor expression following behavioural
sensitization, the results of which are conflicting. Richtand’s group demonstrated that
23
there was a decrease in D3 receptor agonist-induced locomotor inhibition following
chronic amphetamine treatment (Richtand et al., 2003). This same group also
demonstrated that administration of the D3-preferring antagonist, nafadotride, inhibits the
development of behavioural sensitization to amphetamine, presumably by selectively
blocking D3 receptors, so that a persistent down regulation of D3 receptors can not occur
with repeated administration of amphetamine (Richtand et al., 2000). Other studies have
reported findings that support Richtand’s model. One study reported a significant
decrease in both D3 receptor protein binding and D3 mRNA expression following
chronic amphetamine treatment (Chiang et al., 2003). This same group also replicated
the previous finding that D3 antagonists at D3-selective doses, when co-administered
with amphetamine, blocked the development of sensitization, although these drugs had
no effect when administered before amphetamine treatment, or during amphetamine
withdrawal (Chiang et al., 2003). Additionally, it was reported that D3 receptors were
down regulated with chronic cocaine administration (Wallace et al., 1996). And another
group reported a decrease in both D3 receptor binding as well as D3 mRNA expression in
mice sensitized to methamphetamine (Chen et al., 2007).
On the other hand, several other studies have reported findings that contradict this
down-regulation theory by observing either no change or an up regulation of D3
receptors following chronic drug administration. Several studies have reported an
increase in D3 mRNA expression in the striatum in hemiparkinsonian rats that
accompanies behavioural sensitization to chronic L-DOPA (Guillin et al., 2001; van
Kampen & Stoessl, 2003). However chronic L-DOPA was found to have no effect on
24
striatal D3 receptor binding in monkeys (Zeng et al., 2001). Increases in D3 receptor
binding and mRNA expression in the NAcc have been reported to accompany
behavioural sensitization to nicotine in rats (Le Foll et al., 2003) and D3 receptor
expression has also been found to be elevated in the NAcc of human cocaine addicts (D.
M. Segal et al., 1997; Staley & Mash, 1996). Finally preliminary studies performed in
our laboratory suggest that an overall up regulation of D3 receptor binding, but not
mRNA expression may accompany sensitization to ethanol (S. J. Harrison, 2004).
It is uncertain whether these discrepant reports are related to inter-laboratory
differences, methodologies, or parametric variations, or whether there are marked
mechanistic differences in sensitization induced by different drugs of abuse in different
species. However when the results of the aforementioned studies are summarized as they
appear in Table 1, with the exception of the human data, all of the rodent studies point
towards an important mechanistic difference between the different drugs of abuse. On
one hand, when the drug that was chronically administered was d-amphetamine,
methamphetamine or cocaine, all of which are drugs that block the dopamine re-uptake
transporter, there was an overall down regulation of D3 receptor function observed. On
the other hand, when other drugs were administered that either act as indirect dopamine
agonists (L-DOPA) or lead to indirect increases of mesolimbic dopamine (nicotine), there
was an observed up-regulation of the D3 receptor function. Other than the pilot
investigations performed on our laboratory, no other studies to date have examined the
effects of ethanol sensitization on D3 receptor expression or function.
25
Table 1:
Drug D3 Receptor D3 Mediated Species Administered D3 mRNA Protein Behaviours Reference Rat Amphetamine N/A N/C ↓ Richtand et al., 2003 Rat Amphetamine ↓ ↓ ↓ Chiang et al., 2003 Mouse Methamphetamine ↓ ↓ N/A Chen et al., 2007 Rat Cocaine N/A ↓ N/A Wallace et al., 1996 Human Cocaine ↑ N/A N/A Segal et al., 1997 Human Cocaine N/A ↑ N/A Staley et al., 1996 Rat L-DOPA N/A ↑ N/A Guillin et al., 2001 Rat L-DOPA ↑ ↑ ↑ van Kampen et al., 2003
Rat Nicotine ↑ ↑ ↑ LeFoll et al., 2003
Mouse Ethanol N/C ↑ ↑ Harrison, 2004
D3 Receptors Following Sensitization to various drugs of abuse: Summarized above are studies with seemingly conflicting results following sensitization to various drugs of abuse. However with the exception of the human studies, the rodent models appear to follow a mechanistic trend. Drugs acting on dopaminergic uptake transporters such as amphetamine, methamphetamine and cocaine, when used to induce sensitization, result in an overall down-regulation of D3 receptors. Drugs that do not act on dopamine reuptake transporters, such as L-DOPA, nicotine and ethanol, when used to induce sensitization, result in an overall up-regulation of D3 receptors.
26
CHAPTER 3
Differences in Mouse Strains
C57Bl/6 vs. DBA/2
Several studies have examined the psychomotor stimulant effects of ethanol in
mouse models using different strains of mice. Results of these studies have shown that
although variability exists between different inbred mouse strains, most strains including
DBA/2 (DBA) mice become activated in response to acute, low-dose ethanol
administration with the exception of C57Bl/6 (C57) mice (Downing et al., 2003). Studies
have consistently reported either very little locomotor activation or decreased activity in
C57 mice following acute ethanol (Crabbe et al., 1982; Dudek & Tritto, 1995; Tritto &
Dudek, 1994).
Furthermore, studies investigating chronic exposure to ethanol suggest that DBA
mice show a greater susceptibility for the development of ethanol sensitization than C57
mice (Phillips et al., 1994; Phillips et al., 1995). In addition to locomotor activation,
DBA mice also show conditioned place preference for ethanol where C57 show little or
no ethanol-induced place preference suggesting differences in the strains’ sensitivities to
the positive incentive properties of ethanol (Cunningham et al., 1992; Cunningham &
Noble, 1992). On the other hand, C57 mice innately consume larger amounts of alcohol
than DBA mice, which does not appear to be related to overall anxiety levels (Belknap et
al., 1993; Meliska et al., 1995; Misra & Pandey, 2003). One possible reason for the
observed differences in response to ethanol in C57 mice has been attributed to low levels
of activity of the ethanol metabolizing enzyme catalase in C57 mice (Correa et al., 2004)
27
since ethanol consumption was reduced and locomotor activation was increased in these
mice when brain catalase activity was enhanced.
C57 and DBA mice differ in various responses to a number of other drugs of
abuse. For example, in direct contrast to their ethanol responses, C57 mice have been
observed to be more susceptible to amphetamine sensitization as well as to amphetamine-
induced conditioned place preference when compared to DBA mice which are more
resistant to these same behavioural measures (Cabib et al., 2002; Orsini et al., 2005;
Orsini et al., 2004; Ventura et al., 2004). C57s have also been observed to be more
susceptible to morphine-induced sensitization than DBAs (Phillips et al., 1994) as well as
more susceptible to morphine-induced place preference (Orsini et al., 2005). Given that
the relative sensitivities of DBA and C57 mice to various drugs of abuse are not
consistent across drugs, it would therefore seem likely that the effects of these drugs may
be mediated by at least partially divergent neural mechanisms in the two strains of mice.
The neurochemical system most widely attributed with mediation of drug-induced
stimulation and reward is the mesocorticolimbic dopamine system (Kalivas & Stewart,
1991; Wise & Leeb, 1993). In a study by Ventura et al., the hypothesis that an inverse
relationship exists between mesocortical and mesolimbic DA functioning in C57 and
DBA mice was tested (Ventura et al., 2004). The authors measured DA release in the
medial prefrontal cortex (mPFC) and nucleus accumbens (NAcc) with intracerebral
microdialysis in freely moving mice following systemic d-amphetamine. Amphetamine
induced a sustained increase in DA release in the medial prefrontal cortex (mPFC)
accompanied by poor increase of DA in the nucleus accumbens (NAcc) in low-
28
responding DBA mice. On the other hand, in high-responding C57 mice, a low
prefrontal cortical DA outflow accompanied by a high accumbal extracellular DA release
was observed. The results of this study suggest that differences between the two strains
of mice with respect to susceptibility to the stimulating and reinforcing properties of
amphetamine and other drugs of abuse may lie within an unbalanced DA transmission in
the mesocorticolimbic system.
Other differences occurring within the mesolimbic DA system have been
observed between C57 and DBA mice. Misra and Pandey (2003) examined the innate
expression and phosphorylation of cAMP-response element binding protein (CREB) in
various brain structures of C57 and DBA mice. Since these two strains of mice innately
consume differential amounts of alcohol and CREB function has been implicated in
alcohol drinking and drug-taking behaviours (Nestler, 2001; Pandey et al., 1999; Wand et
al., 2001), the authors hypothesized that these two strains of mice might show differences
in basal levels of CREB functioning that reflect differences in alcohol consumption.
Basal CREB expression and phosphorylation levels were found to be significantly
lower in the shell portion of the NAcc in C57 as compared to DBA mice. All other brain
regions measured, including the core region of the NAcc, various cortical regions,
amygdala, hippocampus and striatum showed similar levels of CREB expression and
phosphorylation in the two strains of mice. The authors also measured the expression of
a cAMP-inducible gene, neuropeptide Y (NPY) since it too has been implicated in
alcohol-drinking behaviour (Heilig & Thorsell, 2002; Pandey et al., 2003). NPY levels
29
were significantly lower in the shell, but not the core of the NAcc in C57 mice compared
to DBAs.
Finally, a study was performed in order to investigate the comparative D3 and D1
receptor binding and D3 receptor mRNA expression in C57 vs. DBA mice (R. K.
McNamara et al., 2006a). Using brain homogenates, a 32% reduction in D3 receptor
binding in the ventral striatum (NAcc, islands of Calleja and olfactory tubercle) in C57
mice relative to DBAs was evidenced by a reduced Bmax in C57 mice and lower KD in
DBA mice. D1 receptor binding did not differ between the two strains. The investigators
also measured D3 mRNA expression using real-time reverse transcriptase polymerase
chain reaction (RT-PCR). They observed 26% lower D3 mRNA expression in the
substantia nigra (SN) and ventral tegmental area (VTA) in C57 mice relative to DBAs
with no strain differences in the ventral striatum or prefrontal cortex. Since the D3
receptor sub-type has an inhibitory effect on locomotor activity in rodents, the authors
concluded that the reduced D3 binding and mRNA expression in C57 mice may
contribute to the heightened sensitivity to the locomotor-stimulating effects of
amphetamine in the C57 mouse strain compared to DBAs.
Overall, these studies suggest that one of the key differences between C57 and
DBA mice lies in their opposing behavioural response profiles to various drugs of abuse.
Additionally, these two strains of mice appear to demonstrate an unbalanced if not
inverse relationship between mesocortical and mesolimbic dopamine transmission.
These mice also demonstrate differential levels of CREB expression and phosphorylation
as well as NPY levels in the shell of the NAcc. Finally, C57 mice have been observed to
30
have lower D3 receptor binding in the ventral striatum as well as reduced D3 mRNA
expression in the SN and VTA. All of the above lines of evidence suggest that DBA and
C57 mice have differences in their mesocorticolimbic dopamine systems, with specific
differences that have been shown to lie within the shell region of the NAcc, a key brain
region that is rich in dopamine D3 receptors and widely implicated in reward
mechanisms and drug addiction.
Dopamine Receptor Mutant Mice
Dopamine D1 Receptor Knockouts (D1 KOs)
D1 KO mice show normal appearance, but exhibit extensive growth retardation
and have low survival rates after weaning (Drago et al., 1994; Xu et al., 1994b). This
failure to thrive can be rescued by providing these mice with easy access to soft, palatable
food on the cage floor (Drago et al., 1994). Radioligand binding of the D1 receptor is
negligible in D1 KOs (Friedman et al., 1997). The hyperactivity-inducing effects of D1-
selective agonists and hypoactivity-inducing effects of D1-selective antagonists (but not
by D2-selective agents) are abolished in D1 KOs (Clifford et al., 1999; Drago et al.,
1996; Moratalla et al., 1996; Wong et al., 2003b; Xu et al., 1994a; Xu et al., 1994b). D2
receptor and dopamine transporter binding appear normal in these mice (Xu et al.,
1994b); however there are fewer total dopamine neurons, indicated by fewer tyrosine-
hydroxylase positive cells, in D1 KOs than in wild type controls (Parish et al., 2002).
Midbrain dopamine levels are elevated and dopamine metabolites are altered suggesting a
31
compensatory increase in dopamine synthesis and/or release following inactivation of the
D1 receptor (El-Ghundi et al., 1998; Parish et al., 2002). Lastly, striatal dynorphin and
substance P levels, both of which are synthesized by D1 receptor-expressing neurons, are
reduced in D1 KO mice (Drago et al., 1994; Wong et al., 2003a; Xu et al., 1994b).
While D1 receptor antagonists profoundly reduce locomotor activity, most studies
of D1 KO mice report that these mice exhibit increased locomotor behaviour as well as
reduced habituation in an open field apparatus (Clifford et al., 1998; Crawford et al.,
1997; Karasinska et al., 2000; F. N. McNamara et al., 2003; Wong et al., 2003a; Xu et
al., 2000; Xu et al., 1994a). The reasons for the observed hyperactivity and other
seemingly paradoxical phenotypes demonstrated by these mice are not fully understood.
They do not appear to be the result of strain differences in the parental line, which only
appears to influence the magnitude rather than the nature of these phenotypes (F. N.
McNamara et al., 2003). It is possible then that the hyperactivity observed in D1 KO
mice is, perhaps in response to increased midbrain dopamine levels, the result of
compensatory mechanisms developed from the loss of the D1 receptor (F. N. McNamara
et al., 2003).
In studies of reward-related behaviours, D1 KO mice demonstrate either complete
elimination of, or markedly attenuated hyperactive responses to cocaine (Drago et al.,
1996; Xu et al., 1996), behavioural sensitization to cocaine and amphetamine (Crawford
et al., 1997; Xu et al., 2000) as well as behavioural sensitization to morphine (Becker et
al., 2001). Other reward-related behaviours have been difficult to study in D1 KOs
(perhaps due to their small size and enhanced locomotor activity levels), however one
32
study reported comparable cocaine-induced conditioned place preference in D1 KOs and
wild types (Miner et al., 1995). And in another study D1 KOs were observed to show
reduced EtOH preference and voluntary EtOH consumption as compared to wild type
mice (El-Ghundi et al., 1998).
Dopamine D2 Receptor Knockouts (D2 KOs)
D2 KO mice often exhibit pituitary dysfunction, develop pituitary tumours, show
increased blood pressure and are susceptible to sodium-induced hypertension (Asa et al.,
1999; M. A. Kelly et al., 1997; Li et al., 2001; Ozono et al., 2003; Saiardi & Borrelli,
1998; Saiardi et al., 1997; Ueda et al., 2003). Otherwise, and despite some reports of
slight growth retardation (Diaz-Torga et al., 2002) D2 KO mice appear healthy. With
respect to binding profiles, D2 KOs show a transient postnatal increase in D3 receptor
binding as well as a decrease in D1 receptor binding (Jung et al., 2000; Jung &
Schmauss, 1999; M. A. Kelly et al., 1998).
In studies of locomotor activity the most consistent finding is that D2 KO mice
exhibit significantly lower levels of locomotor activity than wild type controls (Aoyama
et al., 2000; Baik et al., 1995; Jung et al., 1999; M. A. Kelly et al., 1998; Palmer et al.,
2003; Vallone et al., 2002). Some studies have also reported profound deficiencies in
gait, posture and motor coordination in D2 KOs (Aoyama et al., 2000; Baik et al., 1995;
Jung et al., 1999); however, other researchers have hypothesized that this may be largely
related to the background strain of the D2 KO mice. There are marked differences in
33
motor performance on the rotarod, open field and other motor tasks between C57 and 129
strains of mice (Holmes et al., 2002; Tarantino et al., 2000), and it is thought that the
observed motor incoordination phenotype in some D2 KOs may be a false positive
caused by 129 parental genes (Gerlai, 1996) instead of by the loss of the D2 receptor
gene itself. Evidence to support this has been provided by the observation that back-
crossing D2 KOs onto a C57 background strain can “rescue” the motor coordination
deficits seen in these mice (M. A. Kelly et al., 1998).
In studies of reward-related behaviours D2 KO mice have demonstrated abnormal
responses to a number of drugs of abuse as compared to wild type controls. D2 KOs do
not self-administer morphine and fail to develop conditioned place preference to
morphine, while morphine-induced hyperactivity and morphine withdrawal appear
normal in comparison to wild types (Elmer et al., 2002; Maldonado et al., 1997; Smith et
al., 2002). The locomotor stimulant effects of cocaine are reduced in D2 KOs in
comparison to wild types (Chausmer et al., 2002; Chausmer & Katz, 2001); whereas, self
administration of moderate to high doses of cocaine are increased in D2 KOs, an effect
mimicked by D2-selective antagonists but not D3- or D4-preferring antagonists (Caine et
al., 2002). However, unlike wild type mice treated with D2 antagonists, D2 KOs are able
to discriminate cocaine from saline (Chausmer et al., 2002; Chausmer & Katz, 2001).
In studies of ethanol, D2 KOs have been observed to show decreased preference
for, and reduced voluntary consumption of EtOH as compared to wild type mice (Phillips
et al., 1998). D2 KO mice also demonstrate reduced conditioned place preference to
EtOH (Cunningham et al., 2000), as well as decreased operant responding and EtOH self-
34
administration (Risinger et al., 2000). On the other hand, D2 KOs have been observed to
show increased responses to the locomotor stimulating and sensitizing effects of EtOH
(Palmer et al., 2003).
Dopamine D3 Receptor Knockouts (D3 KOs)
D3 KO mice show normal appearance, growth, fertility and no gross neurological
dysfunctions (Holmes et al., 2004; Xu et al., 1997), although some have been found to
develop renin-dependent hypertension (Asico et al., 1998). Brain binding densities of
other dopamine receptors, such as D1 and D2 receptors, as well as neuropeptide gene
expression appear normal in D3 KOs (Accili et al., 1996; Le Foll et al., 2005a; Wong et
al., 2003b; Xu et al., 1997). While most reports of dopamine content, uptake and
neuronal firing appear normal in D3 KOs, some reports have noted higher basal
extracellular dopamine in D3 KOs (Joseph et al., 2002; Koeltzow et al., 1998; Xu et al.,
1997; Zapata et al., 2001).
In locomotor studies of D3 KOs, some researchers report that D3 KOs exhibit
locomotor hyperactivity, while others do not (Boulay et al., 1999; Boyce-Rustay &
Risinger, 2003; Depoortere, 1999; Pritchard et al., 2003; Vallone et al., 2002; Xu et al.,
1999; Xu et al., 1997). A potential reason for this discrepancy is that the increase in
locomotor activity in D3 KOs may be transient and restricted to the early phase of
locomotor testing, which has been interpreted by some as an indication of reduced
neophobia/anxiety-like behaviour in D3 KOs (Accili et al., 1996; Vallone et al., 2002;
35
Xu et al., 1997). Support for this theory has been demonstrated in some studies where
D3 KOs show a reduced anxiety-like phenotype on the elevated plus maze and open field
(Karasinska et al., 2000; Steiner et al., 1997); however these data would also contradict
another finding that certain D3-preferring agonists have demonstrated anxiolytic
properties in laboratory animals (Rogers et al., 2000; Rogoz et al., 2003). An alternative
theory then, is that D3 KOs show a more general failure to inhibit novelty-induced
behavioural responses (Holmes et al., 2004; Pritchard et al., 2003; Xu et al., 1997).
As previously described, a role for the D3 receptor in the modulation of D1/D2
cooperativity has been proposed whereby the D3 receptor is thought to inhibit the
synergistic effects of D1 and D2 receptor co-activation (Glickstein & Schmauss, 2001;
Richtand et al., 2000). Evidence to support this theory has been shown in studies where
D3 KO mice show normal locomotor responses to either D1- or D2-selective agents
when administered alone, but show exaggerated hyperactivity in response to agents that
simultaneously activate D1 and D2 receptors such as cocaine and amphetamine (Accili et
al., 1996; Betancur et al., 2001; R. K. McNamara et al., 2006b; Wong et al., 2003a; Xu et
al., 1997). It should be noted however, that co-administration of separate D1-selective
and D2 selective agonists or antagonists have no effect on the locomotor responses in D3
KOs as compared to wild types (Boulay et al., 1999; Yarkov et al., 2003). Surprisingly
studies of locomotor responses by D3 KOs to amphetamine are lacking. Only one study
published last year, examined the acute locomotor effects of different doses of
amphetamine in D3 KOs (R. K. McNamara et al., 2006b). Results from this study
showed that D3 KOs show significantly higher locomotor activation than wild types at
36
low doses (2.5 mg/kg) of amphetamine, but differences are abolished at higher doses (5-
10 mg/kg). Although D3 KOs have been shown to develop locomotor sensitization to
cocaine, (Betancur et al., 2001) no studies to date have examined the locomotor effects of
chronic amphetamine in D3 KO mice.
With respect to reward-related behaviours D3 KOs have been reported to show
increased conditioned place preference to amphetamine and morphine when compared to
wild types (Frances et al., 2004; Narita et al., 2003; Xu et al., 1997; Zhang & Xu, 2001).
One study also reported that D3 KOs show reduced voluntary ethanol consumption,
coupled with an increased sensitivity to the hypnotic effects of ethanol (Narita et al.,
2002); however other studies have failed to replicate these findings in addition to
observing unaltered conditioned place preference to ethanol as well as operant self-
administration of EtOH in D3 KOs (Benoit et al., 2003; Boyce-Rustay & Risinger, 2003;
McQuade et al., 2003). To our knowledge, no studies to date have examined locomotor
sensitization to ethanol in D3 KO mice.
37
General Objectives
What then, is the role of the dopamine D3 receptor in behavioural sensitization to
ethanol? Although there are many ways to look for answers to this question, we decided
to approach the problem with a three-pronged strategy. Our first set of experiments
utilized transgenic mice lacking the D3 receptor. By employing D3 knockout mice, we
examined how the absence of the D3 receptor in these mice affected their behavioural
responses to chronic ethanol. We also determined how mice lacking the D3 receptor
compared not only to wild type mice, but also to mice lacking the more widely studied
D1 and D2 receptors. Finally, we determined how D3 KO mice respond not only to
repeated systemic ethanol injections, but also to repeated systemic amphetamine
injections in order to observe whether these mice develop sensitization to different drugs
of abuse in a similar or different manner.
