by Maurice Yen Fu Shen A thesis submitted in comformity with … · 2018. 2. 19. · Maurice Yen Fu...
Transcript of by Maurice Yen Fu Shen A thesis submitted in comformity with … · 2018. 2. 19. · Maurice Yen Fu...
The role of the dopamine D1-D2 receptor heteromer in brain reward function: Relevance
to drug addiction and depression.
by
Maurice Yen Fu Shen
A thesis submitted in comformity with the requirements for the degree of Doctor of Philosophy
Graduate Department of Pharmacology and Toxicology
University of Toronto
© Copyright by Maurice Yen Fu Shen (2015)
ii
The role of the dopamine D1-D2 receptor heteromer in brain reward function: Relevance
to drug addiction and depression.
Maurice Yen Fu Shen, Doctor of Philosophy,
Department of Pharmacology and Toxicology, University of Toronto, 2015
Abstract
We have identified a novel dopamine-mediated signaling complex, the D1-D2 receptor
heteromer, which couples to Gq protein to elicit phospholipase C-mediated intracellular Ca2+
release. Activation of the D1-D2 heteromer was further shown to modulate the expression of
proteins implicated in reward in the nucleus accumbens, such as calcium calmodulin kinase II
(CaMKII) and brain derived neurotrophic factor (BDNF), a finding which may be of relevance
in neuropsychiatric disorders that exhibit altered reward perception such as addiction and
depression. Therefore, the purpose of current study was to investigate a role for the D1-D2
heteromer in reward-related behaviours using animal models of cocaine addiction and
depression. The results show that D1-D2 heteromer stimulation by agonist SKF 83959
attenuated addiction-related behaviours including cocaine conditioned place preference (CPP),
the maintenance and reinstatement of cocaine self-administration (SA), and the expression of
locomotor sensitization to cocaine, whereas D1-D2 heteromer inactivation by a selective
disrupting peptide TAT-D1 consistently augmented cocaine-induced behaviours, whereas a
control scrambled peptide had no such effects. Moreover, D1-D2 heteromer stimulation alone
was shown to induce conditioned place aversion, whereas its selective inactivation induced
CPP. In the tests for depression, D1-D2 heteromer stimulation by SKF 83959 induced a pro-
depressive and anxiogenic state in the forced swim test, novelty-induced hypophagia, and the
elevated plus maze, effects that were abolished by pretreatment with TAT-D1 peptide. Lastly,
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it was shown that the D1-D2 heteromer exerts dual modulation on neuronal activity in certain
regions of brain, increasing activity in the NAc, and exerting tonic inhibition in the prelimbic
and infralimbic cortex, orbitofrontal cortex, lateral habenula, and the thalamus, which likely
reflects the dual excitatory/inhibitory capabilities of the GABA/Glu-co-expressing D1R/D2R
MSNs. Collectively, these findings indicate that the D1-D2 heteromer may be a single
molecular entity that could bidirectionally modulate brain reward function depending on its
state of activation. Such an unprecedented function of a receptor complex makes the D1-D2
heteromer an attractive and novel therapeutic target for cocaine addiction and major depression,
two reward-related neuropsychiatric disorders that are currently without effective treatments.
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Acknowledgments
First and foremost, I would like to extend my most sincere gratitude to my supervisor
Dr. Susan George for giving me the opportunity to pursue a doctoral degree in her laboratory.
Her expertise and guidance has been the key to my success during my studies, and I believe the
lessons that I have learned from her will be immensely beneficial to my future career. It has
been an honor and privilege to work under her supervision.
I would like to thank the funding agencies that have supported this research: the
Canadian Institute of Health Research and the National Institure of Health.
Thank you to members of my committee, Dr. Paul Fletcher and Dr. Jose Nobrega, for
providing me with different and insightful perspectives to this work. Particularly, I would like
to thank Dr. Fletcher for lending me the equipment and his expertise for the drug self-
administration study.
I would also like to thank members of the George lab, Dr. Melissa Perreault, Dr.
Ahmed Hasbi, Dr. Gabriela Novak, Dr. Vaneeta Verma, Dr. Brian O’Dowd, Tuan Nguyen,
Theresa Fan, Ryan To, Marco Cheung, and David Yeung for creating a friendly and
inspirational work environment. I loved every moment in the lab with them around. Special
thanks to Dr. Perreault for guiding me with regards to all aspects of my study, ranging from
experimental design, grant applications, to manuscript submissions. My doctoral study
wouldn’t have been as smooth and successful as it has been without her invaluable support.