The second series of experiments used different pharmacological interventions
with a D3 receptor antagonist and agonist. We examined how temporary D3 receptor
blockade or stimulation affected sensitization to ethanol. Furthermore, we selectively
blocked and stimulated D3 receptors during different phases of sensitization in order to
identify whether D3 receptors play a stronger role in mediating the induction or
expression of EtOH sensitization. We also investigated potential D1/D3 receptor
interactions by examining whether D3 receptor activation interfered with D1 agonist-
induced hyperactivity following sensitization to ethanol.
Finally, we examined the brains of mice sensitized to EtOH in order to see
whether changes in D3 receptor binding, if any, could be observed following chronic
38
EtOH or saline. Various literature reports as well as results from a pilot study performed
in our laboratory suggest that a functional up-regulation of the D3 receptor protein is
associated with sensitization to EtOH. By using [125I]-7-OH-PIPAT receptor binding
autoradiography, we visualized the density of the D3 receptor protein in various brain
regions in order to ascertain whether D3 receptor binding is altered in mice sensitized to
ethanol as compared to saline-treated controls.
Therefore, by using transgenic mice, pharmacological interventions and brain
imaging tools, we attempted to elucidate, to some degree, a role for the D3 receptor in
behavioural sensitization to ethanol. Our overall hypothesis was that D3 receptors would
up-regulate in response to repeated ethanol in a manner similar to other drugs of abuse
that do not act directly on the NAcc.
39
CHAPTER 4
Study 1: Examination of the development and expression of sensitization in mice
lacking the D3 dopamine receptor
Purpose
Given its localization in brain areas associated with reward-related behaviours and
the potential role that the D3 receptor plays in sensitization to several drugs of abuse, we
asked the question: Is the D3 receptor necessary for the development and expression of
sensitization to ethanol? One method of examining the role of the D3 receptor in
behavioural sensitization is to observe how mutant mice that have been bred without the
D3 receptor (D3 knockouts- D3 KOs) develop and express locomotor sensitization to
EtOH as compared to animals that lack other dopamine receptors (D1 and D2 KO's) and
normal wild type mice.
Therefore the goal of the first study was to characterize the behaviour of D3 KO
mice over the course of chronic EtOH treatment while directly comparing their behaviour
to that of their better-studied D1 KO, D2 KO and wild type counterparts. Our hypothesis
then was that if D3 receptors have an inhibitory effect on locomotion, and if they are up
regulated during sensitization, then mice lacking the D3 receptor should show enhanced
behavioural sensitization, evidenced by higher levels of locomotor activity as compared
to EtOH-treated wild type mice, and D1 and D2 KOs.
40
Experiment 1A: Comparison of EtOH sensitization in C57 and DBA mice
Materials & Method
Subjects
Several studies have reported that C57 mice are more resistant to the stimulant
and sensitizing effects of EtOH than DBA mice (Crabbe et al., 1982; Phillips et al., 1994;
Tritto & Dudek, 1994). However, even though DBA mice have been reported to show
more robust sensitization than C57 mice, C57s have been observed to demonstrate
increased locomotor activity in response to chronic ethanol, albeit to a lesser extent than
DBAs (Crabbe et al., 1982; Lessov & Phillips, 1998b). Because the background strain of
our knockout mice was C57, it was important to observe whether these mice were
capable of developing and expressing sensitization to EtOH in our hands. Therefore in
order to observe how C57 mice responded to chronic EtOH treatment, we compared the
development and expression of EtOH sensitization behaviour in wild type male C57 and
DBA mice prior to examination of the knockout mouse colonies.
Since it has been demonstrated that intra-strain variability of responding to EtOH
occurs in mice (Masur & dos Santos, 1988) where within a given strain some mice
demonstrate sensitization to EtOH while other individuals remain unresponsive to
chronic treatment, three times as many mice were assigned to receive EtOH as those
receiving saline. Therefore 32 male mice from each of the two strains (C57 and DBA)
aged 5 weeks (Charles River, Canada) were randomly assigned to receive either chronic
EtOH (n=24) or saline (n=8).
41
Drug
Anhydrous ethyl alcohol (Commercial Alcohols, Brampton, ON) was diluted with
physiological saline (0.9% NaCl) to a concentration of 15% weight per volume. Mice
that were assigned to the ethanol group received 2.2 g/kg EtOH i.p. (injection volume 15
ml/kg) during the sensitization period. Control animals received equal volumes of saline
vehicle. On the final test day, all subjects received a test dose of 1.8 g/kg EtOH in order
to examine whether the mice chronically treated with EtOH would express sensitization.
The doses of ethanol used to in this study were chosen based upon literature reports that
have demonstrated this dose range as one that will elicit locomotor stimulant rather than
sedative effects in mice (Masur & Boerngen, 1980; Masur et al., 1986; Phillips et al.,
1991; Phillips et al., 1994; Phillips et al., 1995; Quadros et al., 2002a; Quadros et al.,
2002b).
Apparatus
The test environment consisted of 4 Plexiglas activity monitors (40 x 40 x 35 cm)
(MED Associates Inc., St. Albans, VT.), which detect horizontal locomotion via
interruptions of infrared photoelectric beams. Horizontal movements were recorded as
ambulatory counts. An ambulatory count was defined as an infrared beam-break in the
horizontal axis of the chamber. The activity chambers were interfaced to an IBM PC
computer that provided automated recordings of the locomotor activity, reported as
ambulatory counts, by means of an Activity Monitor Version 5.51 (MED Associates Inc.)
software package.
42
The activity monitors were situated in a quiet room, separate from the colony with
normal ambient lights. Animals were tested individually during the light phase (between
10 am and 2 pm) in the activity chambers for 15 minutes during habituation sessions and
immediately following injections on weekly test sessions. No bedding was present in the
activity chambers, and neither food nor water was available to the animal during the test
sessions.
Ethanol Sensitization (Procedural Overview)
Upon arrival to the animal facilities, mice were allowed to acclimatize to the
colony room for 7 days to adjust to the 14h-10h light-dark cycle (lights on at 8am and off
at 6 pm). Room temperature inside both the colony and activity room was 25ºC.
Standard rat pellets and regular tap water were provided ad libitum throughout the entire
experiment, except when animals were placed in the testing apparatus. Mice were
housed (4 mice per cage) in plastic, shoebox-style cages containing mouse huts, nestlets
and PVC tubing for environmental enrichment. Cages were lined with corncob bedding.
In order to habituate the mice to the activity chambers, mice were exposed to the
test apparatus for three 15-minute sessions prior to the beginning of the experiment.
Each habituation session was spaced 3-4 days apart. On the first two exposures to the
chambers, mice received no injection. On the third habituation session, all mice were
given an intraperitoneal (i.p.) saline injection (15 ml/kg) to allow mice to habituate to
injection handling. From previous experience, this injection does not alter their
43
behaviour as compared to their behaviour on other habituation days. After each 15-
minute habituation session, animals were removed from the chamber, returned to their
home cage and returned to the colony room.
After one week of rest, animals were transported to the activity room for their
acute EtOH injection. Animals were weighed and their injection volume was
individually titrated according to each mouse's weight. Mice received a single injection
of either 15% EtOH (2.2 g/kg i.p., injection volume 15 ml/kg) or equal volumes of saline.
Immediately after injection, mice were placed in the activity chamber, which recorded
their spontaneous horizontal locomotor behaviour for 15 minutes. After the session
ended, mice were returned to their home cages and to the colony room. Animals were
injected twice a week, for a total of 9 sensitizing injections. Injections were spaced out
over the course of days in order to provide an intermittent dosing schedule, which has
been proposed to induce sensitization to drugs of abuse rather than tolerance (Post, 1980).
Furthermore in a pilot study, it was observed that fewer injections (5-9), spaced further
apart (2-3 days) elicited the same EtOH sensitization effect than more injections (14-21)
administered on a daily injection schedule (data not shown). Since the same effect could
be observed in fewer injections spaced further apart, we elected to use a more intermittent
injection schedule since it would be less stressful to the animals as well as agree with the
defining features of behavioural sensitization.
Beginning with the first EtOH injection, mice were tested once a week (on
injections 1, 3, 5 etc.) in activity chambers for 15 minutes. On injection days that mice
were not tested in the activity chambers (injections 2, 4, 6, etc.) mice were transported to
44
the activity room in their home cages, injected with either EtOH or saline, immediately
placed back in their home cage for 15 minutes and returned to the colony room. After the
9th injection mice received 14 days washout when they received no drug. After 14 days
all mice received one test injection of EtOH (1.8 g/kg, i.p.) in order to observe whether
the effects of EtOH sensitization persisted following drug washout. This withdrawal
period was included since sensitization effects are often enhanced following a period of
drug abstinence (Paulson et al., 1991).
Injection sites were rotated on the abdomen so that an animal was not injected
twice consecutively in the same area. Twenty-four hours after the final test injection, all
mice were euthanized via cervical dislocation and brains were removed and frozen for
future analysis. All experiments were approved by the local Animal Care Committee and
were in keeping with the guidelines and practices outlined by the Canadian Council on
Animal Care.
For the sensitization period, locomotor behaviour was analyzed statistically using
a mixed within- and between-subjects analysis of variance (ANOVA) design. Injection
Day was treated as the within-subjects repeated measure, and Drug Treatment Group
(Sensitized vs. Control) as the between-subjects factor. On the final test day, an
independent t-test was performed to compare group means on that day. Statistical
significance was defined as p<0.05.
45
Results
After injection 9, the EtOH-treated groups were sub-divided to include the 8 of 24
highest responders (sensitized), and compared to the 8 saline-treated controls. Overall,
DBA mice expressed more robust sensitization than C57 mice, and a higher percentage of
the EtOH-treated population of DBA mice expressed sensitization. This was indicated
by an overall higher group mean level of locomotor activity demonstrated by EtOH-
treated DBA mice compared to EtOH-treated C57 mice, before mice were sub-divided
into high responders.
After Injection 9, all EtOH-treated DBA mice showed a group average of 2587
ambulatory counts compared to EtOH-treated C57 mice (group mean=1917 ambulatory
counts), demonstrating that overall, EtOH-treated DBA mice showed a significantly
higher level of locomotor activity (25%, p=0.04) than EtOH-treated C57 mice after
injection 9. On the test day, following 14 days of drug withdrawal, all EtOH-treated
DBA mice showed a group average of 2931 ambulatory counts compared to EtOH-
treated C57 mice (group mean=1552 ambulatory counts), demonstrating that EtOH-
treated DBA mice showed significantly higher levels of locomotor activity (48%, p<0.01)
than EtOH-treated C57 mice on the test day. All raw data scores for locomotor activity
levels (reported as ambulatory counts) for individual mice on each injection day are
included in Appendix 1A for DBA mice and 1B for C57 mice.
46
DBA/2 Mice
In DBA mice, by injection 3, EtOH-treated mice showed significant increases
(p<0.05) in locomotor activity as compared to saline-treated controls (Figure 2A).
Progressive increases in locomotor activity were observed until injection 7 where EtOH-
treated mice showed a 300% increase in locomotor levels as compared to saline-treated
controls (p<0.01). This level of locomotor activity appeared to reach saturation and
remained at that same level for injection 9. When sensitized mice were challenged with
EtOH following 2 weeks of drug withdrawal, this sensitization effect was still present.
Over the course of the sensitization period there was a statistically significant
main effect of injection day [F(7,98)=10.45, p<0.001], a statistically significant main
effect of drug treatment [F(1,14)=703.85, p<0.001] and a statistically significant injection
day X group interaction [F(7,98)=17.46, p<0.001]. These results indicate that a group
difference resulted from drug treatment since EtOH-treated animals demonstrated higher
locomotor activity than saline-treated controls. These group differences were also
dependent on the number of injection days, since the amount of drug effect, as
demonstrated by increased locomotion, varied in the drug-treated group over the course
of injection days.
After the 14-day drug withdrawal, EtOH-treated DBA mice continued to
demonstrate a heightened response to a low-dose drug challenge compared to control
animals, since EtOH-treated mice showed the same level of locomotor activity as they
had on injection 9. Results of a t-test analysis revealed that the level of locomotor
47
activity demonstrated by sensitized mice was significantly higher than control mice after
the EtOH challenge [t(14)=4.00, p<0.001].
C57Bl/6 Mice
By injection 3, EtOH-treated C57 mice showed a significant increase in
locomotor activity (p<0.05) when compared to saline-treated controls (Figure 2B). A
progressive increase was observed in EtOH-treated mice until injection 5 (p<0.01). After
injection 5, a significant 150% increase in locomotor activity as compared to controls was
observed and this sensitization effect remained throughout the rest of the sensitization
period. This effect persisted for the 14 days following drug withdrawal as the sensitized
mice demonstrated the same high levels of locomotor activity during the EtOH challenge.
ANOVA results showed that over the course of the injections there was a
significant main effect of injection day [F(7,98)=9.28, p<0.001], a significant main effect
of drug treatment [F(1, 14)]=1145.08, p<0.001], and a significant injection day X drug
interaction [F(7, 98)=15.88, p<0.001]. After the 2-week washout period, sensitized C57
mice continued to show the same level of drug response that was demonstrated on
injection 9, when challenged with a low-dose injection of EtOH. This increased response
to EtOH was significantly higher in sensitized animals than saline-treated controls
[t(14)=9.51, p<0.001].
48
Figure 2:
2A: Behavioural Data from DBA Mice Treated with Chronic EtOH 2B: Behavioural Data from C57 Mice Treated with Chronic EtOH Comparison of EtOH Sensitization in C57 vs. DBA mice: Figures 2A & B shows the average locomotor activity levels (± SEM) for the highest responders (Sens) and control animals in DBA (Fig 2A) and C57 (Fig 2B) strains of mice (n=8). DBA mice showed a more robust sensitization effect (300% increase) than C57 mice (150% increase). After 9 injections (2.2 g/kg EtOH or saline), all mice were withdrawn from EtOH for 14 days and tested with EtOH (1.8 g/kg). In both strains of mice, EtOH sensitization effects persisted even after 14 days of drug washout. * Indicates statistical significance of p<0.05. ** Indicates statistical significance of p<0.01.
0
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49
Overall the results suggested that even though DBA mice showed a more robust
sensitization effect than C57 mice, evidenced by a higher percentage of the drug-treated
DBA population showing higher responses to chronic EtOH than C57s, C57 mice did
indeed express sensitization to chronic EtOH, albeit to a lesser extent than DBA mice.
Discussion
In agreement with the literature reports described in Chapter 3, DBA mice were
more responsive to chronic EtOH treatment than C57 mice. This was demonstrated in
two ways. First of all, by the end of the sensitization period, the sensitized DBA mice
showed a 300% increase in locomotor activity as compared to saline-treated controls,
whereas sensitized C57 mice showed a 150% increase in locomotor activity as compared
to controls. Secondly, a substantially higher proportion of EtOH-treated DBA mice
expressed sensitization as compared to C57 mice, which was demonstrated by an overall
group average that was 25% higher in EtOH-treated DBA mice compared to EtOH-
treated C57 mice on injection 9 and 48% higher on the test day.
Even though sensitization was more pronounced in DBA mice, a sensitization
effect was still observed in the C57 mice. A clear and persistent change in the level of
responding was observed in a sub-population of EtOH-treated mice and this significant
response remained constant even after 2 weeks of drug washout. Because the
background strain of our knockout colonies was C57Bl/6, it was necessary to become
50
familiar with the expected behavioural profile of the C57 mouse strain. With the above-
mentioned limitations in mind, we understood that the expected effect size in these mice
would not be as large as in DBA mice. Additionally, we were aware that at least three
times as many C57 mice would need to be treated with chronic EtOH than saline in order
to obtain a sub-population of EtOH-treated mice that would express sensitization in a
ratio that would match saline-treated controls. Therefore, when designing the
experiments using knockout mice of a C57 background strain, it was important to keep
this behavioural profile in mind as well as to plan for the sub-division of subjects.
51
Experiment 1B: Comparison of EtOH sensitization in dopamine receptor knockouts
Materials & Method
Subjects
Using mice bred from our existing transgenic colonies (originally obtained
commercially from Jackson Laboratories, Bar Harbor, Maine, see appendices 2A-C for
strain and allele details), 36 mice from each colony: D1 KO, D2 KO, D3 KO and 36 wild
type mice (both males and females aged 6-10 weeks with a C57Bl/6 background) were
randomly assigned to one of two experimental groups: EtOH (n=27) or Saline (n=9).
Three times as many mice were assigned to receive EtOH as saline, so that by the end of
the sensitization period, the 9 highest responders from every colony would represent the
sensitized group.
With the exception of the D1 KO colony, all pups were weaned at 21 days of age.
Mice were group-housed with same-sex siblings (3-4 mice per cage), depending on the
number of pups per litter and sex distribution. In keeping with literature reports, D1 KO
pups were observed to be substantially smaller than heterozygote and wild type
littermates by approximately half their body weight. If weaned at 21 days, these mice
died within days. Therefore, the D1 KO mice were left for an additional 2 weeks with
their mothers and their diets were supplemented with wet mash and sunflower seeds to
help them thrive. When weaned at 5 weeks of age, D1 KO pups “adjusted well” to their
new cages. After every three generations, all knockout colonies were backcrossed onto
wild type C57Bl/6 mice to prevent genetic drift.
52
Genotyping
When weaned pups were set up in new cages, all mice received ear tag
identifications and a 0.5 cm tissue sample was removed from the end of each mouse’s
tail. Mice were treated with silver nitrate to cauterize the tail ends. Tail samples were
incubated overnight in a tissue and cell lysis solution (Master Pure DNA Purification Kit,
EPICENTRE Biotechnologies, Madison, WI) with proteinase K, followed by RNAase A.
DNA was precipitated out with an MPC protein precipitation reagent and centrifuged into
a pellet. Extracted DNA from each sample was used in polymerase chain reactions
(PCR) to observe the genotype of each individual mouse. Genotyping protocols and PCR
Master Mix reagents were obtained from Jackson Laboratories (Bar Harbor, Maine).
PCR conditions for each genotype are included in Appendices 2A-C. Detection of
homozygous knockout mice, wild type mice and heterozygotes was established by use of
a 1% agarose gel electrophoresis.
Procedural Overview
Mice were treated exactly as described in Experiment 1A with three times as
many mice receiving EtOH (n=27) as saline (n=9). After three habituation sessions, mice
received 5 EtOH (2.2 g/kg i.p.) or saline injections spaced 3-4 days apart. It was
determined from Experiment 1A that C57 mice reach saturation of the EtOH sensitization
response after injection 5. Therefore, in order to minimize the amount of stress on the
animals, the injection protocol was pre-determined to reach injection 5. Weekly
53
monitoring of locomotor behaviour took place in 15-minute test sessions in activity
monitors as previously described in Experiment 1A. Mice were euthanized via CO2/O2
inhalation 24 hours after the 5th and final injection. Behavioural data from each colony
were analyzed by repeated measures mixed within and between subjects ANOVA design.
Results
Saline-treated Control Animals
In comparing the spontaneous locomotor activity levels of the different genotypic
colonies in the absence of drug, marked differences were observed in the overall levels of
locomotor behaviour demonstrated by the saline-treated animals in each colony (Figure
3). In keeping with previous experiments, saline-treated wild type C57 mice
demonstrated habituation to the activity monitors after the first session, which was
indicated by a decrease in exploratory behaviour in subsequent sessions. After the initial
habituation session wild type mice made on average approximately 1000 ambulatory
counts within the 15-min test sessions. In contrast to this behavioural profile, both saline-
treated D1 and D3 KOs demonstrated an overall higher level of locomotor activity, which
increased rather than habituated over the course of the experiment changing from
approximately 1500-2000 ambulatory counts over the course of all of the test sessions.
And finally D2 KOs showed an overall response pattern that was similar to wild types,
demonstrating a clear habituation to the activity monitors, as evidenced by a decrease in
locomotor activity after the first session. However, D2 KOs demonstrated an overall
54
Figure 3:
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** ** **
****
**** **
**
**
Comparison of Spontaneous Locomotor Activity in Saline-treated Dopamine Knockout Colonies Compared to Wild Type Mice: Average locomotor activity levels (± SEM) for each of the saline-treated dopamine receptor knockout colonies compared to wild type littermates (n=9). Each group in this figure represents only saline-treated and not EtOH-treated animals in order to compare the spontaneous locomotor activity in each colony over time in the absence of drug. Groups that were significantly different from wild types are indicated with ** p<0.01.
55
lower level of locomotor activity that averaged approximately 500 ambulatory counts
during the remaining15-minute test sessions.
ANOVA results indicated a significant main effect of genotype between D1 KOs
and wild type mice [F(1,15)=6.82, p=0.015], between D2 KOs and wild types
[F(1,16)=19.08, p<0.001], and between D3 KOs and wild types [F(1,20)=20.36, p<0.001]
throughout the experiment. These results indicate that mice from every separate
genotypic colony under investigation displayed baseline locomotor behaviour that was
significantly different from wild type mice in the absence of drug. D2 KOs showed
levels of locomotor activity that were always significantly lower than wild type mice,
whereas D1 and D3 KOs showed levels of locomotor activity that were significantly
higher than wild type mice on all test sessions, with the exception of the first habituation
session.
When examining all colonies across all injection days, including the first
habituation session, a significant main effect was observed for injection day
[F(5,195)=2.36, p=0.042] and a significant genotype X injection day interaction was
observed [F(15, 195)= 1.90, p=0.025]. However when examining the baseline locomotor
behaviour without including the first habituation session, there was neither a significant
main effect of injection day (p>0.05) nor a significant genotype X injection day
interaction (p>0.05). These results suggest that the greatest changes in locomotor
behaviour that occurred between injection days for the control mice happened after the
first habituation session, where wild type mice and D2 KOs demonstrated a clear
habituation to the activity chambers, as demonstrated by an overall decrease in locomotor
56
activity between habituation day 1 and 2. In contrast, D1 and D3 KOs did not show
habituation to the activity chambers after the first session, which would account for the
observed genotype X day interaction. The absence of a significant day effect or
interaction on days following the first habituation session suggest that after the initial
exposure to the test chamber, locomotor activity in all control animals remained stable for
the rest of the experiment.
Experimental Animals (Sensitization in Knockout Mice)
Results from the comparative EtOH sensitization study in the D1 KO, D2 KO, D3
KO and wild type colonies of mice are shown in Figure 4. In keeping with the observed
results from Experiment 1A, sensitized wild type mice demonstrated a 150% increase in
locomotor activity as compared to saline-treated controls by the end of injection 5 (Figure
4A). Results of a repeated measure mixed between and within subjects design analysis
revealed a significant main effect of injection day [F(1,18)=32.72, p<0.001], a significant
main effect of drug-treatment group [F(1,18)=14.66, p=0.001] and a significant day X
group interaction [F(1,18)=53.20, p<0.001]. These results directly replicated those
obtained from the previous experiment suggesting that the wild type C57 mice bred in
our home colony did not differ in behaviour from the C57 mice obtained commercially
from Charles River. No significant sex differences were observed between male and
female subjects (p>0.05).
D3 KOs did not show any indication of developing sensitization to repeated EtOH
on any of the injection days (Figure 4D). Both saline- and EtOH-treated D3 KOs showed
57
Figure 4:
Figure 4A: Wild Type Mice
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Figure 4B: D1 Knockouts
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Figure 4C: D2 Knockouts
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****
Figure 4D: D3 Knockouts
0
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HAB 1 HAB2 HAB3 ACUTE INJ 3 INJ 5
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EthanolSaline
Comparison of Locomotor Response to Chronic EtOH in Dopamine Knockout Colonies and Wild Type Mice: Panels A-D show the average locomotor activity levels (± SEM) demonstrated by EtOH- and saline-treated mice (n=9) over the course of injection days for each separate genotypic colony. By injection 5, wild type C57Bl/6 mice treated with ethanol showed a significant 150% increase in locomotor activity compared to controls. Hypoactive D2 KO mice demonstrated a non-significant increase in locomotor activity in response to chronic ethanol by injection 5. Hyperactive D1 and D3 KOs showed no indication of developing sensitization to chronic ethanol. * Indicates statistical significance of p<0.05. ** Indicates statistical significance of p<0.01.
58
an overall increase in locomotor activity across all injection days, but neither group
differed significantly from one another on any injection day.
Results of a repeated measures mixed between and within subjects ANOVA
indicated a significant main effect of injection day [F(1,23)=6.37, p=0.019] indicating
that the behaviour of the D3 KOs varied across injection days. Evidence to support this
can be seen in Figure 4D, where the overall locomotor levels of the D3 KOs increased
over the course of all injection days. On the other hand, there was no significant main
effect of treatment group suggesting no differences between the drug-treated and saline-
treated animals (p>0.05) and there was no significant drug treatment group X injection
day interaction (p>0.05). These results suggest that the EtOH-treated D3 KO mice did
not show an increased response to the drug as compared to saline-treated controls since
no group differences were observed throughout the experiment. Both saline-treated and
EtOH-treated D3 KOs showed an overall increase in locomotor activity with every
injection day that was found to be statistically significant but this difference did not vary
between groups on any day since no interactions were observed. Therefore the data
suggest that D3 KO mice are resistant to the sensitizing effects of EtOH.
The behavioural profile that was observed in the D1 KO mice was similar to that
of the D3 KO mice (Figure 4B). The EtOH-treated D1 KOs showed no differences in
locomotor behaviour in comparison to saline-treated animals on any injection day.
Results of the repeated measures ANOVA revealed no significant main effect of drug
treatment group (p>0.05), no significant main effect of injection day (p>0.05) and no
59
significant group X day interaction (p<0.05). These results indicate that D1 KO mice,
like D3 KOS are resistant to the sensitizing effects of chronic EtOH.
The D2 KO mice showed a behavioural profile that was more similar to the wild
type mice than to either the D1 or D3 KO colonies. D2 KOs that were chronically treated
with EtOH showed a progressive increase in response to the drug with repeated
injections, although this increase in response was not as robust as it was in the wild type
mice (Figure 4C). A repeated measures analysis of variance showed that the differences
between drug-treated and saline-treated groups was close to, but not statistically
significant [F(1,16)=3.82, p=0.068], indicating that the observed increased response in
the EtOH-treated D2 KOs was blunted in D2 KOs as compared to the wild type colony.
Furthermore, no main effect of injection day was observed (p>0.05) and no significant
group X day interaction was observed (p>0.05).
Discussion
In agreement with previous literature reports, the dopamine receptor knockout
mice showed baseline behavioural phenotypes that accurately reflected their respective
genotypes (Accili et al., 1996; Baik et al., 1995; Xu et al., 1994b). Both D1 and D3
knockout mice demonstrated locomotor hyperactivity with no habituation, and the levels
of locomotor activity were significantly higher in both knockout colonies than they were
in wild type controls. Additionally the D2 KO mice showed significant hypoactivity
60
compared to wild types. These behavioural phenotypes accurately reflected those
described previously in Chapter 3 for D1, D2 and D3 KO mice.
Upon examination of the development of EtOH sensitization in the respective
colonies, the wild type mice bred and reared in our facility showed locomotor responses
that were the same as those mice commercially obtained from Charles River, that we had
previously tested in Experiment 1A. The sensitized wild type mice showed a 150%
increase in locomotor activity as compared to saline-treated controls. This observation
agrees with Experiment 1A, confirming that even though C57 mice may not express
EtOH sensitization as robustly as DBA mice, with the planned sub-division of subjects
and expected effect size kept in mind, sensitization to EtOH can be observed in C57
mice.
The D1 KO mice treated with chronic EtOH did not show any indication of
developing sensitization over the course of treatment, as indicated by the lack of
difference between the EtOH- and saline-treated D1 KOs. As previously described, D1
KOs fail to develop sensitization to cocaine, amphetamine and morphine, therefore it was
not surprising that these mice also failed to develop sensitization to EtOH. These
findings are in agreement with reports that the D1 receptor is necessary for the induction
process of behavioural sensitization to various drugs of abuse including amphetamine,
cocaine, and morphine (Kalivas & Stewart, 1991; Vezina, 1996). The observation that
D1 KO mice did not develop behavioural sensitization to EtOH extends those reports to
include EtOH as another drug of abuse that requires a functional D1 receptor in order to
induce sensitization.
61
D2 KO mice, unlike D1 KOs, demonstrated a progressive increase in locomotor
activation in response to chronic EtOH as compared to saline-treated D2 KOs. The
increase in response, however, just missed statistical significance (p=0.068). The pattern
of drug-induced activation was similar in nature to that of the wild type mice, but
considerably blunted in comparison. This overall blunted sensitization response could
suggest that the loss of the D2 receptor diminished, but did not eliminate, the overall
expression of sensitization to EtOH, since the level of locomotor activation did not reach
that of wild type mice.
Our results here are in contrast to a previous report that D2 KO mice show
enhanced sensitization to EtOH as compared to wild type mice (Palmer et al., 2003).
Perhaps differences in laboratory handling conditions and injection protocols between the
two studies affected the level of responding in each case. In the Palmer study, EtOH was
administered daily whereas in our study, EtOH was administered in a more intermittent,
twice per week injection regime. A role for the D2 receptor has been proposed in the
context-dependent expression of sensitization to drugs of abuse rather than the
development or induction process of sensitization itself (Kalivas & Stewart, 1991; Pierce
& Kalivas, 1997; Weiss et al., 1989). Therefore it is possible that D2 KOs in both studies
responded differently to variable environmental cues and injection protocols.
Interestingly though, in the Palmer study, little role for the D2 receptor was found when a
genetic background more permissive to EtOH sensitization (the 129 strain background)
was used. This latter observation was more similar to our current findings with D2 KOs.
In comparison to the other mouse colonies in our study, since the D2 KOs did show an
62
augmented locomotor response to chronic EtOH, although blunted in comparison to wild
types, it would appear as though the D2 receptor does not a play as large of a role in the
development of sensitization to EtOH as the D1 receptor.
Finally, in contrast to D2 KOs, but similar to the D1 KOs, the D3 KO mice did
not show any indication of developing sensitization to EtOH. The similar levels of
locomotor activity observed between EtOH- and saline-treated D3 KOs alike
demonstrated this finding. Our observation was in direct contrast to our hypothesis,
which had predicted enhanced locomotor activation in response to chronic EtOH as a
result of the loss of the inhibitory effects of the functional D3 receptor. More
specifically, one important part of our hypothesis included an assumption that D3
receptor up-regulation may accompany sensitization to EtOH, perhaps as a compensatory
response to chronic D1/D2 activation. If this were the case, and the possibility for an
accompanying D3 receptor up-regulation was precluded in D3 KO mice, then we would
expect an exaggerated sensitization effect in these mice. Despite the observed baseline
hyperactivity in D3 KO mice, these mice did not demonstrate a potentiated sensitization
effect compared to wild type mice following chronic EtOH administration. In fact the D3
KOs showed no sensitization to EtOH to any degree.
To our knowledge this was the first study to examine EtOH sensitization in D3
KO mice. Although the response shown by these mice was in opposition to our own
prediction, it was not contradictory to a previous report which indicated that chronic D3
receptor blockade with a pharmacological antagonist blocks the development of
amphetamine sensitization in normal mice (Chiang et al., 2003). Surprisingly, however,
63
an examination of amphetamine sensitization in D3 KO mice has not been reported in the
literature, and it is not known how EtOH sensitization and amphetamine sensitization
may compare to one another.
Perhaps the D3 receptor plays a more complex role in the development of
sensitization to EtOH, than merely an inhibitory response to D1/D2 activation. From the
observed behavioural profiles of our knockout colonies, it appeared as though D3
receptors, like D1 receptors, have a stronger role in the induction process of EtOH
sensitization, more so than D2 receptors. As described earlier, D1 and D3 receptors are
co-expressed on the same neurons in the shell region of the NAcc, as well as the islands
of Calleja. Perhaps with respect to EtOH sensitization, D1 and D3 receptors interact
more closely with each other than they do with D2 receptors in order to facilitate the
induction process of sensitization to EtOH. Although the D1 receptor has been well
established in playing a role in the induction process of sensitization with both antagonist
studies and D1 KO studies alike, such studies with D3 KOs are considerably less
abundant in the literature.
It is important to note that research involving the use of transgenic mice is not
without its limitations. Although the concept of studying phenotypic differences in mice
that do and do not express a particular gene is attractive, there are a number of examples,
as described in Chapter 3 where the phenotypic differences between knockout mice and
wild types can be different from those predicted by pharmacological intervention. These
differences can be qualitative or quantitative. For example, D2 KO mice show overall
levels of hypoactivity compared to wild type mice, as we observed in our experiment,
64
even though administration of D2 antagonists to wild type mice can result in profound
neuoleptic/cataleptic effects when high doses are used to block this receptor (Baik et al.,
1995). This quantitative difference in locomotor activity in D2 KOs suggests that these
mice have developed a compensatory mechanism from birth to allow these mice to
ambulate even in the absence of D2 receptors. Additionally, as described in Chapter 3,
D1 KO mice demonstrate an overall hyperactive phenotype, again observed in this
experiment, which is in contrast to the hypoactive-inducing effects of D1 antagonist
administration in wild type mice. Again it thought that this qualitative difference in
phenotype might be the result of a compensatory response to the elevated midbrain
dopamine levels that have developed in these mice (F. N. McNamara et al., 2003).
These phenotypic discrepancies observed in KO mice suggest that adaptive
changes have occurred in KO mice as a result of lifelong deletion of the receptor in
question. Such adaptive changes are clearly undesirable when the goal of the research is
to understand how a receptor normally functions. It is now widely accepted that
phenotypic differences observed in KO mice must be interpreted with this caveat in mind
and that KO mice do not represent a research panacea (Holmes et al., 2004). Thus
studies involving the examination of dopamine receptor knockout mice are most
effectively utilized when employed in conjunction with other techniques, such as
pharmacological intervention. In this manner one could then generate converging lines
of evidence regarding dopamine receptor function.
In summary both D1 and D3 KO mice showed spontaneous locomotor
hyperactivity as compared to wild types whereas D2 KO mice were hypoactive. The
65
behavioural profiles of the knockout colonies suggest that D1 and D3 KO mice are
resistant to EtOH sensitization and show a more similar response pattern to chronic
ethanol when compared to wild type and D2 KO mice. These results support the
involvement of both D3 and D1 receptors in the development of EtOH sensitization, with
an apparent lesser involvement of the D2 receptor subtype. In order to test whether these
observations are the result of specific receptor loss or whether these changes are
associated with compensatory mechanisms in transgenic animals, pharmacological
blockade with antagonists for D1 and D3 receptors should be tested in the future.
66
Experiment 1C: Examination of amphetamine sensitization in D3 knockout mice
Purpose
In Experiment 1B, D3 KO mice were found to be resistant to the sensitizing
effects of chronic ethanol. This observation raised the question of whether D3 knockouts
would show the same resistance to sensitization following the administration of other
drugs of abuse or whether the observed response, or lack thereof, was unique to ethanol
sensitization. Amphetamine is a dopaminergic stimulant that has long been known to
induce behavioural sensitization in laboratory animals. Therefore the purpose of the
following experiment was to see whether D3 KOs were also resistant to amphetamine
sensitization. If D3 KO mice showed the same resistance to the development and
expression of amphetamine sensitization as EtOH sensitization, then a role for the D3
receptor sub-type may underlie the neural mechanisms of behavioural sensitization to
several drugs of abuse. However if D3 KO mice do sensitize to amphetamine, then
perhaps a role for the D3 receptor may be unique to the sensitizing effects of EtOH.
Materials & Method
Subjects
Fourteen male D3 KOs and 14 male wild type mice aged 6-10 weeks with a
C57Bl/6 background strain were randomly assigned to receive either chronic
amphetamine or saline vehicle (n=7). All mice were bred in-house and weaned at 3
weeks of age. Weaned pups were genotyped by the transgenic staff as described
67
previously in Experiment 1B. Food and water were provided at all times throughout the
experiment with the exception of the 60-minute test sessions when animals were in the
activity monitors. Because C57 mice have been previously described to be highly
responsive to amphetamine, animals were not sub-divided into high and low responders
and all subjects were included in the behavioural analysis. Behavioural data were
analyzed by a repeated measures mixed within and between subjects design, with
injection day as the repeated measure, and both drug treatment group (Amphetamine vs.
Saline) and genotype (D3 KO vs. Wild type) as the between subjects factors.
Drug
D-amphetamine HCl (US Pharmacopeia) was dissolved in 0.9% physiological
saline for an injection volume of 10ml/kg. During the sensitization period mice received
1.5 mg/kg amphetamine (i.p.) or equal volumes of saline vehicle. On the final test day,
all mice received 0.5 mg/kg amphetamine (i.p.).
Procedural Overview
After three habituation sessions D3 KO and wild type mice were injected with
either amphetamine (1.5 mg/kg, i.p.) or equal volumes of saline. Mice were then placed
immediately into the activity monitors for a 60-minute test session. Locomotor activity
was recorded as previously described in Experiment 1A, with the exception of the longer
test session (60 minutes instead of 15 minutes). Mice were injected every second day for
a total of 6 sensitizing injections. On the injection days that mice were not tested,
68
(injections 2-5) mice were transported in their home cages to the test room, injected with
amphetamine or saline and placed immediately back into their home cage. Home cages
were kept in the testing room for 60 minutes before they were returned to the colony
room. After the 6th sensitizing injection, mice were tested in the activity monitors for 60
minutes to observe whether behavioural sensitization to amphetamine had been induced.
Mice then received 14 days of withdrawal, during which time the animals received no
drug. After the washout period, all mice received a single test injection of 0.5 mg/kg
(i.p.) and were tested in the activity chambers for 60 minutes.
Results
The results from the amphetamine sensitization study are shown in Figure 5.
Upon acute injection of the drug, both amphetamine-treated wild type and D3 KO mice
showed an increased locomotor response compared to the saline-treated mice. This
increased response to amphetamine was more pronounced by injection 6, suggesting that
a progressive increase in responding, or sensitization to amphetamine had taken place in
both wild type and D3 KO mice alike. When all animals were tested with a low dose of
amphetamine (0.5 mg/kg) following a 14-day washout period, sensitized wild types and
D3 KOs continued to show higher responses to amphetamine than the control animals
that were receiving the drug for the first time, although this effect was more pronounced
in the wild type mice than the D3 KOs, since the D3 KO mice showed a higher locomotor
response to the test dose than the wild types.
69
Figure 5:
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Comparison of amphetamine sensitization in wild type and D3 KO mice: Mean ambulatory counts (±SEM) for D3 KO and wild type mice during a 60-minute test session following amphetamine injections after the acute injection and injection 6 (1.5 mg/kg) and after the final test session where all mice were injected with 0.5 mg/kg. Overall chronic amphetamine resulted in a significant increase in responding (sensitization) to amphetamine compared to saline-treated controls irrespective of genotype. ** Indicates statistical significance of p<0.01.
70
ANOVA results revealed a significant main effect of drug treatment group
[F(1,24)=16.91, p<0.001] a significant main effect of injection day [F(2,48)=7.36,
p=0.002] but no significant main effect of genotype (p>0.05), which suggested that wild
type and D3 KO responded similarly to one another on the various injection days. There
was a significant injection day X drug interaction [F(2,48)=5.86, p=0.005] which
suggested that the drug effects varied significantly between injection days. There was no
significant injection day X genotype interaction (p>0.05), no significant genotype X drug
interaction (p>0.05) and no significant injection day X drug X genotype interaction
(p>0.05). These results indicate that amphetamine significantly increased locomotor
activity in both wild type and D3 KO mice alike, and this increase varied across injection
days. Whether the mice were wild type or knockout had no significant impact on the
response to chronic amphetamine.
Discussion
In this experiment we observed that chronic administration of amphetamine
resulted in a progressive increase in locomotor activity in all drug-treated mice regardless
of genotype. Upon acute administration of the drug, both wild type and D3 KO mice
showed relatively the same level of locomotor activation that did not differ significantly
between groups. After 6 sensitizing injections both drug-treated groups demonstrated an
enhanced response to amphetamine, which again, did not differ significantly between
genotypic groups. Finally after the 14-day drug withdrawal, when tested with a low dose
71
of amphetamine, both drug-treated groups once again demonstrated levels of locomotor
activity that were higher than saline-treated control animals that were receiving the test
dose for the first time. Taken together these data suggest that D3 KOs sensitize to
amphetamine in a manner similar to wild type mice.
To our knowledge, this is first study examining locomotor responses to chronic
amphetamine in D3 KO mice. Only one other study examined in detail the locomotor
responses of D3 KO mice to acute amphetamine (R. K. McNamara et al., 2006b) and our
results have some similarities and differences to that study. First of all in their study,
McNamara et al. found that D3 KOs showed an increased locomotor response to a low
dose of amphetamine (2.5 mg/kg) but showed equal activation to wild types at higher
doses (5-10 mg/kg) suggesting that D3 KOs were more sensitive to lower doses of the
drug than wild type mice. In our study, the doses used were much lower than the
McNamara study, and in contrast to their findings, our sensitizing dose of 1.5 mg/kg, did
not elicit differential responses between wild type and D3 KO mice upon acute injection.
Furthermore in a pilot study where we first used 2.5 mg/kg as the sensitizing dose, a dose
which proved to be too high for chronic treatment (eliciting high levels of stereotypy and
low levels of locomotor activity in all groups), we once again did not observe a difference
in response between wild types and D3 KOs upon acute administration of this dose (data
not shown).
However, upon examination of the final test day, the D3 KOs that had been
previously treated with saline, and were receiving 0.5 mg/kg of amphetamine for the first
time, showed a higher level of activation than the wild type mice receiving the same dose
72
for the first time. Thus in keeping with the McNamara observation, a low dose of
amphetamine appeared to elicit a higher response from the D3 KOs than the higher dose.
The key difference between studies lies in the dose range of the drug, and this may be
related to the background strain of the mice used for the studies. Our knockout mice
have been repeatedly backcrossed to the C57Bl/6 strain, where the mice used in the
McNamara study were derived directly from the 129 strain. It has been previously
described that the background strain of mice may alter the magnitude of a phenotypic
response (F. N. McNamara et al., 2003), and since C57 mice are also highly sensitive to
amphetamine, the D3 KO mice derived from the C57 background may be more sensitive
to amphetamine at lower doses than those derived from the 129 strain.
We also observed that the saline-treated D3 KOs did not exhibit locomotor
hyperactivity when compared to saline-treated wild types on either the acute injection, or
on injection 6. This finding was in contrast to the behaviour observed in our EtOH
sensitization study where the saline-treated D3 KOs demonstrated baseline levels of
activity that were significantly higher than wild type controls. This discrepancy was not
surprising considering that the locomotor hyperactivity that is often observed in D3 KOs
is transient and restricted to the early phases of locomotor testing (Holmes et al., 2004).
In this experiment animals were tested in the activity chambers for 60 minutes, and in our
previous EtOH studies, mice were tested for 15 minutes. When we looked at the
locomotor activity in the D3 KOs within the first 10 minutes of testing, D3 KOs once
again showed higher levels of locomotion as compared to wild types. The differences in
baseline locomotor levels eventually evened out over the course of the remaining hour in
73
the activity chamber. This observation was in agreement with the literature reports that
D3 KOs show increased novelty-induced activation in response to changes in
environments.
The results from this experiment indicate that similar to wild type C57 mice, D3
KO mice develop and express sensitization to chronic amphetamine. This finding is in
contrast to our previous experiment where D3 KO mice were resistant to EtOH
sensitization, even though wild type mice showed sensitization to chronic EtOH. These
results could suggest that the D3 receptor plays a more important role in the development
and expression of sensitization to EtOH than to amphetamine, which may point toward
the possibility for a unique neural mechanism that underlies EtOH sensitization,
distinguishing this model from other drugs of abuse.
74
CHAPTER 5
Study 2: Examination of the induction and expression of ethanol sensitization following pharmacological blockade with a D3R antagonist
Purpose
In Study 1 we observed that D3 KO mice were resistant to the sensitizing effects
of chronic EtOH when compared to their wild type littermates. To observe whether the
lack of the D3 receptor function has a true effect on chronic EtOH administration or
whether the observed resistance to EtOH sensitization was a compensatory mechanism in
knockout mice, we asked the question, would temporary blockade of the D3 receptor by
pharmacological intervention in wild type mice yield the same resistance to EtOH
sensitization as observed in D3 KOs?