Also special thanks to Theresa for her technical expertise on animal surgeries, and to Zhao-xia
Li and Tony Ji for their technical support during my time at the Center for Addiction and
Mental Health for the self-administration work.
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Last but not least, I would like to express my utmost gratitude to my family and friends
for always being there for me, and never doubting my ability to finish a prestigious degree.
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TABLE OF CONTENTS
i. Abstract 4
ii. Acknowledgments 6
iii. List of Publications 8
iv. List of Figures 9
v. List of Abbreviations 11
1. General Introduction 16
1.1 G protein-Coupled Receptors (GPCRs) 16
1.1.1 GPCR Structure 16
1.1.2 GPCR Activation 18
1.1.3 G-proteins 19
1.1.4 GPCR Oligomerization 20
1.2 Dopamine Receptors 22
1.2.1 The Genetic and Structural Properties of Dopamine Receptors 22
1.2.2 Dopamine Receptor Expressions 24
1.2.3 Dopamine Receptor Signalling 29
1.2.3.1 The Gαs-cAMP-PKA-DARPP-32 Pathway 29
1.2.3.2 The MEK-ERK1/2 Pathway 32
1.2.3.3 Calcium Signalling via Gαq-coupling 33
1.2.4 Dopamine Receptor Oligomerization 35
1.3 The Dopamine D1-D2 Receptor Heteromer: A Novel Dopamine Receptor Coupled
to Gq-PLC-Ca2+
36
1.3.1 The D1R and D2R Form Heteromeric Complex in vitro 38
1.3.2 Expression of the D1-D2 Heteromer in the Brain 40
1.3.3 A Unique Functional Role for the D1-D2 Heteromer 42
1.3.4 Regulation of the D1-D2 Heteromer Signalling 44
1.3.5 Generation of a Novel Antagonist for the D1-D2 Heteromer: The TAT-D1
Peptide 45
1.3.6 Physiological Effects of the D1-D2 Heteromer 46
1.4 The Role of Dopamine in the Brain Reward System 49
1.4.1 The Neurocircuitry of the Brain Reward System 50
1.4.1.1 The Nucleus Accumbens (NAc) 52
1.4.1.2 The Ventral Tegmental Area (VTA) 54
1.4.2 The Role of Mesolimbic Dopamine in Depression and Drug Addiction 58
1.5 The D1-D2 Heteromer as a Modulator of Brain Reward Function: Relevance to
Addiction and Depression 60
1.5.1 CaMKII 61
1.5.2 BDNF 62
1.5.3 GAD67 63
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1.6 Research Rational and Objectives 65
1.6.1 Hypotheses 66
2. The role of the dopamine D1-D2 receptor heteromer in cocaine reward. 67
3. Rapid anti-depressant and anxiolytic activities following dopamine D1-D2
receptor heteromer inactivation. 110
4. The dopamine D1-D2 receptor heteromer exerts tonic inhibitory effect on the
expression of amphetamine induced locomotor sensitization. 139
5. Regulation of c-fos expression by the dopamine D1-D2 receptor heteromer. 164
6. General Discussion 184
6.1 The D1-D2 heteromer is a negative modulator of psychostimulant and natural
reward 185
6.2 D1-D2 heteromer stimulation induced a depressive-like state 188
6.3 The D1-D2 heteromer modulation of reward: A potential role for BDNF and
GAD67 191
6.4 The D1-D2 heteromer is a novel molecular substrate for aversions 195
6.5 Significance and Conclusion 197
7. Future Directions 200
8. References 203
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List of Publications
1. Shen M.Y.F., Perreault M.L., Fan T., George S.R. The dopamine D1-D2 receptor
heteromer exerts a tonic inhibitory effect on the expression of amphetamine-induced
locomotor sensitization. Pharmacol Biochem Behav. 2015 Jan;128:33-40.
2. Perreault M.L.*, Shen M.Y.F.*, Fan T., George S.R. Regulation of c-fos expression by the
dopamine D1-D2 receptor heteromer. Neuroscience. 2015 Jan 29;285:194-203. *These
authors contributed equally to this work
3. Shen M.Y.F.*, Perreault M.L.*, Bambico F.R., Jones-Tabah J., Cheung M., Fan T.,
Nobrega J.N., George S.R. Rapid antidepressant and anxiolytic actions following
dopamine D1-D2 heteromer inactivation. Eur. J. Neuropsychopharm. Manuscript under
revision. *These authors contributed equally to this work
4. Hasbi A., Perreault M.L., Shen M.Y.F., Zhang L., To R., Fan T., Nguyen T., Ji X.,
O'Dowd B.F., George S.R. A peptide targeting an interaction interface disrupts the
dopamine D1-D2 receptor heteromer to block signaling and function in vitro and in vivo:
effective selective antagonism. FASEB J. 2014 Nov;28(11):4806-20.