D3 KOs show normal appearance and no gross neurological dysfunctions
(Holmes et al., 2004; Xu et al., 1997). D3 KOs have been most widely cited as
displaying spontaneous locomotor hyperactivity in novel environments, which is in
keeping with the putative role for the D3 receptor in inhibiting locomotor activity
(Boyce-Rustay & Risinger, 2003; Pritchard et al., 2003; Xu et al., 1999; Zhang & Xu,
2001). The D3 receptor is limited in its expression throughout the brain, and D3 KO
mice are phenotypically similar to wild types. It is possible that the lack of observed
EtOH sensitization in these KO mice is due primarily to the loss of functional D3
receptor and not due to a compensatory mechanism that has developed since birth.
Therefore it was our hypothesis that a temporary blockade of D3 receptors with a D3-
75
selective antagonist in wild type mice would, in a similar manner to D3 KOs, interfere
with either the development or expression of locomotor sensitization to ethanol.
As previously described, behavioural sensitization can be divided into two main
phases: induction and expression. In this study we examined these different phases
separately in order to ascertain in which phase the D3 receptor may play a role. In the
first experiment (Experiment 2A), we induced EtOH sensitization in wild type mice and
tested to see whether acute D3 receptor blockade interfered with the expression of EtOH
sensitization once sensitization had been induced. In the second experiment (Experiment
2B), we chronically blocked D3 receptors during the induction phase of sensitization, by
co-administering the D3 antagonist with every EtOH injection to see whether chronic D3
receptor blockade interfered with the induction of EtOH sensitization. Since D3
receptors are located primarily in the nucleus accumbens, a brain region associated with
mediating the expression of behavioural sensitization, our hypothesis was that D3
receptors would play a stronger role in mediating the expression rather than induction of
EtOH sensitization
76
Experiment 2A: Effects of D3 antagonist on the expression of EtOH sensitization
Materials & Method
Subjects
Since this experiment did not call for the use of transgenic mice, we were not
restricted by the background strain of the knockout colonies. Therefore, to maximize the
sensitization effect obtained after chronic EtOH administration, we elected to examine
DBA mice that demonstrate more robust sensitization to EtOH in addition to the C57
mice that we had been previously studying for the knockout experiments.
In order to observe whether blockade of D3 receptors with an antagonist would
interfere with the expression of sensitization, 32 male C57 and 32 male DBA mice aged 5
weeks (Charles River, Canada) received the same sensitization injection protocol as
previously described in Experiment 1A with the following exceptions: Due to housing
restrictions, there was not sufficient room in the facility to accommodate three times as
many EtOH-treated mice as saline-treated controls using the two strains of mice.
Therefore EtOH-treated mice could not be sub-divided into high and low responders, and
all EtOH-treated mice were included in the challenge data, high and low responders alike.
Additionally, it was also determined from the original sensitization data obtained from
Experiment 1A that DBA mice show saturation of the sensitization response by injection
7. Therefore, all mice in the study received 7 sensitizing injections of EtOH or saline in
order to achieve the maximum response from the DBA mice.
77
Drug
2,3-Dihydro-5,6-dimethoxy-N,N-dipropyl-1H-inden-2-amine maleate (U99194A
maleate) was obtained commercially from Tocris Bioscience (Ellisville, MO). The drug
was dissolved in 0.9% physiological saline to an injection volume of 10 ml/kg. Since D3
receptor antagonists are known to have drug-dependent biphasic effects on locomotor
activity, a dose-response curve was run to determine the ideal test dose for achieving D3-
selective blockade. This D3-selective dose would be indicated by stimulation of
locomotor activity without blocking D2 receptors, which would be indicated by decreases
in locomotor activity.
Results of the dose response curve can be seen in Figure 6 and the time course of
the drug over the 15-minute test session can be seen in Figure 7. Figure 6 shows that
significant increases in locomotor activity compared to saline-treated animals were
observed at both the 10- and 15-mg/kg doses. Figure 7 shows that the drug was fast
acting, since the 15-mg/kg dose elicited significant increases in locomotor activity within
3 minutes of administration. At 10 mg/kg, significant increases in locomotor activity
were not observed until 6 minutes following administration of the drug, therefore it was
determined that the drug should be administered at least 6 minutes prior to EtOH
administration to ensure prior D3 blockade before EtOH administration.
After careful consideration, it was decided that 10 mg/kg would be the chosen
dose to stimulate locomotor activity as a result of D3 receptor blockade, without risk of
additional D2 receptor blockade. Although acute administration of the 15-mg/kg dose,
78
Figure 6:
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Dose-response curve for the D3 receptor antagonist U99194A: DBA mice (n=6 mice per drug dose) were injected subcutaneously and tested for 15 minutes. 10 mg/kg was found to significantly increase locomotor activity in mice (p<0.05), and 15 mg/kg was also found to significantly increase locomotor behaviour (p<0.01). A dose of 10 mg/kg was chosen as the test dose since a significant difference in behaviour could be elicited at this dose without saturating the response curve.
79
Figure 7:
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Time Course for Acute U99194A Across 15-minute Test Session: DBA mice received subcutaneous injections of the D3 antagonist U99194A (0-15 mg/kg). Average locomotor activity levels (± SEM) were recorded in 3-minute bins for the 15-minute session. Significant increases in activity levels compared to saline-treated mice were observed within 3 minutes for the 15 mg/kg dose and after 6 minutes for both the 15 mg/kg and 10 mg/kg doses. In order to avoid saturation of D3 receptors with the chronic administration of the antagonist, the 10 mg/kg dose was chosen in order to block only D3 receptors without concomitantly blocking D2 receptors.
80
resulted in a greater stimulation of locomotor activity than the 10 mg/kg-dose, we were
concerned whether this high dose, when administered chronically, would saturate D3
receptors and begin to block D2 receptors. Furthermore it was important to keep the dose
consistent between the acute and chronic studies. We therefore elected to use the lower
10-mg/kg dose of the antagonist in this study that would elicit a D3-selective effect
without risk of concomitantly blocking D2 receptors when administered chronically.
Procedural Overview
Thirty-two male DBA and 32 male C57 mice were randomly assigned to receive
chronic EtOH or saline (n=16). All mice received 7 bi-weekly injections of EtOH (2.2
g/kg) or saline as previously described in Experiment 1A. After the sensitization period
animals received no drug for 14 days. After this washout stage, EtOH-treated and control
mice (n=16) were further sub-divided into 4 groups where sensitized (EtOH-treated) and
control mice (saline-treated) were challenged with the D3-preferring antagonist U99194A
(10 mg/kg s.c.) or equal volumes saline vehicle (n=8) 6 minutes prior to the EtOH test
injection (1.8 g/kg, i.p.).
A 2 X 2 analysis of variance was carried out for each set of strain-specific data,
with pretreatment (Sensitized vs. Control) and challenge (U99194A vs. Vehicle) as the
fixed factors. The dependent variable was locomotor activity within the 15-minute test
session, when animals were challenged with the D3 antagonist or saline vehicle
preceding the final EtOH injection.
81
Results
Results from the D3 antagonist challenge following sensitization to EtOH can be
seen in Figure 8. Figure 8A shows the results from the C57 mice where Figure 8B shows
the results from the DBA mice. The most noticeable difference between the two strains
of mice is the apparent lack of sensitization in the C57 mice. After the sensitization
period, mice were not subdivided into high and low responders and all subjects were
included in the analysis. As it was reported in Experiment 1A, a considerably smaller
proportion of the C57 population expresses sensitization to EtOH than in the DBA
population (30 vs. 70% respectively). Thus the amount of low-responding C57 mice
remaining in this analysis would lower the overall averages of the EtOH-treated C57
mice, as observed in this experiment. Nonetheless, in the current experiment, the EtOH-
treated C57 mice, as a group, expressed significantly higher levels of locomotor activity
as compared to their saline-treated counterparts.
Results of the ANOVA for the C57 group demonstrated a significant main effect
of pretreatment [F(1,52)=11.94, p=0.001] suggesting that EtOH-treated animals were
significantly different from saline-treated controls. There was no significant main effect
of challenge (p>0.05) and no significant group X challenge interaction (p>0.05),
indicating that the D3 antagonist, U99194A, had no significant impact on the behaviour
expressed by either the saline- or EtOH-treated C57 mice.
The same findings were observed in the DBA mice. Results of the 2 X 2-way
ANOVA revealed a significant main effect of pretreatment group [F(1,43)=96.71,
p<0.001] indicating that the EtOH-treated animals showed locomotor levels that were
82
Figure 8: Figure 8A: Acute U99194 Challenge in Sensitized vs Control C57 mice
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Figure 8B: Acute U99194 Challenge in Sensitized Vs Control DBA mice
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Expression of EtOH Sensitization in C57 and DBA Mice Following Acute Challenge with the D3 Antagonist U99194A: Following sensitization and 14 days washout, sensitized and control C57 (Figure 8A) and DBA (Figure 8B) mice were challenged with U99194A (10 mg/kg s.c.) or saline vehicle (n=8), followed by a test injection of EtOH (1.8 g/kg i.p.). Mean locomotor activity (±SEM) during test session showed that DBA mice expressed an overall higher level of EtOH sensitization than C57 mice, but U99194A had no effect on the expression of EtOH sensitization in either strain of mice. ** Indicates statistical significance of p<0.01.
83
significantly higher than saline-treated controls. Additionally, there was no significant
main effect of challenge (p>0.05) and no significant pretreatment group X challenge
interaction (p>0.05) suggesting once again that the D3 antagonist had no effect on the
expression of sensitization in either EtOH-sensitized or control DBA mice. Overall these
results suggest that acute blockade of D3 receptors with an antagonist had no effect on
the expression of sensitization, once mice have already been sensitized to EtOH. This
held true for both strains of mice tested in this experiment.
Discussion
When EtOH-treated mice were not sub-divided into high and low responders, the
DBA mice responded much more strongly to chronic EtOH as compared to C57 mice,
which was indicated by the DBA mice, as an entire group, expressing higher levels of
locomotor sensitization than the C57 mice (Figures 8A and 8B). These results highlight
the importance of sub-dividing out high responders in C57 mice when using them in
studies of EtOH sensitization. However, despite the differences in magnitude, both
strains of mice did express statistically significant levels of locomotor sensitization when
compared to saline-treated controls (p<0.01).
After 14 days withdrawal, acute challenge with the D3 antagonist U99194A did
not have a significant effect on the locomotor behaviour in either strain of mice. In both
populations, when U99194A was administered to saline-treated control mice, the
locomotor responses were not altered in comparison to mice challenged with saline
84
vehicle. As indicated from the dose-response curve generated in our laboratory, the dose
of U99194A used in this study (10 mg/kg) did elicit a significant increase in locomotor
activity compared to saline-treated mice when the drug was administered by itself. Given
that the 10-mg/kg dose did not saturate the response curve, we purposefully chose this
dose knowing that it lay at the lower end of the response curve, where D3 specificity of
binding was assured. In this current experiment, all mice received the antagonist or
vehicle challenge preceding a test dose of EtOH (1.8 mg/kg) and it is entirely possible
that the lack of observed acute D3 antagonist-induced locomotor activation in the control
mice was a result of EtOH co-administration.
In sensitized mice, acute challenge with the D3 antagonist once again had no
impact on the expression of locomotor sensitization in either strain of mice. Following
14 days washout, mice that were chronically treated with EtOH showed levels of
locomotor activity that were significantly higher than control mice. Whether these mice
were challenged with the D3 antagonist or saline vehicle, no subsequent differences in
locomotor activity levels were observed. This observation indicates that acute blockade
of D3 receptors does not interfere with the expression of EtOH sensitization once it has
been induced.
It is conceivable that using the lower 10-mg/kg dose of the D3 antagonist, rather
than the higher 15-mg/kg dose resulted in only a partial, rather than full blockade of D3
receptors. However, as previously described, the decision was made to stay well within
the D3-specific range both in this acute experiment as well as in the planned experiment
85
with repeated dosing of the antagonist (Experiment 2B), to avoid the risk of D2 receptor
blockade with chronic administration of the drug.
Our results from this experiment are nonetheless in line with previous studies
where acute D3 antagonist challenge failed to block the expression of sensitization to
psychostimulants once it had been induced. In one study, U99194A induced no
locomotor effects in comparison to vehicle when administered to rats that had been
previously sensitized to amphetamine and challenged 10 days post washout (Chiang et
al., 2003). And in another study, the D3 antagonist nafadotride had no effect on the
expression of cocaine sensitization at a low dose (0.2 mg/kg) as compared to vehicle
challenged controls; however at a higher dose (0.4 mg/kg) nafadotride enhanced the
expression of locomotor sensitization in rats that were previously sensitized to cocaine
(Filip et al., 2002). Whether the enhanced expression of sensitization in the second study
was a result of nafadotride’s mild selectivity for the D3 receptor, or occurred because
cocaine sensitization differs from the other models (especially EtOH sensitization) is not
certain. However, taken together, the observations made in these studies lend support to
our finding that acute blockade of D3 receptors does not block the expression of EtOH
sensitization in animals that have been chronically treated with EtOH, once sensitization
has already been induced.
86
Experiment 2B: Effects of D3 antagonist on the induction of EtOH sensitization
Materials & Method
Procedural Overview
To observe whether chronic blockade of D3 receptors with an antagonist would
interfere with the induction of sensitization to EtOH, 32 male C57 and 32 male DBA
mice aged 5 weeks (Charles River, Canada) received 7 bi-weekly injections of either the
D3 antagonist U99194A (10mg/kg s.c) or saline vehicle 6 minutes prior to every EtOH
(2.2 g/kg i.p.) or saline injection (n=8) during the sensitization period. Locomotor
activity was observed and recorded in weekly 15-minute test sessions. Behavioural data
were analyzed by three-way ANOVA with injection day as the repeated measure, group
(EtOH vs Saline) as the first between subjects factor and D3 antagonist (U99194A vs
Vehicle) as the second between subjects factor.
Results
C57Bl/6 Mice
Results from the chronic U99194A study are shown in Figure 9A for C57 mice
and 9B for DBA mice. C57 mice showed no EtOH sensitization, most likely due to small
numbers (n=8). However, C57 mice that received chronic U99194A showed locomotor
sensitization to the D3 antagonist itself. Co-administration of U99194A with chronic
EtOH appeared only to delay the development of sensitization to the D3 antagonist
87
Figure 9:
Chronic Co-Administration of D3 Antagonist U99194A with Chronic EtOH in C57 and DBA Mice: C57 and DBA mice received U99194A (10 mg/kg s.c.) or saline administered before every EtOH injection (2.2 g/kg i.p.). Locomotor activity was recorded weekly in 15-min test sessions. C57 mice showed sensitization to the D3 antagonist itself whereas in DBA mice, the induction of EtOH sensitization was blocked by the co-administration of the D3 antagonist. * Indicates statistical significance of p<0.05. ** Indicates statistical significance of p<0.01.
Figure 9B: Chronic Administration of U99194 in DBA/2 Mice
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Figure 9A: Chronic U99194 Administration in C57Bl/6 Mice
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**
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88
(Figure 9A). Thus both saline- and EtOH-treated mice that received chronic co-
administration of the D3 antagonist U99194A showed progressive increases in locomotor
activity compared to both EtOH- and saline-treated groups that received chronic co-
administration of saline vehicle.
Results from the ANOVA indicated a significant main effect of injection day
[F(7,140)=10.43, p<0.001] and a significant main effect of the D3 antagonist
[F(1,20)=36.10, p<0.001] but no significant main effect of ethanol (p>0.05). The D3
antagonist X injection day interaction was also significant [F(7,140)=10.25, p<0.001].
There was no significant group X injection day interaction (p>0.05), no significant group
X D3 antagonist interaction (p>0.05) and no significant group X D3 antagonist X day
interaction (p>0.05).
DBA/2 Mice
Chronic administration of the D3 antagonist in DBA mice had no behavioural
effect when administered only with a saline vehicle (Figure 9B). EtOH-treated DBAs
showed sensitization to chronic EtOH, as previously observed in other experiments, and
this EtOH sensitization was completely blocked by the co-administration of U99194A.
In contrast to C57 mice, in DBA mice a statistically significant main effect of ethanol
was observed [F(1,26)=4.88, p=0.036]. Unlike C57 mice, there was no significant main
effect of D3 antagonist (p>0.05) indicating that overall the D3 antagonist did not induce
overt effects on behaviour in the DBA mice.
89
There was a significant group X day interaction [F(6,156)=3.21, p=0.005] and a
significant D3 antagonist X day interaction [F(6,156)=3.83, p=0.01]. These data indicate
that a significant variation in response occurred over the course of injection days between
EtOH- and saline-treated animals, as well as between U99194A- and vehicle-treated
mice. Finally a significant injection day X group X D3 antagonist interaction was
observed [F(6, 156)=4.42, p=0.01]. This suggests that the effect of U99194 across
injection days was different between saline and ethanol-treated mice.
Discussion
The most prominent result from this experiment was the observation that in DBA
mice, chronic administration of the D3 antagonist completely eliminated the development
of sensitization to EtOH, whereas on its own (in saline-treated mice) the D3 antagonist
had no effect on locomotor activity in these animals. The group that received only
chronic EtOH developed sensitization as previously observed in other experiments. And
the group that received chronic co-administration of both U99194A and EtOH also
showed the same level of locomotion as saline-treated controls.
These results are in agreement with those observed in Experiment 1B, where D3
KOs, mice lacking a functional D3 receptor, failed to demonstrate sensitization to EtOH.
What was not clear from that particular study was whether the absence of a functional D3
receptor interfered with either the expression or induction of behavioural sensitization to
EtOH. We observed in Experiment 2B that acute blockade of D3 receptors had no effect
90
on the expression of sensitization to EtOH, once sensitization was already established. In
this current experiment, the data strongly suggest that chronic blockade of the D3
receptors, much like the chronic absence of functional D3 receptors in KO mice,
interferes with the induction of sensitization to EtOH.
Although our observations were in contrast to our hypothesis that D3 receptor
blockade would play a stronger role in the expression rather than the induction of EtOH
sensitization, our findings in this experiment as well as in the previous experiment were
consistent with another study performed in Taiwan (Chiang et al., 2003). In their report,
Chiang et al observed that chronic co-administration of either the D3 antagonist
U99194A or GR103691 along with amphetamine blocked the induction of amphetamine
sensitization in rats. They also observed that these drugs, when administered acutely 10
days after amphetamine sensitization had been induced, had no subsequent effect on the
expression of amphetamine sensitization. This report is in agreement with our
observations that acute administration of the D3 antagonist U99194A had no effect on the
expression of sensitization to EtOH once sensitization had been induced, but that chronic
co-administration of the D3 antagonist eliminated the development of sensitization from
occurring.
The picture is not as clear when results from the C57 mice are considered. First,
EtOH-treated C57 mice in this particular study did not develop locomotor sensitization to
any observable degree. In our previous studies, C57 mice still demonstrated locomotor
activation to some degree, although the effect was not as robust as in DBA mice. We
have estimated that approximately 30% of the C57 population exhibits locomotor
91
sensitization to EtOH. In this study, only 2 out of 8 (25%) mice receiving only EtOH
showed locomotor activation in response to the drug, albeit to non-significant levels. The
rest of the mice remained unresponsive to chronic EtOH. It is entirely possible that of
this small population of C57 mice, most of these mice were and remained resistant to the
stimulating effects of chronic EtOH.
On the other hand, the C57 mice that received only chronic U99194A showed
progressive increases in locomotor response to the challenge drug itself. This was in
direct contrast to the DBA mice where no locomotor activation was observed in response
to the D3 antagonist in either the saline- or EtOH-treated groups. It was previously
described that DBA and C57 mice differ and often show opposite responses to several
drugs of abuse, especially dopaminergic agents. These mice have also been described in
several studies as having marked differences in their mesolimbic dopamine systems as
well as differences in basal levels of D3 receptor expression (See Chapter 3 for review).
Perhaps then, in addition to the above-mentioned factors and since DBA mice are more
responsive to EtOH, whereas C57s are more responsive to dopaminergic stimulants such
as amphetamine, it would not be surprising to observe a heightened response to the D3
antagonist in the C57 mice.
Overall, the results of these experiments suggest that in addition to having
differential effects in two genetically different strains of mice, D3 antagonists do not
block the expression of EtOH sensitization once it has already been induced. However,
chronic blockade of D3 receptors with an antagonist may be useful in preventing the
induction of EtOH sensitization, but only in individuals that are perhaps genetically more
92
susceptible towards developing sensitization to EtOH. This observation is in keeping
with the previously observed D3 KO data, where D3 KOs failed to develop and thus
express sensitization to EtOH.
93
CHAPTER 6
Study 3: Examination of the expression of ethanol sensitization following pharmacological stimulation of D3 receptors with an agonist
Experiment 3A: Effects of D3 agonist on expression of EtOH sensitization
Purpose
After examination of how acute and chronic D3 receptor blockade affected the
development and expression of EtOH sensitization, we became interested in examining
how acute and chronic D3 receptor stimulation could affect EtOH sensitization. We
hypothesized that D3 agonist administration would yield results opposite to those that we
observed with D3 antagonist administration.
Additionally, in a pilot study, brains from mice sensitized to EtOH and controls
were removed and examined for D3 receptor mRNA expression and D3 receptor binding
using in situ hybridization and receptor binding autoradiography respectively. Results
from this preliminary study suggested that there was no change in D3 mRNA expression
in sensitized mice compared to controls, but an overall trend towards increased D3
receptor binding in the sensitized brains was observed. We were interested in confirming
this putative up-regulation of D3 receptors following EtOH sensitization and asked
whether this potential increase in D3 receptor binding resulted in a functional change that
may be expressed behaviourally. Our hypothesis then was that if D3 receptors up-
regulate as a result of EtOH sensitization, and if this up-regulation results in a functional
change in the D3 receptor, then mice sensitized to EtOH should be more sensitive to D3
receptor stimulation than control mice.
94
Materials & Method
Procedural Overview
Since DBA mice express EtOH sensitization in a more reliable manner than C57
mice, and since we were limited by our housing facilities such that we could not treat
enough animals to sub-divide our C57 population into high and low responders, we chose
only to use DBA mice for this study. Thirty-two male DBA mice aged 5 weeks (Charles
River, Canada) received chronic EtOH or saline (n=16) as previously described in other
experiments followed by 14 days washout. After the washout period sensitized and
control mice were further sub-divided into challenge groups receiving either the D3-
preferring agonist PD128907 (0.01 mg/kg s.c.) or saline vehicle (n=8) 6 minutes prior to
a test injection of EtOH (1.8 g/kg). Behavioural data obtained from the challenge were
analyzed by a 2 X 2 way ANOVA, with pretreatment group (EtOH vs. Saline) and
challenge group (PD128907 vs. Vehicle) as fixed factors.
Drug
The D3 dopamine receptor-preferring agonist (+/-)-(4aR,10bR)-3,4,4a,10b-
tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin-9-ol hydrochloride (PD
128907 HCl), (Sigma-Aldrich Canada, Oakville, ON) was dissolved in physiological
saline (0.9% NaCl) for an injection volume of 10 ml/kg. The test dose of PD128907 was
determined from a dose-response curve run in our laboratory (Figure 10). The time
course of the drug effects over the 30-minute test session is shown in Figure 11. A test
95
Figure 10:
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Dose-response Curve for the D3 Receptor Agonist PD128907: DBA mice (n=6 mice per drug dose) were injected subcutaneously and tested for 30 minutes. Significant agonist-induced decreases in locomotor activity were observed at doses higher than and including 0.05 mg/kg. At 0.01 mg/kg decreases in locomotor activity were not statistically significant. This dose was chosen for the test dose in order to ascertain whether an up-regulation of D3 receptors accompanying EtOH sensitization would be reflected in an increased sensitivity to D3 agonists at doses otherwise not observed to be high enough to induce a significant effect alone.
96
Figure 11:
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Time Course for the D3 Agonist PD128907 Over 30-Minute Test Session: DBA mice received subcutaneous injections of the D3 agonist PD128907 (0-1.00 mg/kg). Average locomotor activity levels were recorded in 3-minute bins for the 30-minute session. Error bars were omitted for visual simplification. Significant decreases in activity levels compared to saline-treated mice were observed within 6 minutes for the 0.1, 0.5 and 1.0 mg/kg doses. Significant differences between saline and drug-treated mice were not observed after 24 minutes. The dose of 0.01 mg/kg was chosen as the challenge dose since it did not produce significant decreases in locomotor activity compared to saline-treated mice.
97
dose of 0.01 mg/kg was chosen for use in this experiment as this dose was found to be
high enough to elicit a physiological response (agonist-induced inhibition of locomotor
activity) without reaching statistical significance. We chose a dose of the D3 agonist that
would not significantly alter the behaviour of saline-treated control animals, but would
significantly alter the behaviour of those mice sensitized to EtOH, if in fact, EtOH
sensitization results in an up-regulation of D3 receptors and therefore increased
sensitivity to D3 receptor stimulation.
Results
Results from the D3 agonist (PD128907) challenge following sensitization to
EtOH can be seen in Figure 12. Following 14 days drug washout, EtOH-treated mice
that received no drug challenge (Sensitized + Vehicle) continued to demonstrate levels of
locomotor activity that were significantly higher than all other groups. As expected,
saline-pretreated control animals did not differ in their response to the D3 agonist
challenge. On the other hand EtOH-treated mice that had been challenged with the D3
agonist (Sensitized + PD128907) showed levels of locomotion that were similar to saline-
treated animals.
ANOVA results indicated a significant main effect of pretreatment (Sensitized vs.
Control) [F(1,41)=7.20, p=0.010], a significant main effect of challenge (PD128907 vs.
Vehicle) [F(1,41)=6.71, p=0.013] and no significant interaction between these two
factors (p>0.05).
98
Figure 12:
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Acute PD128907 Challenge in sensitized vs. control DBA mice: Average ambulatory counts (±SEM) for each group following acute PD128907 or vehicle challenge in sensitized and control animals (n=8). Male DBA mice received 7 injections of either EtOH (2.2 g/kg i.p.) or saline followed by 14 days washout. Mice then either received PD128907 (0.01 mg/kg s.c) or saline before a test injection of EtOH (1.8 g/kg i.p.). In sensitized mice only, PD128907 attenuated the EtOH-induced hyperactivity but had no significant effect in controls animals. # Represents statistically significant reduction in locomotor activity in sensitized mice challenged with D3 agonist compared to those challenged with saline vehicle, p<0.05.
99
Despite the lack of a significant interaction, we made two mean comparisons
following the ANOVA. We compared the differences in response to the D3 agonist
challenge in control and sensitized animals. In the control animals, the agonist-
challenged mice showed a non-significant 21% reduction in locomotor activity as
compared to vehicle-challenged controls (t test, p>0.05). However in the sensitized
animals, agonist-challenged mice showed a significant 43% reduction in locomotor
activity [t(20)=-2.32, p=0.031], which was comparable to control animal levels that
received no EtOH treatment. These results indicated that the sensitized animals were
more responsive to the D3 agonist than control animals.
Discussion
In order to observe whether mice sensitized to EtOH would have a heightened
response to acute agonist administration, we elected to use a dose of D3 agonist, obtained
from our dose-response curve, that would elicit a small but non-significant response in
control animals. As indicated by the saline-pretreated control animals, acute
administration of low dose D3 agonist PD128907 induced a small and non-significant
inhibition of locomotor activity compared to saline-challenged controls. However, in
sensitized mice, this same low dose of PD128907 induced a significant attenuation of
locomotor activity as compared to sensitized mice challenged with vehicle. These results
suggest that mice that have been sensitized to EtOH are more responsive to D3 ligands
than control animals. This increased responsivity to D3 ligands in sensitized mice could
100
also potentially be suggestive of a functional up-regulation of D3 receptors
accompanying sensitization to EtOH.
Furthermore in comparison to our previous results from Experiment 2A, where
acute administration of the D3 antagonist U99194A had no effect on the expression of
EtOH sensitization once it had been induced, in this experiment, acute administration of
the D3 agonist PD128907 significantly attenuated the expression of EtOH sensitization
once it had been induced.
Our results here are supported by a previous study, that reported that acute
administration of the D3 agonist 7-OH-PIPAT, but not the D3 antagonist nafadotride,
blocked the expression of cocaine sensitization in rats once it had already been induced
(Filip et al., 2002). Taken together then, administration of D3 agonists, unlike D3
antagonists, may interfere with the expression of sensitization.
Results from this study showed that sensitized mice, when challenged with the
D3-preferring agonist, PD12907 showed a greater inhibition of locomotor activity than
saline-treated controls. These data suggest not only that D3 agonists, in direct contrast to
D3 antagonists, may block the expression of EtOH sensitization once it has been induced,
but also that a functional up-regulation of D3 receptors may accompany EtOH
sensitization making these animals more sensitive to the inhibitory effects of D3 agonists.
101
Experiment 3B: Effects of D3 agonist on induction of EtOH sensitization
Purpose
We have previously observed that acute blockade of D3 receptors had no effect
on the expression of EtOH sensitization, but chronic blockade of D3 receptors appears to
interfere with induction of EtOH sensitization in DBA mice. On the other hand, acute
administration of a D3 agonist appeared to interfere with the expression of EtOH
sensitization in DBA mice. These results have led us to ask the question: How would
chronic D3 receptor stimulation by an agonist affect the induction of sensitization to
EtOH? Since we observed that D3 KOs fail to develop or express sensitization to EtOH,
and since we observed that mice receiving chronic D3 antagonist administration also fail
to develop or express sensitization, we hypothesized that mice receiving chronic D3
receptor stimulation with an agonist, may develop either normal or potentiated
sensitization to EtOH in a manner opposite to chronic D3 receptor blockade.
Materials & Method
Procedural Overview
Thirty-two male DBA mice aged 5 weeks (Charles River, Canada) were randomly
assigned to receive 7 bi-weekly injections of either the D3 agonist PD128907 (0.01
mg/kg, s.c.) or saline vehicle 6 minutes prior to every EtOH (2.2 g/kg, i.p.) or saline
injection. Locomotor activity was recorded in activity monitors in weekly 15-minute test
sessions as previously described. All mice were euthanized via CO2/O2 inhalation 24
102
hours after their 7th and final injection. Behavioural data obtained from these mice were
analyzed with a 3-way mixed within and between subjects design, with injection day as
the within subjects repeated measures factor, and group (EtOH vs. Saline) as the first
between subjects factor, and D3 agonist (PD128907 vs. Vehicle) as the second between
subjects factor.
Results
Results from the chronic co-administration of the D3 agonist experiment can be
seen below in Figure 13. Control mice that received chronic D3 agonist PD128907
demonstrated a consistent agonist-induced inhibition of locomotor activity that remained
constant throughout the experiment. Mice that received chronic EtOH co-administered
with vehicle demonstrated the highest levels of locomotor activity. Mice that received
chronic co-administration of EtOH and D3 agonist demonstrated locomotor sensitization
to EtOH, although the entire response curve appeared to be driven down by the co-
administration of the D3 agonist in a manner consistent with the control animals
receiving chronic PD128907. In other words, it appeared as though chronic PD128907
inhibited locomotor activity equally in EtOH-treated and control animals alike.
Results from this analysis revealed a significant main effect of injection day
[F(6,144)=6.82, p<0.001] indicating that the behaviour of the mice varied significantly
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Figure 13:
Chronic co-administration of the D3 agonist PD128907 with EtOH sensitization: Plotted above are the average ambulatory counts (±SEM) of the mice receiving chronic co-administration of either D3 agonist PD128907 or Vehicle with either chronic EtOH or saline (n=8). Chronic administration of the D3 agonist PD128907 appeared to drive down responding equally in Saline- and EtOH-treated mice alike. Statistical difference from saline + vehicle-treated controls is indicated with * p<0.05, and ** p<0.01. Chronic co-administration of the D3 agonist PD128907 with EtOH sensitization: Plotted above are the average ambulatory counts (±SEM) of the mice receiving chronic co-administration of either D3 agonist PD128907 or Vehicle with either chronic EtOH or saline (n=8). Chronic administration of the D3 agonist PD128907 appeared to drive down responding equally in Saline- and EtOH-treated mice alike. Statistical difference from saline + vehicle-treated controls is indicated with * p<0.05, and ** p<0.01.
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***
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104
from day to day, over the course of the experiment. A significant main effect for group
was also found [F(1,24)=21.03, p<0.001], which suggested that the EtOH-treated and
saline-treated mice differed significantly from each other throughout the experiment.
Both the EtOH + PD128907 and EtOH + vehicle groups evidenced this by showing
higher levels of locomotor activity throughout the experiment than both of the saline-
treated groups. No significant main effect was observed for the D3 agonist (p>0.05)
indicating that the chronic co-administration of the D3 agonist did not significantly alter
the behaviour of the mice. There was a significant group X day interaction
[F(6,144)=18.38, p<0.001] indicating that the response to EtOH varied significantly
across injection days. There was also a significant D3 agonist X day interaction
[F(6,144)=3.87, p=0.001], which suggested that the response to the D3 agonist varied
across injection days as well. This interaction was demonstrated by the D3 agonist-
treated mice showing behaviour in response to the drug that was different from that
shown during the habituation sessions. No other significant interactions were observed.
There was no significant group X D3 agonist interaction (p>0.05) and no significant
injection day X group X D3 agonist interaction (p>0.05).
105
Discussion
In this current experiment, chronic co-administration of the D3 agonist PD128907
had an overall inhibitory effect on locomotor behaviour over the course of injection days.
In saline-treated animals, acute administration of the D3 agonist, in agreement with our
previous results, did not significantly inhibit locomotor activity, but with repeated
administration induced a significant agonist-induced inhibition of locomotor activity that
remained otherwise constant throughout the rest of the experiment. In a similar fashion,
the chronic co-administration of PD128907 along with chronic EtOH resulted in an
overall diminished expression of locomotor sensitization in those mice. It should be
noted however; that the diminished activity levels observed in both the saline- and EtOH-
treated mice was relatively constant and not augmented over the course of injection days.
Furthermore the magnitude of locomotor inhibition was the same in both EtOH- and
saline-treated mice alike. It appeared then as though the chronic agonist-induced
stimulation of the D3 receptors produced a constant subtractive effect in both PD128907-
challenged groups regardless of whether the animals received chronic EtOH or saline.
In a previous study, Mattingly et al co-administered the D3 agonist 7-OH-DPAT
during the development of sensitization to both cocaine and apomorphine (Mattingly et
al., 2001). The authors reported that acute co-administration of 7-OH-DPAT with
cocaine resulted in reduced locomotor behaviour in rats, and this agonist-induced
response had no effect on subsequent responding. However when the D3 agonist was
chronically co-administered with either cocaine or apomorphine, rats continued to
106
develop sensitization to both drugs. Therefore in agreement with our own findings,
chronic co-administration of the D3 agonist did not block sensitization to cocaine or
apomorphine, even though acute administration resulted in pronounced locomotor
inhibition.
The combined results of these agonist studies suggest a role for D3 receptor
stimulation in mediating expression as opposed to the induction of sensitization to EtOH.
Evidence to support this theory came with the observation that with chronic
administration of the D3 agonist PD128907, sensitization to EtOH still developed in
DBA mice, albeit diminished compared to mice receiving only EtOH. This agonist-
induced inhibition effect was of the same magnitude in both EtOH- and saline-treated
animals and was not augmented across injection days. These results suggest that the
observed inhibition in these mice was a result of no more than an overall subtractive
effect induced by the D3 agonist equally in both groups. Otherwise, these mice appeared
to develop normal sensitization to EtOH. On the other hand, when sensitization to EtOH
had already been induced, acute administration of the D3 agonist significantly attenuated
the sensitized response, or blocked the expression of EtOH sensitization. In this case,
there was not simply the same subtractive effect occurring equally between sensitized and
control groups, since control animals did not show significant inhibition of locomotor
activity, whereas sensitized mice did show a significant reduction in activity levels.
These data further suggest that sensitized mice may therefore be more responsive to D3
ligands than control mice. Our results here are also complementary to our previous
observations for D3 receptor blockade with an antagonist, where acute blockade with an
107
antagonist did not affect the expression of sensitization but chronic blockade of D3
receptors resulted in a failure to induce sensitization to EtOH.
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CHAPTER 7
Study 4: Examination of D1/D3 receptor interactions in a pharmacological challenge
following sensitization to ethanol
Purpose
Upon examination of the knockout mice chronically treated with EtOH in Study
1, we observed that neither D1 nor D3 knockout mice expressed behavioural sensitization
to EtOH. Furthermore these two colonies showed behavioural profiles that were more
similar to each other than they were to wild type or D2 knockout mice. It has been
previously described that, as opposed to the distribution of D2 and D3 receptors that
share complementary rather than overlapping brain distribution, D1 and D3 receptors are
highly co-expressed in both the islands of Calleja and shell portion of the NAcc. This co-
localization of D1 and D3 receptors has led researchers to be interested in the possible
functional consequences of such receptor distribution and the possibility of receptor
interactions. We were therefore interested in investigating whether or not such possible
receptor interactions would play any role in sensitization to EtOH.
A previous study reported that an acute injection of the D3-preferring agonist
PD128907 attenuated D1 agonist-induced hyperlocomotion with SKF81297, but had no
effect on mice that had been injected with a saline vehicle (Mori et al., 1997). This
previous study was performed with acute administration of both drugs and did not
investigate how chronically treated animals would behave when administered the same
D3 agonist. We were therefore interested in examining how sensitized (chronically-
109
treated) and non-sensitized animals differentially respond to both the D1 and D3 agonists.
Our hypothesis was that the D3 receptor-preferring agonist PD128907 would have no
effect on mice chronically treated with saline, but that the hyper locomotion induced by
either acute administration of a D1 agonist or ethanol sensitization would be attenuated
by the D3-preferring agonist PD128907.
Materials & Method
Procedural Overview
Sixty-four male DBA mice aged 5 weeks (Charles River, Canada) were randomly
assigned to receive chronic EtOH (2.2g/kg, i.p.) or equal volumes of saline (N=32), as
previously described. Animals sensitized to EtOH and controls were challenged 14 days
post-washout with the D1 agonist SKF81297, the D3 agonist PD128907, both drugs
together, or saline vehicle (n=8). Locomotor behaviour was observed for 30 minutes to
assess whether any changes in locomotor activity occurred upon administration of these
drugs. Since we were interested in isolating the effects of the D1 and D3 agonists
themselves, a test injection of EtOH was not included in the challenge session. Vehicle-
challenged mice (sensitized and controls) were sacrificed by cervical dislocation and their
brains removed for autoradiographic analysis in Study #5. All other mice were sacrificed
via CO2/O2 inhalation. Behavioural data obtained from the challenge was analyzed by a
2 X 2 X 2-way analysis of variance with pretreatment (Sensitized vs. Control) as the first
fixed factor, D1 challenge (SKF81297 vs. Vehicle) as the second fixed factor, and D3
challenge (PD128907 vs. Vehicle) as the third fixed factor.
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Figure 14:
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Dose-response curve for the D1 receptor agonist SKF81297: DBA mice (n=6 mice per dose) were injected intraperitoneally and tested for 90 minutes. Doses higher than and including 5 mg/kg induced significant increases in locomotor activity. 5 mg/kg was chosen as the test dose since this dose significantly altered locomotor behaviour without saturating the response curve.
111
Figure 15:
Time Course for the D1 Agonist SKF81297 Across 90-minute Test Session: DBA mice received acute intraperitoneal injections of the D1 agonist SKF81297 (0-20 mg/kg) and were observed for 90 minutes. Average locomotor activity levels were recorded in 5-minute bins across the 90-minute test session. Error bars were omitted for visual simplification. Significant increases in locomotor activity as compared to saline-treated animals were observed after 15 minutes for the 10-mg/kg dose and after 20 minutes for the 5- and 20-mg/kg doses. D1-agonist induced hyperactivity was no longer observed after 50 minutes for the 5-mg/kg dose and after 65 minutes for the 10- and 20-mg/kg doses.
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112
Drugs
The D1 receptor agonist 6-chloro-7,8-dihydroxy-2,3,4,5-tetrahydro-1-phenyl-1H-
3-benzazepine hydrobromide (SKF81297 HBr) was obtained commercially from Tocris
Bioscience (Ellisville, MO). The drug was dissolved in 0.9% physiological saline to an
injection volume of 10 ml/kg. A dose-response curve was carried out in experimentally
naïve DBA mice (n=6 mice per dose) and results can be seen in Figure 14. The time
course of the D1 agonist’s effects over the 90-minute session can be seen in Figure 15.
Results of the time course analysis revealed that the D1 agonist takes at least 15-20
minutes to have behavioural effect and therefore should be administered at least 20
minutes prior to the D3 agonist in order to see whether D3 agonist administration would
interfere with D1 agonist-induced hyperactivity.
A test dose of 5mg/kg was chosen from the dose-response curve to be used in the
experiment, since this dose was shown to reliably induce locomotor hyperactivity in
DBA mice, but was not a dose high enough to have saturated the response curve. By
choosing this dose, we would be able to determine whether sensitization to EtOH would
attenuate or potentiate the D1-agonist induced hyperactivity.
After 14 days withdrawal, sensitized and control mice received either the D1
receptor agonist SKF81297 (5 mg/kg i.p) or saline vehicle, followed 20 minutes later by a
second injection of either the D3 agonist PD128907 (0.01 mg/kg) or saline vehicle.
Locomotor activity was assessed for 30 minutes in the activity monitors.
113
Results
The results from the D1/D3 interaction experiment can be seen in Figure 16. In
both control (Figure 16A) and sensitized animals (Figure 16B), acute administration of
the D1 agonist SKF81297 resulted in an approximate 200% increase in locomotor
activity as compared to vehicle-challenged counterparts. The degree of D1-induced
hyperlocomotion did not differ between sensitized and control animals. On the other
hand, acute administration of the D3 agonist PD128907, in keeping with our results from
Experiment 3A, did not significantly affect the behaviour in non-sensitized control
animals, but did significantly inhibit locomotor activity in mice that were sensitized to
EtOH (p<0.05). In agreement with the Mori study, the D3 agonist attenuated the D1
agonist-induced hyper locomotion in both sensitized and control animals, however
differences between sensitized and control animals could not be detected.
Results from this analysis showed a significant main effect of D1 challenge [F(1,
51)=54.72, p<0.001] suggesting that the D1 agonist SKF81297 had a significant effect on
locomotor behaviour. A significant main effect was also observed for D3 challenge [F(1,
51)=10.03, p=0.003] suggesting that PD128907 also had a significant effect on the
locomotor behaviour. No significant main effect of pretreatment group was observed
(p>0.05) indicating that there was no variation between sensitized and control mice in
their responses to the drug challenges. No significant interactions were observed
(p>0.05).
114
Figure 16: Figure 16A: Control Mice
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Control + Veh/Veh ry Control + SKF/Veh Comparison of EtOH Sensitized and Control animals following administration of D1 and D3 receptor agonists: In both sensitized and control animals, the D1 agonist SKF81297 induced a 200% increase in locomotor activity compared to controls. Sensitized mice showed a greater inhibitory response to the D3 agonist PD128907 than control mice. In combination, the D3 agonist attenuated D1-induced hyperactivity, this effect was not different between sensitized and control animals.
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Figure 16B: Sensitized Mice
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115
Discussion
The results from this experiment confirmed several previous findings as well as
providing additional information into the mechanisms involved in EtOH sensitization.
First of all, our results from the control animals, those pretreated with saline and therefore
receiving acute challenges of the D1 and D3 agonists, replicated the findings reported in
the Mori study (Mori et al., 1997). In both the Japanese experiment and ours, the acute
administration of the D3 agonist PD128907 at low dose had no effect on locomotor
activity by itself, but attenuated the hyperactivity induced by the D1 agonist SKF81297.
In addition to these observations, the Japanese group also found that the κ-receptor
agonist U50,488H had no effect on D1-induced hyperactivity, suggesting that the
attenuation of locomotor hyperactivity was mediated by D3 and not κ-opioid receptors.
The authors concluded that D3 agonists, when administered at a low dose, might
negatively influence D1-receptor mediated behaviours via D3 receptors. Our data from
the control animals support this theory.
However we were also interested in observing how sensitization to EtOH might
alter any or all of these effects. We observed that mice challenged with the D1 agonist
alone, showed the same amount of locomotor activation in response to the drug whether
the mice were sensitized to EtOH or not. These results suggest that acute D1-agonist
induced hyperactivity is not mediated by the same mechanisms underlying EtOH
sensitization, since the sensitized and control mice did not differ in their responses to
SKF81297. On the other hand, although the D3 agonist did not significantly alter the
behaviour in control mice, in sensitized mice an enhanced D3-agonist induced inhibition
116
of locomotor activity was observed. This finding is in keeping with the previous
observation from Experiment 3A where mice sensitized to EtOH showed greater
inhibition of locomotor activity in response to PD128907 than control mice. These
results replicated and confirmed the previous observation that mice sensitized to EtOH
are more responsive to D3 ligands, suggesting a functional up-regulation of D3 receptors
accompanying EtOH sensitization.