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List of Figures
Figure 1 The basal ganglia circuitry in rats. 26
Figure 2 A potential third neuronal pathway within the basal ganglia. 28
Figure 3 The regulation and function of DARPP-32 phophorylations. 31
Figure 4 The activation of the MEK-ERK pathway is dependent on the
stimulation of both the D1R and the NMDAR. 34
Figure 5 The D1R and the D2R interact via electrostatic interactions to form the
D1-D2 heteromer. 39
Figure 6 The neurocircuitry of the brain reward system. 51
Figure 7 The proposed role of the D1-D2 heteromer in the regulation of brain
reward function. 199
Figure I-1 The effects of D1-D2 heteromer stimulation and inactivation on basal
conditioned place preference. 80
Figure I-2 The effects of D1-D2 heteromer stimulation and inactivation on cocaine-
induced CPP. 81
Figure I-3 The effect of Cdk5 inhibitor roscovitine on SKF 83959-induced CPA. 82
Figure I-4 The effects of D1-D2 heteromer stimulation and inactivation on
locomotion induced by acute and repeated cocaine treatment. 84
Figure I-5 The effects of D1-D2 heteromer stimulation and inactivation on the
expression of cocaine-induced locomotor sensitization. 86
Figure I-6 The effect of D1-D2 heteromer stimulation on cocaine self-
administration under the FR5 schedule. 88
Figure I-7 The lever-pressing behaviour during extinction training. 90
Figure I-8 The effects of D1-D2 heteromer stimulation and inactivation on cocaine-
induced reinstatement. 91
Figure I-9 The effect of D1-D2 heteromer stimulation on cue-induced
reinstatement. 93
Figure II-1 The effects of D1-D2 heteromer stimulation or inactivation on the
latency to and total immobility time in the forced swim test in rats. 122
Figure II-2 D1-D2 heteromer stimulation or inactivation had bidirectional effects on
anxiety-like behaviours in the elevated plus maze. 124
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Figure II-3 D1-D2 heteromer stimulation or inactivation had bidirectional effects on
anxiety-like behaviours in the novelty-induced hypophagia test. 126
Figure II-4 The effects of TAT-D1 peptide treatment on anhedonia-like reactivity
induced by chronic unpredictable stress. 128
Figure II-5 The effects of TAT-D1 peptide treatment on anxiety-like reactivity
induced by chronic unpredictable stress. 130
Figure III-1 The effects acute D1-D2 heteromer stimulation and inactivation on basal
and amphetamine-induced locomotor activity. 148
Figure III-2 The effects of subchronic D1-D2 heteromer stimulation and inactivation
on basal and amphetamine-induced locomotor activity. 150
Figure III-3 The time course of basal or amphetamine-induced locomotor responses
following D1-D2 heteromer stimulation or inactivation during the course
of repeated treatment.
153
Figure III-4 The effects of D1-D2 receptor heteromer stimulation and inactivation on
the expression of amphetamine-induced locomotor sensitization. 155
Figure IV-1 Regulation of c-fos expression in rat nucleus accumbens by the
dopamine D1-D2 receptor heteromer. 171
Figure IV-2 Grooming induced by SKF 83959 in rats is mediated by the dopamine
D1-D2 heteromer. 173
Figure IV-3 Regulation of c-fos expression in rat caudate putamen by the dopamine
D1-D2 receptor heteromer. 174
Figure IV-4 Effects of dopamine D1-D2 heteromer disruption on c-fos
immunoreactivity in rat cortex. 176
Figure IV-5 Effect of dopamine D1-D2 heteromer disruption on c-fos expression in
rat lateral habenula and thalamus. 178
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List of Abbreviations
7-TM Seven Transmembrane
AAV Adeno-associated Virus
AC Adenylyl Cyclase
AH α-helical
AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
AP-1 Activator Protein-1
BAC Bacterial Artificial Chromosomes
BDNF Brain-derived Neurotrophic Factor