In both control and sensitized animals, we observed a D3 agonist-induced
attenuation of D1 agonist-induced hyperactivity, although this effect was not as robust in
the sensitized mice as compared to the control mice. Since there was no significant main
effect of pretreatment and no interactions between pretreatment, D1 challenge and D3
challenge, we could not conclude that the sensitized mice showed significant differences
in the D3 agonist-induced inhibition of D1 agonist-induced hyperactivity as compared to
controls.
One possible reason for the lack of observed difference between sensitized and
control mice here, may be due to the fact that D1 receptors have been observed, both in
literature reports and in our previous knockout data, to play a role in mediating the
induction process of sensitization. In this experiment, mice were challenged with both
agonists after the development of sensitization to EtOH had already occurred. Therefore
it is possible that the attenuation of D1-induced hyperactivity by a D3 agonist is mediated
by neural mechanisms that are not the same as, nor have any effect on those mechanisms
that mediate the expression of EtOH sensitization, but may be more strongly related to
the neural mechanisms that underlie the induction of EtOH sensitization.
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CHAPTER 8
Study 5: Examination of changes in D3 receptor binding following sensitization to
ethanol in the mouse brain
Purpose
As described earlier, conflicting reports exist as to whether D3 receptors are up or
down-regulated with sensitization to various drugs of abuse. While some studies have
reported a down-regulation in D3 receptors following amphetamine and cocaine
sensitization, other studies have observed an up-regulation in D3 receptors following
sensitization to nicotine and L-DOPA. Behavioural data from our laboratory indicate that
mice sensitized to ethanol are more sensitive to the locomotor inhibiting effects of D3
agonists than saline-treated controls. These data suggest that an up-regulation of D3
receptors may accompany sensitization to EtOH. Furthermore in our own pilot study, D3
receptor binding was examined in EtOH-sensitized and non-sensitized mouse brains
using [3H]-7-OH-DPAT autoradiography. Results suggested an increase in D3 receptor
binding in sensitized brains compared to controls in all regions examined although the
increase was not significant. The images obtained from the 7-OH-DPAT-
autoradiography study, although demonstrating specificity in binding for the D3 receptor
over the D2 receptor, lacked visual resolution. We have since increased visual resolution
by refining the assay for, and performing a receptor binding autoradiography analysis
using [125I]-7-OH-PIPAT. Our hypothesis remained such that using this binding assay;
we would observe an increase in D3 receptor binding in the EtOH-sensitized brains
compared to saline-treated controls.
118
Materials & Method
Brain Sectioning
Sensitized and control DBA mice from Study #4 were sacrificed via cervical
dislocation and their brains were removed and immediately frozen. The subjects used
from Study #4 were only those mice challenged with saline vehicle, so that the mice used
in this analysis were only treated with EtOH or saline (n=8). Whole brains were
embedded in Tissue-Tek Optimal Cutting Temperature (O.C.T.) mounting medium
(Sakura Finetek USA Inc., Torrance, CA.) and sectioned on a Leica CM-3000 cryostat
microtome (Leica, Germany). Coronal sections were cut from rostral to caudal end at a
thickness of 10 μm. For each brain, approximately 40 representative sample sections
were taken containing regions known to be rich in D3 receptors, spanning brain regions
from the olfactory tubercle through to the cerebellum. All sections were thaw-mounted
onto glass microscope slides (Superfrost-plus slides, Fisher Scientific) and stored in
airtight containers at -80°C.
D3 Receptor Binding Autoradiography
Thawed slides were pre-incubated for 30 minutes at 30°C in 50 mM TRIS buffer
(pH 7.4) containing 100 mM NaCl, and 50 mM guanylyl-imido-diphosphate to remove
endogenous dopamine. Slides were not dried between the pre-incubation rinse and the
incubation in radio-ligand.
119
For total binding, slides were incubated for 90 minutes at room temperature in a
50 mM TRIS buffer (pH 7.0) containing 40 mM NaCl, 50 mM guanylyl-imido-
diphosphate (Sigma-Aldrich, Canada, Oakville, ON) 5 mM 1,3-di(2-tolyl)guanidine
(DTG) (Sigma-Aldrich) and 0.2 nM [125I]-7-OH-PIPAT (PerkinElmer Life Sciences,
Boston, MA). For non-specific binding, representative slides were incubated with 10
mM unlabelled 7-OH-DPAT HBr (TOCRIS Bioscience, Ellisville, MO) added to the
radio-ligand solution and incubated under the same conditions as for the total binding
sets.
At the end of the incubation period, the slides were washed three times in 50 mM
TRIS buffer (pH 7.4) at 4°C for 30 minutes each. Rinsed slides were then dipped in ice-
cold distilled water for 10 seconds to remove excess salts, and dried under a steady
stream of cool air. Dried slides along with calibrated plastic standards ([11C] Micro-
scales, Amersham, U.K.), were exposed on Kodak Biomax MR-1 film for 3 days and
then developed. The films were given to an objective third person that coded the subject
labels in order to keep the experimenter blind to the group identity of each subject.
Image Analysis
After the films were developed, densitometric analysis was performed using the
MCID software program (Imaging Research Inc., St. Catherine’s, ON, Canada). A
standard curve was generated that relates optical density to known quantities of [125I]
120
(μCi/g tissue). Images obtained from the exposed films could then be quantified for each
brain by analyzing the films with the MCID software.
Data was then sampled from over 10 brain regions (and sub-divisions thereof) in
the limbic system, midbrain and cerebellar regions including the nucleus accumbens
(NAcc), islands of Calleja (ICj), ventral pallidum (VP) and lobules 9 and 10 of the
cerebellum (Cer 9 & Cer 10). The regions were defined according to the atlas of the
mouse brain (Franklin & Paxinos, 1997). The regions chosen to be sampled, were the
regions that were most strongly labeled to a "blind" reader and appeared consistently
throughout multiple brain sections sampled, suggesting target-specificity in the radio-
ligand binding. Furthermore, these were the same regions that corresponded to the brain
areas that have been reported to be rich in D3 dopamine receptors. Several samples were
taken from each region and a mean value was generated for that region within each brain.
When all brains were sampled, the subjects were de-coded and sorted into their
respective treatment groups. A mean value for radio-ligand binding was generated per
region, per group. Data were analyzed using a t-test for each region separately. Mean
values for sensitized and control groups were compared independently at each of the
regions sampled. Group treatment was the independent variable and the mean value of
radio-ligand reported in μCi /g tissue was the dependent variable. Statistical significance
was taken at p<0.05.
121
Results
Sample autoradiogram images generated from the [125I]-7-OH-PIPAT
autoradiography can be viewed in Figure 17. These images demonstrate the D3 receptor
protein distribution in the mouse brain. We found that the images obtained from this
analysis showed D3 binding signals that were in agreement with previous literature
reports for the distribution of the D3 receptor in vivo. Our signal was also reliably
displaced by the addition of another un-labelled D3 ligand (7-OH-DPAT). We were
therefore confident that the assay performed was specific to D3 receptor binding.
Results from the [125I]-7-OH-PIPAT autoradiography binding analysis are
summarized in Table 2. The table includes a list of all brain regions sampled, the mean
binding-density values reported in μCi/g of tissue for both sensitized and control animals,
as well as the p-value results from each t-test performed. Results of the t-test analyses
indicated no significant differences in binding densities between sensitized and control
brains in any of the regions tested (p>0.05). These data indicate that changes in D3
receptor binding density do not accompany sensitization to EtOH.
122
Figure 17:
A B
C D
E F
G
NAcc-Ant-Pol
Olf-TubICj ICj
ICj-MNAcc-Sh
CPu-DL
CPu-VM
VP
CPu-DM
CPu-VL
Thal-CM
Hippocampus
Habenula
Mam-Thal-tr
SNc
IP
Mam-Teg-tr
Cer-Lob-9
Cer-Lob-10
Sample autoradiogram images obtained from [125I-]7-OH-PIPAT receptor binding autoradiography demonstrating D3 receptor protein distribution in the mouse brain: Panels A-G above are sample autoradiograms showing D3 receptor protein distribution in the brains of mice. The images above were generated by [125I]-7-OH-PIPAT receptor binding autoradiography. D3 receptor binding densities were highest in the limbic forebrain region, including ICj, Olf tub, and NAcc-Ant Pol &-Sh. Moderate binding was also observed in the VP, Thal-CM, SNc and Cer-Lob9 & 10. Very low to no signal was detected in the CPu-DL or Cortex. Visual resolution of images was greatly enhanced using [125I]-7-OH-PIPAT as the radioligand in comparison to [3H]-7-OH-DPAT. Abbreviations for brain regions are defined in the legend for Table 2.
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Table 2:
Region Sensitized (μCi/g Tissue) Control (μCi/g Tissue) p-value Olf Tub 0.72 ± 0.06 0.76 ± 0.02 0.45 NAcc-Ant-Pol 0.92 ± 0.08 1.10 ± 0.05 0.16 NAcc-Sh 1.53 ± 0.09 1.53 ± 0.06 0.96 NAcc-C 0.83 ± 0.07 0.83 ± 0.06 0.96 ICj 2.29 ± 0.12 2.28 ± 0.04 0.90 ICj-M 2.50 ± 0.07 2.38 ± 0.10 0.39 CPu-Ant 0.34 ± 0.04 0.42 ± 0.07 0.42 CPu-DL 0.11 ± 0.01 0.11 ± 0.01 0.90 CPu-DM 0.20 ± 0.01 0.19 ± 0.01 0.61 CPu-VL 0.21 ± 0.01 0.22 ± 0.01 0.58 CPu-VM 0.45 ± 0.01 0.43 ± 0.01 0.39 VP 1.09 ± 0.11 1.01 ± 0.07 0.54 Thal-CM 0.24 ± 0.02 0.22 ± 0.03 0.56 Mam-Thal-Tr 0.27 ± 0.03 0.27 ± 0.02 0.98 Mam-Teg-Tr 0.21 ± 0.02 0.19 ± 0.01 0.54 SNc 0.40 ± 0.03 0.32 ± 0.03 0.06 IP 0.30 ± 0.04 0.25 ± 0.01 0.24 ATg 0.21 ± 0.01 0.21 ± 0.01 0.99 Cer-10 0.18 ± 0.01 0.17 ± 0.01 0.44 Cer-9 0.22 ± 0.01 0.21 ± 0.02 0.61
Summary of Results from [125I]-7-OH-PIPAT Binding Autoradiography: Listed above are the mean binding-density values in μCi/g of tissue for sensitized and control animals, as well as the p-value results from the t-test analyses. No significant differences in D3 binding densities were observed between sensitized and control mice for any region sampled. Abbreviations for brain regions are as follows: Olfactory tubercle (Olf Tub), nucleus accumbens (NAcc)-anterior pole (Ant-Pol), -shell (Sh), -core (C), islands of Calleja (ICj), -major (M), Caudate and Putamen (CPu), -anterior (Ant), -dorsolateral (DL), -dorsomedial (DM), -ventrolateral (VL), -ventromedial (VM), ventral pallidum (VP), thalamus-central medial nucleus (Thal-CM), mammillo-thalamic tract (Mam-Thal-Tr), mammillo-tegmental tract (Mam-Ted-Tr), substantia nigra pars compacta (SNc), interpeduncular nucleus (IP), anterior tegmentum (ATg), cerebellum (Cer), -lobule 10 (10), -lobule 9 (9).
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Discussion
In two of our previous studies, we observed that mice sensitized to EtOH
demonstrated an enhanced response to D3 receptor ligands. This observation, coupled
with supporting reports in the literature suggested that a functional up-regulation of D3
receptors might accompany sensitization to EtOH. We were therefore interested in
examining brain changes associated with this putative up-regulation of D3 receptors and
if possible to obtain images of such changes.
In a previous study we examined the expression of D3 mRNA in the brains of
sensitized and non-sensitized mice using in situ hybridization. Results of this experiment
showed that no changes in D3 mRNA expression were associated with sensitization to
EtOH as compared to control animals (S. J. Harrison, 2004). These data indicated that if
a functional up regulation of the D3 receptor system is associated with EtOH
sensitization, the changes that occur are not detectable at the level of mRNA
transcription, suggesting that EtOH sensitization does not result in increased (or
decreased) production of new D3 receptors.
Following our D3 mRNA study, we were interested in examining whether
changes in D3 receptor protein binding were associated with behavioural sensitization to
EtOH. Therefore we performed a receptor binding autoradiography assay using [3H]-7-
OH-DPAT as the D3-preferring radioligand. Results from this analysis demonstrated an
overall trend towards increased D3 receptor binding in sensitized brains as compared to
controls, however these changes were not statistically significant. Furthermore, the
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images obtained from this assay lacked visual resolution. The only brain regions we
could view included the NAcc and islands of Calleja. For sample images generated from
this binding analysis and the D3 mRNA analysis, please refer to Figure 1, Chapter 2, on
page 14. In order to clarify whether changes in D3 receptor binding do in fact
accompany sensitization to EtOH, it was important to perform the D3 receptor binding
autoradiography using a radioligand that would generate images with higher visual
resolution and therefore refine the image analysis.
We refined the technique for and performed the D3 receptor binding
autoradiography using [125I]-7-OH-PIPAT instead of [3H]-7-OH-DPAT as the
radioligand. The reasons for this change were twofold. First of all 7-OH-PIPAT is a D3
receptor-preferring agonist with higher affinity for the D3 receptor over the D2 receptor
than 7-OH-DPAT (Stanwood et al., 2000a; Waters et al., 1993). However, and more
importantly 125I is a far more sensitive radiolabel than 3H, and the images obtained from
this assay using [125I]-7-OH-PIPAT provided those with far greater visual resolution than
the previously generated images using [3H]-7-OH-DPAT. Sample autoradiograms from
both analyses can be seen in Figures 1 and 17. The images obtained from this more
recent assay allowed a more comprehensive analysis to be performed on additional brain
regions including the ventral pallidum, substantia nigra and archicerebellum. In the end,
we were confident that the images obtained from this analysis would be able to
demonstrate changes in D3 receptor binding density if they were, in fact, associated with
sensitization to EtOH. Results of our analysis demonstrated clearly that sensitization to
EtOH is not accompanied by significant changes in D3 receptor binding.
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Given that we have observed that mice sensitized to EtOH show enhanced
sensitivity to D3 ligands, a putative functional up regulation of the D3 system remains to
be determined. Because changes in D3 receptor binding were not found to be associated
with sensitization to EtOH does not necessarily mean that no other changes occur
elsewhere within the functional pathway related to dopamine D3 receptors. The receptor
binding analysis detects changes that occur at the cellular membrane level, but cannot
give any information about what may be happening within the internal cellular or second
messenger systems. D3 receptors are coupled to G proteins and until recently, the signal
transduction pathway associated with the D3 receptor has remained poorly understood
(Missale et al., 1998; Shafer & Levant, 1998). Recent reports, however have indicated
that the D3 receptor may be associated with the Akt/glycogen synthase kinase 3 (GSK3)
pathway (Beaulieu et al., 2007; Chen et al., 2007).
Akt is a serine/threonine kinase that is a downstream target of dopamine receptor
signaling and is inhibited/dephosphorylated in response to direct and indirect dopamine
receptor agonists (Beaulieu et al., 2005). Inactivation of Akt in response to dopaminergic
agents results in the activation of GSK3, which in turn contributes to the expression of
dopamine-associated behaviours (Beaulieu et al., 2004). In a recent paper, Beaulieu et al
reported that D3 receptors participate in this signaling pathway, where D3 KO mice show
a reduced sensitivity of Akt-mediated signaling in response to dopaminergic drugs
(Beaulieu et al., 2007). Furthermore Chen et al reported a decrease in Akt/GSK3 signal
in the limbic forebrains of D3 KO mice compared to wild types following sensitization to
methamphetamine (Chen et al., 2007). These reports indicate that not only is the D3
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receptor associated with this signaling pathway, but also that changes in this pathway can
be induced in response to dopaminergic agents as well as following sensitization.
It is conceivable that changes in the neural mechanisms underlying sensitization
to EtOH may not necessarily occur at the level of the membrane bound receptor, but
further downstream in this signal transduction pathway. Our observation that animals
sensitized to EtOH are more responsive to D3 ligands than controls suggests that an up
regulation of the D3 pathway has occurred. Because we did not observe a change in D3
receptor binding between sensitized and control animals, it is more likely that the
heightened behaviour observed in sensitized mice is a result of changes that we could not
detect at the receptor binding level, but perhaps further downstream within this
Akt/GSK3 pathway.
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CHAPTER 9
General Discussion
As outlined in the introduction we examined the role of the dopamine D3 receptor
in behavioural sensitization to EtOH by taking a three-pronged approach. We first used
D3 knockout mice to examine how the overall loss of the D3 receptor affected the
behavioural responses in mice chronically treated with EtOH as compared to wild type
mice as well as D1 and D2 knockouts. We also examined amphetamine sensitization in
D3 knockout and wild type mice. Our second approach was to employ pharmacological
interventions with a D3 receptor antagonist and agonist to temporarily block and
stimulate D3 receptors at different stages of EtOH sensitization. Additionally, we
investigated potential D1/D3 receptor interactions following EtOH sensitization using D1
and D3 receptor agonist challenges. Finally we examined brain changes associated with
D3 receptors following sensitization to EtOH by measuring D3 receptor binding using
[125I]-7-OH-PIPAT autoradiography.
D3 Knockout Mice
In examining the role of the dopamine D3 receptor in EtOH sensitization, our data
support a strong role for the D3 receptor in mediating this response, most likely through a
modulatory mechanism in conjunction with D1 and to a lesser extent, D2 receptor
subtypes. This suggestion is supported by the observation that mutant mice lacking the
D3 receptor were resistant to the sensitizing effects of ethanol when compared to their
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wild type counterparts. The resistance to ethanol sensitization in D3 knockout mice was
more similar to the D1 knockout colony of mice than it was to the D2 knockout colony.
Where hypoactive D2 knockout mice showed a small increase in response to repeated
administration of EtOH, both hyperactive D1 and D3 knockout colonies failed to show
any change in response to repeated EtOH. These results suggest that D1 and D3
receptors play a more important role in mediating ethanol sensitization in mice than D2
receptor sub-types.
It is of importance to note that the results of our studies apply specifically to
EtOH sensitization. D3 KOs developed sensitization to amphetamine in a manner similar
to wild type mice. The finding that D3 KO mice were resistant to EtOH but not
amphetamine sensitization suggests that the observations made in these studies are not
necessarily common to all drugs of abuse and may, to some extent, be unique to EtOH
sensitization.
It is likely that the difference in response of the D3 KOs to EtOH and
amphetamine is due in part to the mechanistic differences between the two drugs of
abuse. Although both drugs have been reported to ultimately lead to increases in
accumbal extracellular dopamine they do so by different mechanisms (Di Chiara &
Imperato, 1988; Koob, 1992; Wise, 2002). Acute EtOH, working via GABAa receptors,
disinhibits the mesolimbic pathway, stimulating the release of dopamine in the NAcc (Di
Chiara & Imperato, 1985; Imperato & Di Chiara, 1986), whereas amphetamine acts by
both blocking dopamine reuptake transporters in the NAcc as well as reversing the
direction of the transporters, facilitating dopamine release into the NAcc (Carboni et al.,
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1989). In Chapter 2 (Table 1) we observed that rodents sensitized to drugs acting on the
dopamine reuptake transporter, such as cocaine and amphetamine, demonstrated an
overall down-regulation of D3 receptor function (Chen et al., 2007; Chiang et al., 2003;
Richtand et al., 2003; Wallace et al., 1996). On the other hand, rodents sensitized to
drugs that do not act upon dopamine transporters but either act to directly increase
accumbal dopamine by stimulating dopamine receptors such as L-DOPA or indirectly
lead to increased dopamine release such as nicotine and ethanol, showed an overall up-
regulation of D3 receptor function (Guillin et al., 2003; S. J. Harrison, 2004; Le Foll et
al., 2003; van Kampen & Stoessl, 2003). Given this trend in the literature reports, it is
possible that these mechanistic differences may dictate either the modulatory strength or
even the direction of modulatory effect that the D3 receptor plays in mediating the
behavioural sensitization response to different drugs of abuse, i.e., whether D3 receptor
function is up- or down-regulated following sensitization.
Since we observed in Study 1 that D3 KOs demonstrated behavioural
sensitization to amphetamine but not EtOH, we would suggest that the D3 receptor plays
a more important role in mediating sensitization to EtOH, indicating a feature of this
model that distinguishes it from amphetamine sensitization. Furthermore given that
ethanol increases accumbal dopamine without acting on dopamine reuptake transporters,
we would predict that ethanol sensitization results in an up- rather than down-regulation
of D3 receptor function. It is important to note that since we observed no changes in D3
receptor binding associated with EtOH sensitization, however, this putative increase in
D3 receptor function may involve mechanisms that are not necessarily reflected at the
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level of the membrane bound receptor, but perhaps are mediated by other intracellular
mechanisms.
Pharmacological Intervention
In a series of pharmacological challenges, we suggested that chronic D3 receptor
blockade interferes with the induction, but not expression of EtOH sensitization, whereas
D3 receptor stimulation can block the expression, but not induction of sensitization to
EtOH. This was shown in a series of experiments where acute D3 receptor blockade had
no effect on the expression of EtOH sensitization once it had been induced; however,
chronic blockade of D3 receptors eliminated the induction of ethanol sensitization but
only in mice that were susceptible to the stimulating effects of EtOH. These findings
were in agreement with our previous observation that D3 KO mice were resistant to
EtOH sensitization. On the other hand, D3 receptor stimulation with an agonist
interfered with the expression of sensitization once it had been induced, but chronic D3
receptor stimulation did not eliminate the progressive increase in response to repeated
EtOH administration. Taken together these data suggest that D3 receptors may play a
modulatory role in sensitization to ethanol where stimulation of D3 receptors mediates
the expression and D3 receptor blockade mediates the induction of sensitization.
The observation that D3 receptor stimulation interferes with the expression of
EtOH sensitization is in agreement with what researchers know about the expression of
behavioural sensitization to other drugs of abuse. First of all a large number of D3
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receptors are located in the shell portion of the NAcc, a terminal projection site of the
mesolimbic dopamine pathway (Levesque et al., 1992; Sokoloff et al., 1990). As
previously described, the NAcc has been reported to mediate many of the neurochemical
changes that are associated with the expression but not induction process of behavioural
sensitization to psychostimulants (Cador et al., 1995; Perugini & Vezina, 1994; Vezina &
Stewart, 1990). When drugs of abuse such as amphetamine and cocaine are locally
infused into the NAcc instead of the VTA, an increase of locomotor activity is observed
in a manner similar to acute administration; however this increased level of responding is
not augmented across time indicating that the NAcc is more important for the expression
but not induction of sensitization (Cador et al., 1995).
In our third study, we examined how acute and chronic D3 receptor stimulation
would affect EtOH sensitization. The results of this study showed that acute
administration of the D3 receptor agonist PD128907 blocked the expression of EtOH
sensitization by inhibiting locomotor hyperactivity in sensitized but not control mice.
When the D3 agonist was chronically co-administered with EtOH, an overall agonist-
induced inhibition of locomotor activity was observed throughout the experiment. This
inhibitory effect however, remained constant and was not augmented across time. And
even though a constant subtractive effect elicited by the D3 agonist was observed across
injections days, the development of locomotor sensitization, indicated by a progressive
increase in locomotor activity was still observed in all mice receiving chronic EtOH.
These results point heavily towards a role for D3 receptor activation in inhibiting the
expression but not induction of sensitization to EtOH. Since D3 receptors are present in
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the NAcc, it is likely that receptor activation in this brain region, resulting in locomotor
inhibition, mediates this response. Such a role may exist to serve as a protective
mechanism to act, as previously proposed by Richtand, as a brake on the
hyperlocomotion induced by the synergistic actions of D1 and D2 receptors during
behavioural sensitization.
As previously described in Chapter 1, induction of sensitization to
psychostimulants occurs primarily in the VTA and is mediated by D1 receptors (Bjijou et
al., 1996; Cador et al., 1995; Kalivas et al., 1993; Kalivas & Stewart, 1991; Pierce &
Kalivas, 1997; Vezina, 1996). If the neural mechanisms underlying sensitization to
psychostimulants are the same as those underlying sensitization to ethanol, then a role for
the D3 receptors in the induction process may seem more puzzling, given their primary
localization in the NAcc, rather than the VTA. However as we observed in Study 1, D3
KO mice showed a resistance to developing EtOH sensitization but not to amphetamine
sensitization, indicating an important difference in sensitization processes mediated by
ethanol versus psychostimulants. Furthermore one literature report has demonstrated
another very important difference between EtOH and psychostimulant sensitization.
Where sensitization to psychostimulants is associated with an enhanced release of
dopamine in the NAcc (Kalivas & Duffy, 1990; Kalivas & Stewart, 1991; Pierce &
Kalivas, 1997; Robinson et al., 1988), it has recently been reported that behavioural
sensitization to ethanol is not associated with elevated release of dopamine in the NAcc
(Zapata et al., 2006), even though acute ethanol has been reported to elevate dopamine in
the NAcc (Di Chiara & Imperato, 1985, 1988; Imperato & Di Chiara, 1986). The
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authors suggested that dissociation exists between the mechanisms mediating acute
locomotor stimulation to EtOH and the development of sensitization to EtOH. The
observations reported by Zapata et al., in addition to our own indicate that the neural
mechanisms underlying sensitization to ethanol may be quite different from those
underlying sensitization to psychostimulants.
The above-mentioned observed and reported differences between EtOH and
amphetamine sensitization however, do not directly address the induction processes
mediating EtOH sensitization. In our knockout study, the permanent inactivation of the
D3 receptor made it difficult to ascertain whether the lack of D3 receptor function had an
impact on either the development or expression of sensitization to EtOH. Furthermore,
with respect to findings of Zapata et al., previously reported elevated dopamine levels in
the NAcc are thought to mediate the more enduring neural changes that are associated
with the expression of behavioural sensitization. Zapata et al., recognized that the
induction process mediating EtOH sensitization can still be associated with an increased
EtOH-evoked firing of dopamine neurons in the VTA (Brodie, 2002) and suggested a
possible temporal dissociation between EtOH-induced excitation of dopaminergic cell
bodies in the VTA and decreased release of dopamine from the terminals in the NAcc. In
other words, Zapata et al., still look towards the VTA as an important site for mediating
the initiation process of EtOH sensitization.
Since D3 receptors exist primarily in the NAcc and other terminal sites of the
mesolimbic dopamine pathway, it is not immediately clear how D3 receptor blockade can
interfere with the induction of EtOH sensitization, when their presence is lacking from
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the VTA. Reports of whether the D3 receptor exists in the VTA as well as its putative
role in this brain region remain somewhat controversial in the literature.
Sokoloff’s group originally found that D3 mRNA signals could not be detected in
the VTA or susbstantia nigra (SN) using in situ hybridization; however, D3 signals were
detected in these areas using northern blot and polymerase chain reaction (PCR) analysis
after 32 but not 25 cycles. Furthermore, when these dopaminergic neurons were
degenerated using 6-hydroxydopamine (6-OHDA-a toxin that selectively ablates
catecholaminergic neurons), there was a marked reduction in the PCR-generated D3
mRNA signal in both the SN and VTA, suggesting presynaptic localization of the signal
(Sokoloff et al., 1990). Because of these and other findings the authors then proposed a
potential role for the D3 receptor as a presynaptic autoreceptor in these regions. Since
then however, several other studies have had difficulty in confirming the existence of D3
mRNA in midbrain neurons (Landwehrmeyer et al., 1993; Meador-Woodruff et al.,
1994; Richtand et al., 1995).
When investigating the localization of the D3 receptor protein itself, original
binding studies using [3H]-7-OH-DPAT autoradiography revealed a very low signal in
the SN but no presence of D3 receptor protein in the VTA was reported (Levesque et al.,
1992). Furthermore when D3 receptor binding techniques were refined and [125I]-7-OH-
PIPAT autoradiography generated images with higher visual resolution, additional brain
regions were reported to contain D3 receptors but the VTA was not one of these reported
regions (Stanwood et al., 2000a). However a different group using [125I]-7-OH-PIPAT
binding did report a D3 signal in the VTA (Diaz et al., 2000). In this report however, the
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authors point toward an area that they refer to as the border between the SN and VTA and
refer to this region in general as the SN/VTA with no further delineation between the
regions. In contrast to this Diaz et al. report but not to the Stanwood study, the images
generated in our laboratory do not reveal a D3 signal in the VTA even though a clear
signal is present in the SN (see Figure 14F).
In Study 2 the observation that chronic D3 receptor blockade in DBA mice
prevents the induction of sensitization was quite striking. It would seem unlikely that
such a strong effect could be mediated simply by the presence of D3 receptors in the
VTA, when the presence of the receptor protein in this region is so dubious. It is more
likely that the prevention of induction by D3 receptor blockade occurs through a more
complex mechanism, perhaps one involving interactions with the D1 receptors.
In a study performed by Schwartz et al., hemiparkinsonian rats were produced
with unilateral lesions of dopamine neurons using 6-OHDA (Schwartz et al., 1998a).
This ablation of dopaminergic input resulted in a marked reduction of D3 receptor
density in the denervated NAcc (Levesque et al., 1995; Schwartz et al., 1998a; Schwartz
et al., 1998b). These hemiparkinsonian rats were then sensitized to L-DOPA, which
produced not only a recovery but also an overexpression of D3 receptors in the
denervated NAcc but not on the control side. Additionally an ectopic overexpression of
D3 receptors was observed in the denervated dorsal striatum, an area that does not
normally contain D3 receptors. This ectopic induction of D3 receptors in the striatum
had several characteristics. First of all it was D1-mediated since the ectopic
overexpression of D3 receptors in the striatum could be replicated with D1 agonists and
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blocked by D1 antagonists. The increase in D3 receptor density in this region across time
paralleled the development of behavioural sensitization to L-DOPA. Both the ectopic
induction of D3 receptors as well as the development of behavioural sensitization to L-
DOPA could be blocked by the D3 antagonist nafadotride. And finally the ectopic
induction of D3 receptors in the striatum, but not the normotic overexpression of D3
receptors in the NAcc was transient and disappeared after cessation of drug treatment.
The observation in the Schwartz study that these neurochemical changes were
both transient and D1-mediated agrees with what researchers know to hold true for the
induction process of behavioural sensitization to many drugs of abuse. That the induction
of behavioural sensitization to L-DOPA was blocked by the D3 antagonist nafadotride in
the Schwartz study is in agreement with our observation that the D3 antagonist U99194A
blocked the induction of behavioural sensitization to EtOH. It should also be noted that
in the Schwartz study, the overexpression of D3 receptor density in the NAcc remained
stable, perhaps reflecting a neurochemical change in the NAcc that is more persistent and
thus mediating the expression rather than induction of behavioural sensitization to L-
DOPA. It is also interesting to note that this change in the D3 receptors in the denervated
side was reflective of an overall upregulation of D3 receptors, even though changes in the
D3 receptor binding were not apparent on the control side. Given the similarities
between our some of our observations and those made by Schwartz et al., it may be
possible that the role for the D3 receptor in the induction of behavioural sensitization to
EtOH occurs via similar mechanisms, which will be discussed further in a proposed
model of receptor induction.
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D1/D3 Interactions
In Study 4, an examination of D1/D3 receptor interactions, it was confirmed that
sensitized mice have a greater response to D3 agonist administration than saline-treated
control animals, suggesting an increased sensitivity to D3 ligands following sensitization
to ethanol. This finding confirmed our previous observation from Study 3 that sensitized
mice were more responsive to D3 agonist administration than control animals, again
suggesting that EtOH sensitization results in an upregulation of D3 receptor function.
There was no difference in response to D1 agonist-induced hyperactivity between
sensitized and non-sensitized groups. Furthermore, the D3 agonist attenuated the D1
agonist-induced hyperactivity in both sensitized and control mice to a similar degree.
Even though the D3 agonist appeared to interact with the D1-induced behaviour,
no differences were observed between sensitized and control animals in response to the
combined challenges. These results could suggest that the neural mechanisms underlying
D3 agonist-induced attenuation of D1 agonist-induced hyperactivity are different from
the neural mechanisms that underlie sensitization to EtOH. Alternatively the lack of
observed difference in response between sensitized and control mice in this experiment
may be due to the fact that these challenges took place after induction of sensitization had
already occurred. As previously discussed, the D1 and D3 receptors appear to interact
more strongly during the induction phase of sensitization and in this experiment we
challenged the mice when they were already expressing sensitization to EtOH. It is
possible that differences in sensitivities to D1/D3 interactions may only be detected
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during the initiation of EtOH sensitization and may no longer show augmentations once
sensitization is expressed.
Brain Analysis
In our fifth study we attempted to image an anatomical substrate for the putative
increase in D3 receptor function as a result of EtOH sensitization. We examined
potential brain changes associated with EtOH sensitization by measuring D3 receptor
binding in sensitized and control brains using [125I]-7-OH-PIPAT autoradiography.
Results of this analysis did not reveal any changes in D3 receptor binding levels between
sensitized and control mice. This finding does not necessarily mean that no changes in
D3 receptor function are associated with EtOH sensitization, but only that these changes
are not detectable at the level of the membrane bound receptor and its ability to bind
ligands. However a neural substrate that may reflect an up regulation of D3 receptor
function has yet to be identified.
It is possible that an up regulation of D3 receptor function takes place at the
intracellular level, downstream from the receptor itself, and it is likely that these changes
may occur in the Akt/GSK3 signal transduction pathway. Modifications in the Akt and
GSK3 proteins can be readily measured and quantified using Western immunoblot
analysis. It would therefore be an interesting and practical area for future research to
examine and quantify potential changes in this Akt/GSK3 pathway following
sensitization to EtOH. Such changes, if any, may help to identify a neural substrate for a
functional change in the D3 receptor pathway in response to chronic EtOH.
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The D3 Receptor in Behavioural Sensitization to Ethanol: A Proposed Model of Receptor Induction
Based on the above, it seems possible to suggest a model whereby the
development and expression of behavioural sensitization to EtOH is mediated, at least in
part by changes in D3 receptors, some of which may be similar to those described by
Schwartz and could occur as follows. Initially, repeated EtOH administration, through
GABAergic disinhibition of the mesolimbic pathway, leads to stimulation of D1
receptors in the VTA. Since there are no dopaminergic afferents to the VTA, the only
source of dopamine here is dendritic release (Ranaldi & Wise, 2001). There are no D1
receptors on dopaminergic neurons however; D1 receptors in the VTA are located on the
terminals of glutamate and GABA afferents to the VTA (M. B. Harrison et al., 1990; Lu
et al., 1997). Thus decreases in GABAergic inputs evoked by ethanol can lead to
dendritic release of dopamine in the VTA. When stimulated by dendritic dopamine, D1
receptors can affect local glutamate and GABA concentrations, which can, in turn control
activation of dopaminergic neurons arising from the VTA (Kalivas & Duffy, 1995; M.
Starr, 1987). This overall activation of the mesolimbic dopamine pathway results in an
upregulation of D3 receptors. This upregulation of D3 receptors is likely to occur in the
shell portion of the NAcc and could also possibly coincide with the induction of D3
receptor expression in another region such as the dorsal striatum (possibly by co-
activation of the nigrostriatal pathway).
This induction or overexpression of D3 receptors may be transient in the striatum
(or elsewhere) and more persistent in the NAcc. This transient induction of D3 receptors
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may be necessary for the initiation of behavioural sensitization to EtOH, such that
blockade of D3 receptors during induction would result in a failure to develop EtOH
sensitization as we observed in Experiment 2B in DBA mice. Since these putative
changes are transient and reversible then blockade of D3 receptors after the induction
process has taken place would have no effect on EtOH sensitization as we observed in
our Experiment 2A, when acute challenge with the D3 antagonist had no effect on the
expression of sensitization after it had been induced. The induction of EtOH sensitization
then would be dependent on both D1 and D3 receptors and therefore D1 and D3 KO mice
would not, as we observed in Experiment 1B, develop behavioural sensitization to EtOH.
More persistent changes in D3 receptor functionality may take place in the NAcc
and would therefore mediate the expression of sensitization. An upregulation of D3
receptor function in the NAcc would therefore leave sensitized mice more responsive to
D3 agonists than control mice. Since D3 receptor stimulation leads to inhibition of
locomotor activity, then sensitized mice would demonstrate greater locomotor inhibition
in response to D3 agonists than control mice as we observed in Experiment 3A as well as
Experiment 4, when mice sensitized to EtOH showed a greater D3 agonist-induced
inhibition of locomotor activity than control mice. Thus D3 receptor stimulation with an
agonist would block the expression of behavioural sensitization to EtOH once it had been
induced. Since this putative upregulation of D3 receptor function would be persistent, we
would not expect a response to chronic D3 agonist administration to augment across time
and remain fixed, as we observed in Experiment 3B, when chronic D3 agonist
administration resulted in a constant inhibitory effect across injection days.
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It is important to note that the induction of D3 receptor overexpression arising
from D1 activation in the VTA may not necessarily depend upon release in the NAcc.
Diaz et al., reported that during development D3 receptor expression is dependent upon
innervation of dopamine neurons (Diaz et al., 1997). Interestingly, lesioning dopamine
cells, blocking axonal transport, or decreasing the firing rate of dopaminergic neurons can
block the expression of D3 receptors, but D3 receptor expression is unaffected by
removal of dopamine or its related co-transmitters (Levesque et al., 1995). These authors
suggested that D3 receptor regulation is under the influence of a separate anterograde
trophic factor released from dopamine neurons. This factor has since been identified as
brain derived neurotrophic factor (BDNF) (Guillin et al., 2001; Guillin et al., 2003;
Sokoloff et al., 2002). The release of BDNF from activated dopaminergic cells into the
NAcc could then be responsible for the induction of D3 overexpression in response to
ethanol, irrespective of dopamine release in the NAcc. This theory would not conflict
with the reported observation that sensitization to EtOH is not necessarily accompanied
by an increased dopamine release in the NAcc (Zapata et al., 2006). Thus the initiating
step of key importance in this model is the activation of mesolimbic dopamine cells
arising from the VTA, but not necessarily the release of dopamine in the NAcc.
In this suggested model the D3 receptor would mediate the induction and
expression of behavioural sensitization through temporally and perhaps even
anatomically distinct mechanisms. Chronic EtOH would lead to a transient increase in
D3 receptor expression responsible for the initiation of behavioural sensitization to
EtOH, which may either precede or coincide with a persistent up-regulation of D3
143
receptor function that may in turn participate in the expression of EtOH sensitization.
Whether or not any or all of these changes in D3 receptors occur in the NAcc, or whether
they may occur in another brain region such as the dorsal striatum is purely speculative.
These regions were included in this current model based on the reported findings of
Schwartz et al., where a similar pattern of D3 expression occurred in those regions during
sensitization to L-DOPA. Since L-DOPA, a metabolic precursor to dopamine, also acts
to directly stimulate the nigrostriatal pathway, the observation of increased D3 receptor
induction in the striatum may occur in response to chronic L-DOPA but may not
necessarily occur in response to chronic EtOH.
However in our own experiments we did not observe a change in D3 receptor
binding in either of these regions following sensitization to EtOH. This may have
occurred for several reasons. First of all, we only examined the changes in D3 receptor
binding after sensitization had been induced. We did not examine brain changes that
were occurring during the initiation of sensitization, and therefore we may have missed a
window of opportunity to study these potential changes in D3 receptor expression during
this phase. Additionally, as we have previously discussed, although the increased
response to D3 ligands suggests that a functional up-regulation of D3 receptors has
occurred, this functional up-regulation may not necessarily be evident at the level of the
membrane-bound receptor, but perhaps by another intracellular mechanism. Thus it is
also possible that either transient or persistent increases in D3 receptor function resulting
from chronic EtOH may not necessarily be detectable with receptor binding
autoradiography. Finally in the Schwartz study, both the reported normotic
144
overexpression of D3 receptors in the NAcc and ectopic induction of D3 receptors in the
striatum were only observed in the dopamine depleted side, but not the control side of the
brain. If these changes in D3 receptor expression do in fact accompany sensitization to
either L-DOPA or ethanol, then it is possible that these changes may be masked by the
presence of an otherwise functional dopamine system and may require the depletion of
dopamine in order to unmask and fully observe such changes.
Since activation of the VTA is a necessary function for the above described set of
events to occur, we would predict that drugs of abuse that act to increase VTA activity,
either through excitatory inputs such as nicotine or through disinhibition of the VTA such
as EtOH, would set in motion this sequence pattern, resulting in an upregulation of D3
receptor function. On the other hand, drugs of abuse that act primarily in the NAcc, such
as cocaine and amphetamine which block dopamine transporters, may not necessarily
lead to this same pattern of response and may even lead to a down regulation of D3
receptor function through a different, otherwise unidentified mechanism.
145
DopamineGlutamateGABAAcetylcholine
Figure 18:
mPFC
Dorsal Striatum2) Potential transient, ectopic
over-expression of D3Rs(Induction)
The D3 Receptor in Behavioural Sensitization to Ethanol: A Proposed Model for Receptor Induction: Many drugs of abuse can lead to D1R stimulation in the VTA either by increasing excitatory inputs or removing inhibitory inputs. EtOH is thought to work by the latter mechanism, by disinhibiting GABAergic inputs from the striatum. Excitation of the VTA, leads to increased activity of the mesolimbic pathway (and possibly nigrostriatal pathway) having two potential consequences on D3Rs: a normotic over expression of D3Rs in the NAcc-Sh and a possible ectopic over expression of D3Rs in the dorsal striatum. These events may be transient and mediated only during the induction phase of EtOH Sens. After drug cessation, over expression of D3 receptors may no longer be detectable but D3Rs in the NAcc remain functionally up regulated. This functional up-regulation may be long-lasting, and thus reflect at least one neural mechanism involved in mediating the expression of EtOH Sens.
Of particular importance to this model is that VTA activation is a necessary step in precipitating these events. Drugs of abuse that act primarily in the NAcc by blocking the DA reuptake transporter, such as amphetamine and cocaine, would essentially by-pass this preceding step and would therefore not be expected to set in motion the same pattern of response. However other drugs of abuse such as nicotine, which activates the VTA via excitatory cholinergic inputs, would be expected to follow the same pattern of activation as described above for EtOH.
Heavy lines in the diagram highlight pathways discussed in the body of the text; lighter lines indicate other possible pathways of reward circuitry not necessarily presented in the described model.
NAcc(Sh)2) Normotic over-expression
of D3Rs (Induction)
3) Increased D3R functionremains stable after drugwithdrawal (Expression)
PPTg/LDTg
VTA1) Various drugs ofabuse can lead toD1R stimulation
+
+
+
+
_
_
+/-
+/-
SNc
+/-
XEtOH disinhibits
GABAergic inputsto VTA
Increased activation of mesolimbic pathway
146
One caveat of this model is that although we observed that chronic blockade of
D3 receptors blocks the induction of behavioural sensitization to EtOH, we only observed
this in DBA but not C57 mice. In Experiment 2B, DBA mice developed clear
sensitization to chronic EtOH that was blocked by the chronic co-administration of the
D3 antagonist U99194A. However C57 mice were resistant to EtOH sensitization, but
developed a progressive increase in response to the D3 antagonist itself. This difference
in response between strains raises several issues that might be considered when applying
the above-described model. C57 and DBA mice differ in a number of ways previously
described in Chapter 3. Of particular importance, C57 mice are less susceptible to EOH
sensitization (Phillips et al., 1994; Phillips et al., 1995) and have overall lower levels of
basal D3 receptors than DBA mice even though D1 receptor expression is the same in
both strains (R. K. McNamara et al., 2006a). If D3 receptors are necessary for the
induction of EtOH sensitization, then it is entirely possible that C57 mice, demonstrating
lower levels of D3 receptors in the NAcc, are more resistant to EtOH sensitization than
DBAs due to lower basal D3 receptor expression.
It may be possible that the induction of sensitization to EtOH requires a certain
threshold level of D3 receptors to be recruited before the induction of EtOH sensitization
takes place. If this is the case, then perhaps C57 mice with lower basal levels of D3
receptors are more resistant to EtOH sensitization because these threshold numbers are
not met, even after chronic EtOH induces a putative up-regulation of D3 receptors. The
up-regulation induced in these mice may not reach threshold levels in order to initiate
EtOH sensitization. It could therefore be theoretically possible that the basal level of D3
147
receptor expression may determine whether or not induction of EtOH sensitization takes
place.
As previously described in Chapter 2, Neil Richtand has proposed that
behavioural sensitization results from an imbalance of D3 receptors relative to D1 and D2
receptors (Richtand, 2006; Richtand et al., 2003; Richtand et al., 2001). He further posits
that an imbalance of D3 receptors in certain individuals may serve as a genetic marker for
a predisposition towards developing behavioural sensitization and that an individual’s
basal D3 receptor expression may account for a reason why some individuals are more
likely to be susceptible to drug abuse while others are more resistant. C57 and DBA
mice differ with respect to their sensitivity to EtOH as well as their basal levels of D3
receptors, lending support to Richtand’s hypothesis. It could therefore be conceivable
that the model suggested above applies only to those individuals that are susceptible to
developing behavioural sensitization to EtOH, whereas individuals that are resistant to
EtOH sensitization may not necessarily demonstrate this same pattern of response.
Whether a pre-existing imbalance in the levels of D3 receptors is the determining
factor in whether sensitization to ethanol develops or whether alterations in D3 receptors
balance are a result of ethanol sensitization is difficult to determine. It is entirely possible
that given the amount of within-strain variation in behavioural responses to ethanol, that a
similar within-strain variability may exist with respect to the expression of D3 receptors.
Therefore in one respect, those animals expressing EtOH sensitization may have higher
levels of D3 receptors than those that do not sensitize to EtOH. Alternatively, higher
levels of D3 receptors may be a consequence of the development of sensitization to
148
ethanol. The only way to be certain whether a pre-existing level of basal D3 receptors
determines whether sensitization to EtOH takes place would be to quantify these
receptors in individual mice before drug treatment has begun, and then after drug
treatment has taken place in these same individuals. Such a task would involve the use of
in vivo imaging tools, which although currently exist for human subjects, are currently
not suitable for accurately imaging the small brains of rodents, especially mice. Until
such technology exists, this question will remain unanswered.
149
Experimental Limitations and Future Directions
One of the limitations of these studies was that an anatomical substrate for
changes in D3 functionality was not identified. Despite the increased sensitivity to D3
ligands demonstrated by sensitized mice, alterations in D3 receptors were not observed
following D3 receptor binding autoradiography. Although we had refined this technique
to our satisfaction, the tool itself was most likely not useful for detecting the functional
changes associated with the D3 receptor on an intracellular level. As previously
described changes in D3 receptor function may not necessarily have been reflected by
changes in D3 receptor binding at the membrane level. Examination of a more
downstream mechanism such as the intracellular signaling pathway associated with the
D3 receptor may help to identify an anatomical mechanism that leads to a functionally
more responsive system. It may therefore be useful to examine modifications in the
Akt/GSK3 pathway using Western immunoblot analysis in order to detect changes in the
signaling pathway associated with the D3 receptor following sensitization to EtOH.
Conversely potential transient changes in D3 receptor binding may have indeed
taken place during the induction phase of sensitization, however we did not perform our
receptor binding analysis until after EtOH sensitization was fully expressed and after 2
weeks withdrawal from the drug. It is therefore possible that a window of opportunity to
examine immediate and transient alterations in D3 receptor binding was missed in our
experiments. It may be useful in the future to examine whether an increase in D3
receptor binding accompanies the initiation of sensitization to EtOH. Therefore a time-
course study could be of use to compare how D3 receptor binding (in addition to changes
150
in the Akt/GSK3 pathway) may progress throughout the initiation and expression phases
of EtOH sensitization when brains are removed at different time points throughout the
course of drug administration. Furthermore, given that the D3 agonist PD128907 did not
produce an augmented response across the course of our experiments, it may also be of
interest to extend the length of time that this drug is administered in order to see whether
or not an augmented response develops after long-term administration of the D3 agonist.
Such an experiment would certainly have clinical relevance when considering prescribing
D3 agonists to patients over a long period of time.
As previously described, studies using transgenic mice are not without their
limitations. Knockout mice provide a useful tool to examine how permanent loss of a
particular receptor will result in phenotypic differences between mice lacking that
receptor and wild type mice that possess the functional receptor in question. However
mice that have lived their entire lives without a particular receptor can adapt to this loss
by developing compensatory mechanisms to account for the overall deficits that may
occur in the system. There is always a risk in working with knockout mice that the
differences observed in these knockout mice compared to wild types may not be directly
due to the loss of the receptor in question, but may be an artifact that has arisen through
the development of an otherwise unidentified compensatory mechanism. Therefore
researchers using transgenic mice should always keep this caveat in mind and design
experiments that can be accompanied with other techniques to examine receptor function
in order to provide converging lines of evidence.
151
In our current set of experiments we employed pharmacologic interventions to
examine how temporary and reversible blockade of D3 receptors could compare to our
results from the D3 knockout study. Our overall findings from the D3 antagonist
experiments were in agreement with our knockout study providing encouraging results
that the effects observed were in fact, due to the loss of the receptor and not to
compensatory mechanisms. However pharmacological studies are also not without their
limitations. The introduction of any drug into the system can bring with it non-specific
changes that are not relevant to the receptor in question, but may bring about unrelated
alterations in behaviour. Furthermore, drugs that are not completely selective for one
particular receptor may interact with other receptors, which again, may lead to
behavioural changes not selectively associated with the receptor in question. Finally
without the addition of receptor occupancy data, which currently can only be performed
in ex vivo studies, it is difficult to ascertain whether full receptor blockade has been
achieved with antagonist administration and for how long the blockade will last.
Although this temporary and reversible characteristic of pharmacological intervention is
one of its strengths, it can also be a limiting factor when it is uncertain how many
receptors are blocked and for how long the blockade will last. Alternative tools to gene
targeting are also available including antisense knockdown of dopamine receptors
(Sibley, 1999) as well as viral-mediated RNA interference of dopamine function
(Hommel et al., 2003), which can provide the selectivity of receptor loss, that may not be
provided by pharmacological intervention, and the reversibility of receptor function not
associated with knockout mice.
152
Although the results of our pharmacological interventions support the results from
our knockout study and agree with several literature reports, providing a good framework
upon which to base future studies, we used only one D3 agonist (PD128907) and one D3
antagonist (U99194A) in our studies. Additionally, we deliberately chose to use a low
dose of the antagonist in order to stay within a D3-selective range. Consequently our
observations may have been the result of partial rather than full blockade of D3 receptors
in vivo. Although we cannot be certain whether full saturation of D3 receptors would be
necessary to alter our findings, it might be interesting to examine whether a higher dose
of the D3 antagonist would yield results different from those observed our experiment.
The D3 ligands used in our experiments were chosen because they are
commercially available, thus easily obtained and have been widely studied in a number of
experiments, providing a wide literature base for comparing drug effects. Although these
factors may be attractive when designing pilot experiments, the compounds used in these
studies are neither the most selective compounds known to date, nor clinically relevant.
There are other, more recently developed D3 ligands that are now becoming more widely
studied and have a higher selectivity for the D3 receptor. It would be interesting to
compare how other D3 ligands such as the more selective agonist PHNO, the clinically
relevant pramipexole, or the highly selective antagonist SB-277011A produce responses
that may be similar or unique from the D3 ligands used in these studies. It could also
prove interesting to see how the D3 partial agonist BP-897 shapes the pharmacological
profile obtained from these experiments.
153
The observations made in these studies were limited to the locomotor activating
or inhibiting effects of EtOH and the D3 ligands. Although locomotor sensitization in
rodents has been used as a model for compulsive drug-seeking behaviour our results do
not speak directly to the rewarding effects of these drugs. The fact that D3 agonists
attenuate the locomotor activating effects resulting from chronic EtOH administration
does not necessarily mean that the rewarding effects of these drugs are diminished in
these animals as well. We have not tested how D3 receptor agonists and antagonists
could influence the rewarding or motivating effects of EtOH either before, during or after
sensitization. Perhaps an area for future research would be to examine how sensitized
and non-sensitized mice develop conditioned place preference to EtOH, voluntary EtOH
consumption, preference or self administration, and how D3 ligands may shape any or all
of these behaviours. If the rewarding or motivational effects of these D3 ligands agree
with our locomotor data, then these drugs could potentially be used as
phamacotherapeutic agents to treat alcohol abuse in humans.
The results obtained from these studies have brought to light some important
findings that could have potential clinical implications. The finding that D3 receptor
agonists and antagonists can have opposing modulatory effects on the development and
expression of EtOH sensitization could be extended for use in the treatment and/or
prevention of alcohol abuse in humans. In theory, if the development of EtOH
sensitization is a predictive indicator of alcohol abuse, then D3 antagonist administration
might be useful in the prevention of alcohol abuse in those individuals who are
susceptible or genetically predisposed to such behaviours. However the identification of
154
individuals that may be susceptible to alcohol abuse before abuse has begun, may be
difficult. Also the administration of chronic D3 receptor antagonists to these individuals
may prove to be an impractical, if not unethical task. On the other hand, administration
of D3 agonists to block the expression of behavioural sensitization to EtOH may be a
theoretically less appealing but more practical option to treat alcohol abuse once it has
developed.
The results of these studies are important because a better understanding of the
role for the D3 receptor in sensitization to ethanol may help not only to identify some of
the underlying neural mechanisms of behavioural sensitization to drugs of abuse, but also
to help in the identification of treatment strategies for patients that may be susceptible to
drug abuse and relapse to substance abuse.
155
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Appendix 1A
DBA Mouse ID Group Hab 1 Hab 2 Hab 3 Acute Inj #3 Inj #5 Inj #7 Inj #9 TEST M02 EtOH 836 1170 1012 729 2280 4391 2627 1697 2240
M03 EtOH 1458 943 1393 1019 3149 2958 2627 3314 3765
M04 EtOH 1087 1333 1500 1949 3535 2133 5113 2084 2028
M05 EtOH 1860 1236 663 1056 2291 498 1517 2639 3181
M06 EtOH 2199 1749 2098 1275 1855 4014 3686 4629 3636
M08 EtOH 1748 1171 1308 542 2102 2096 3261 3269 2777
M10 EtOH 2166 1638 1104 997 2693 1732 1231 1296 991
M11 EtOH 1510 1469 832 602 Deceased Deceased Deceased Deceased Deceased
M12 EtOH 2105 1480 1569 483 2798 1355 5667 3284 4146
M13 EtOH 2210 2008 892 1303 3113 4779 1335 2674 4080
M15 EtOH 1483 1371 1254 1528 2106 1193 1613 1972 5154
M16 EtOH 1454 793 911 1360 3689 3618 4992 2698 5794
M18 EtOH 1973 1186 1300 1688 2573 4237 5296 4734 5862
M19 EtOH 1396 1510 1477 2094 2210 2196 1104 1307 2600
M20 EtOH 1650 1098 1441 375 2273 1943 2013 3557 1841
M21 EtOH 2108 2166 1993 1094 1391 3035 3172 2879 3367
M23 EtOH 2109 2047 2254 850 1414 2904 3417 3812 2512
M24 EtOH 1462 1199 1475 1832 4033 3672 5162 4590 2681
M25 EtOH 1188 733 562 116 636 403 74 292 871
M26 EtOH 1270 1374 750 56 543 698 298 623 617
M27 EtOH 2722 1959 1782 504 1474 1370 1912 1387 2615
M29 EtOH 2273 2346 1307 2311 2595 3226 2277 2993 3148
M30 EtOH 2231 1623 933 817 2118 2996 3233 3106 2796
M31 EtOH 1383 1357 1451 582 2088 3219 3423 676 723
M01 Saline 1353 1303 768 664 859 836 674 623 981
M07 Saline 1781 1335 458 381 793 837 775 782 1716
M09 Saline 2029 1394 1123 1059 1344 1089 1439 898 1443
M14 Saline 1849 1226 1091 836 733 1013 1102 856 1673
M17 Saline 2010 1579 1483 1064 997 987 834 642 1624
M22 Saline 1472 1856 1936 1629 1279 1088 883 698 654
M28 Saline 1518 1709 1482 865 772 1101 1134 1098 2311
M32 Saline 2261 843 1116 702 788 832 1069 823 3985
Raw Ambulatory Count Data for DBA mice during EtOH sensitization: Individual scores for each DBA mouse on all session days. Each score represents the total number of ambulatory counts (horizontal beam breaks) made by each mouse within the 15-minute test session. One mouse died after the acute ethanol injection. After Injection #9 mice were sub-divided so that the 8 highest responders (top 30%) were designated as sensitized and plotted against saline-treated controls in Figure 2A.
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Appendix 1B
C57 Mouse ID Group Hab 1 Hab 2 Hab 3 Acute Inj #3 Inj #5 Inj #7 Inj #9 Test M33 EtOH 1691 1434 750 1162 1511 2106 3105 1942 2571
M34 EtOH 1786 1365 1040 866 575 4075 4198 3798 3200
M36 EtOH 1283 1327 757 889 1775 2336 2361 2285 1962
M37 EtOH 1658 1636 978 413 575 1476 1688 1624 560
M39 EtOH 1692 1788 1249 1344 2049 2471 2421 2665 2976
M42 EtOH 1248 919 729 710 986 1358 1861 1507 816
M43 EtOH 1439 1525 684 1227 1276 1504 2291 2515 1963
M45 EtOH 1551 871 584 732 1279 2171 2449 2357 748
M46 EtOH 2095 2463 1411 584 1214 791 734 1176 1275
M47 EtOH 1880 1911 1135 845 365 1940 2587 1564 1276
M49 EtOH 1754 1278 830 813 1571 624 1825 655 709
M50 EtOH 2458 1449 1284 605 725 489 478 423 717
M51 EtOH 1897 1413 1061 559 Deceased Deceased Deceased Deceased Deceased
M52 EtOH 1809 1217 734 590 212 1323 1423 1721 1525
M53 EtOH 2067 2003 1430 616 1145 626 1536 2925 624
M54 EtOH 2408 2399 1701 640 1507 2862 2480 1937 2074
M56 EtOH 1733 1539 1065 836 2413 1107 3126 1251 1465
M57 EtOH 1800 1978 1709 1365 2386 2580 2831 2134 2277
M58 EtOH 2448 1863 2031 911 920 2340 360 2013 1642
M60 EtOH 1730 1012 617 208 1058 1439 954 1247 530
M61 EtOH 1679 1342 566 938 1675 3385 2699 2971 2418
M63 EtOH 1731 1293 1242 1008 2165 2268 1562 2397 2010
M40 EtOH 1265 1175 717 741 1064 1017 896 1071 802
M35 Saline 1267 2096 640 481 952 1092 578 704 1031
M38 Saline 1851 1121 558 473 796 1059 1125 751 1661
M41 Saline 1504 1292 806 688 895 662 557 1051 534
M48 Saline 1444 900 1013 884 997 710 684 538 869
M55 Saline 1831 1602 1592 1029 842 936 1003 763 782
M59 Saline 1353 976 1028 920 1142 1292 679 1017 746
M62 Saline 1920 1446 1239 1172 1384 1139 1233 914 939
M64 Saline 1781 1192 1029 807 823 845 628 871 667
Raw Ambulatory Count Data for C57 mice during EtOH sensitization: Individual scores for each C57 mouse on all session days. Each score represents the total number of ambulatory counts (horizontal beam breaks) made by each mouse within the 15-minute test session. One mouse died after the acute ethanol injection. After Injection #9 mice were sub-divided so that the 8 highest responders (top 30%) were designated as sensitized and plotted against saline-treated controls in Figure 2B. Overall C57 mice had a lower response to chronic EtOH than DBAs.
181
Appendix 2A PCR reaction components, cycling conditions and primer sequences for the dopamine D1 receptor knockout mice genotyping protocol from Jackson Laboratories. Drd1atm1Jcd, Version 2 Gene & Allele Details Allele Symbol Drd1atm1Jcd
Allele Name targeted mutation 1, John Drago Common Name(s) D1-; D1A-; Dlr-; Mutation Made By John Drago, National Institutes of HealthStrain of Origin 129S4/SvJae ES Cell Line Name J1 ES Cell Line Strain 129S4/SvJae Gene Symbol and Name Drd1a, dopamine receptor D1A Chromosome 13 Products Gel Image
+/+ = 350bp +/- = 350bp & 172bp -/- = 172bp
Cycling Conditions
Cycling Reaction A Step Temp Time Note 1 94 °C 3 min 2 94 °C 20 sec 3 64 °C 30 sec *-0.5 C per cycle 4 72 °C 35 sec Go to step 2, 12 times 5 94 °C 20 sec
6 58 °C 30 sec
182
7 72 °C 35 sec Go to step 5, 25 times 8 72 °C 2 min 9 10 °C
Separated by gel electrophoresis on a 1.5% agarose gel. Primers oIMR0158 5'- CTG AAT
GAA CTG CAG GAC GA -3' Tm = 60.0 °C [Tm calc'd by Primer 0.5]
20-mer A=7, C=4, G=6, T=3
neo primer
oIMR0159 5'- ATA CTT TCT CGG CAG GAG CA -3' Tm = 60.0 °C [Tm calc'd by Primer 0.5]
20-mer A=5, C=5, G=5, T=5
neo primer
oIMR0190 5'- AAA gTT CCT TTA AgA TgT CCT -3' Tm = 51.6 °C
21-mer A=6, C=4, G=3, T=8
Drd1A Drago forward primerJ.D.27
oIMR0191 5'- Tgg Tgg CTg gAA AAC ATC AgA -3' Tm = 63.9 °C
21-mer A=7, C=3, G=7, T=4
Drd1A Drago reverse primer JD.26. 350 bp product w\ oIMR190.
All of the above information can be found on the Jackson Laboratories Website for the genotyping protocol for the D1 receptor knockout mouse, stock #002322. http://jaxmice.jax.org/strain/002322.html
183
Appendix 2B PCR reaction components, cycling conditions and primer sequences for the dopamine D2 receptor knockout mice genotyping protocol from Jackson Laboratories. Drd2tm1, Version 1 Gene & Allele Details Allele Symbol Drd2tm1Low
Allele Name targeted mutation 1, Malcolm J Low Common Name(s) D2-; D2KO; Drd2-; Mutation Made By Malcolm Low, Oregon Health Sciences University Strain of Origin 129S2/SvPas ES Cell Line Name D3 ES Cell Line Strain 129S2/SvPas Gene Symbol and Name Drd2, dopamine receptor 2 Chromosome 9 Products Gel Image+/+ = 105 bp +/- = 280 bp + 105 bp -/- = 280 bp
N/A
Cycling Conditions
Cycling Reaction A Step Temp Time Note 1 94 °C 3 min 2 94 °C 20 sec 3 64 °C 30 sec *-0.5 C per cycle 4 72 °C 35 sec Go to step 2, 12 times 5 94 °C 20 sec 6 58 °C 30 sec 7 72 °C 35 sec Go to step 5, 25 times
8 72 °C 2 min
184
9 10 °C
Separated by gel electrophoresis on a 1.5% agarose gel. Primers oIMR0013 5'- CTT ggg Tgg AgA
ggC TAT TC -3' Tm = °C
20-mer A=3, C=3, G=8, T=6
neo generic primer
oIMR0014 5'- Agg TgA gAT gAC Agg AgA TC -3' Tm = 54.0 °C
20-mer A=7, C=2, G=8, T=3
neo generic primer
oIMR0991 5'- TgT gAC TgC AAC ATC CCA CC -3' Tm = 56.0 °C
20-mer A=5, C=8, G=3, T=4
Drd2 wildtype primer
oIMR0992 5'- gCg gAA CTC AAT gTT gAA gg -3' Tm = 56.0 °C
20-mer A=6, C=3, G=7, T=4
Drd2 wildtype primer
All of the above information can be found on the Jackson Laboratories Website for the genotyping protocol for the D2 receptor knockout mouse, stock #003190. http://jaxmice.jax.org/strain/003190.html
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Appendix 2C PCR reaction components, cycling conditions and primer sequences for the dopamine D3 receptor knockout mice genotyping protocol from Jackson Laboratories. Drd3tm1Dac, Version 1 Gene & Allele Details Allele Symbol Drd3tm1Dac
Allele Name targeted mutation 1, Domenico Accili Common Name(s) D3-; delta148(+30); Mutation Made By Sara Fuchs, National Institutes of HealthStrain of Origin 129S4/SvJae ES Cell Line Name J1 ES Cell Line Strain 129S4/SvJae Gene Symbol and Name Drd3, dopamine receptor 3 Chromosome 16 Products Gel Image
+/+ = 137 bp +/- = 137 bp and 200 bp -/- = 200 bp
Cycling Conditions
Cycling Reaction A Step Temp Time Note 1 94 °C 3 min 2 94 °C 20 sec 3 64 °C 30 sec* *-0.5 C per cycle 4 72 °C 35 sec Repeat steps 2-4, 12 times. 5 94 °C 20 sec
6 58 °C 30 sec
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7 72 °C 35 sec Repeat steps 5-7, 25 times. 8 72 °C 2 min 9 4 °C
Separated by gel electrophoresis on a 1.5% agarose gel. Primers oIMR0215 5'- gCA gTg gTC
ATg CCA gTT CAC TAT CAg -3' Tm = 68.9 °C
27-mer A=6, C=7, G=7, T=7
Drd3 KO. amplifies a 137bp band from the wildtype allele with oIMR216.
oIMR0216 5'- CCT gTT gTg TTg AAA CCA AAg Agg AgA gg -3' Tm = 70.2 °C
29-mer A=9, C=4, G=10, T=6
Drd3 KO. amplifies a 137bp band from the wildtype allele with oIMR215.
oIMR0217 5'- Tgg ATg Tgg AAT gTg TgC gAg -3' Tm = 65.4 °C
21-mer A=4, C=1, G=10, T=6
Drd3 KO. amplifies a 200bp band from the targeted allele with oIMR218.
oIMR0218 5'- gAA AgC AAA gAg gAg Agg gCA ggA C -3' Tm = 68.4 °C
25-mer A=11, C=3, G=11, T=0
Drd3 KO. amplifies a 200bp band from the targeted allele with oIMR217.
All of the above information can be found on the Jackson Laboratories Website for the genotyping protocol for the D3 receptor knockout mouse, stock # 002958. http://jaxmice.jax.org/strain/002958.html
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