BLA Basolateral Amygdala
BRET Bioluminescence Resonance Energy Transfer
CaMKII Calcium Calmodulin Kinase II
cAMP Cyclic Adenosine Mono-phosphate
Cdk5 Cyclin-dependent Kinase 5
CeA Central Amygdaloid Nucleus
CK1 Casein Kinase 1
CK2 Casein Kinase 2
Co-IP Co-immunoprecipitation
CP Caudate Putamen
CPA Conditioned Place Aversion
CPP Conditioned Place Preference
CREB cAMP Response Element Binding Protein
DAG Diacylglycerol
DARPP-32 Dopamine and cAMP-regulated Protein Phosphatase (32kDa)
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DAT Dopamine Reuptake Transporter
DREADDs Designer Receptors Exclusively Activated by a Designer Drug
DYN Dynorphin
ECL Extracellular Loop
ENK Enkephalin
EPN Entopeduncular Nucleus
ER Endoplasmic Reticulum
ERK Extracellular Signal-related Kinase
FR Fixed-Ratio
FRET Fluorescent Resonance Energy Transfer
GABA γ-Aminobutyric Acid
GAD Glutamate Decarboxylase
GAP GTPase Accelerating Protein
GDP Guanine Nucleotide Di-phosphate
GP Globus Pallidus
GPCR G Protein-coupled Receptor
GRK G Protein-coupled Receptor Kinase
GSK-3 Glycogen Synthase Kinase 3
GTP Guanine Nucleotide Tri-phosphate
HEK Human Embryonic Kidney
HSV Herpes Simplex Virus
ICL Intracellular Loop
ICSS Intracranial Self-stimulation
IFC Infralimbic Cortex
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IGF Insulin-like Growth Factor
IP3 Inositol 1,4,5-trisphosphate
LDTg Laterodorsal Tegmentum
LHb Lateral Habenula
LTD Long-term Depression
LTP Long-term Potentiation
MAPK Mitogen-activated Protein Kinases
MEK Mitogen-activated Protein Kinase Kinase
MSN Medium Spiny Neurons
mTOR Rictor-mammalian Target of Rapamycin
NAc Nucleus Accumbens
NLS Nuclear Translocation Sequence
NMDA N-methyl-D-aspartate
OFC Orbitofrontal Cortex
PD Parkinson’s Disease
PDK1 Phosphoinositide-dependent Kinase-1
PFC Prefrontal Cortex
PH Plekstrin Homology
PI3K Phosphoinositide 3 Kinases
PIP2 Phosphatidylinositol 4,5-bisphosphate
PKA Protein Kinase A
PKC protein Kinase C
PLCβ Phospholipase C β
PP-1 Protein Phosphatase-1
xiv
PP2A Protein Phosphatase 2A
PP2B Protein Phosphatase 2B
PPTg Pedunculopontine Tegmentum
PR Progressive Ratio
PTX Pertussis Toxin
RGS Regulators of G-protein Signalling
RH Regulator of G-protein Signalling Homology
RH RGS Homology
RKIP Raf-kinase Inhibitor Protein
RMTg Rostromedial Tegmental Nucleus
SA Self-administration
SKF 81297 (±)-6-Chloro-2,3,4,5-tetrahydro-1-phenyl-1H-3-benzazepine hydrobromide
SKF 83822 6-Chloro-2,3,4,5-tetrahydro-1-(3-methylphenyl)-3-(2-propenyl)-1H-3-benzazepine-7,8-diol
hydrobromide
SKF 83959 6-Chloro-2,3,4,5-tetrahydro-3-methyl-1-(3-methylphenyl)-1H-3-benzazepine-7,8-diol
SNc Substantia Nigra pars compacta
SNr Substantia Nigra pars reticulata
SRF Serum Response Factor
STEP Striatal-enriched Tyrosine Phosphatase
STN Subthalamic Nucleus
TH Tyrosine Hydroxylase
TM Transmembrane
VGLUT-1 Vesicular Glutamate Transporter-1
VGLUT-2 Vesicular Glutamate Transporter-2
xv
VP Ventral Pallidum
vSub Ventral Subiculum
VTA Ventral Tegmental Area
1
1. General Introduction
1.1 G-protein Coupled Receptors (GPCRs)
G-protein coupled receptors (GPCR), also known as seven transmembrane receptors,
are crucially involved in the transduction of various extracellular stimuli including light,
hormones, and neurotransmittors into specific cellular responses (Müller et al., 2008;
Rosenbaum et al., 2009). Abberant GPCR signalling has been implicated in various disease
states, ranging from cardiovascular diseases to neuropsychiatric disorders (Moreno et al., 2013;
Zalewska et al., 2014), and more than 30% of commercially available drugs target GPCRs
(Williams and Hill, 2009), making these receptors ideal candidates for the discovery of novel
therapeutic targets. As a result GPCRs have been widely studied targets for drug discovery
over several decades.
1.1.1 GPCR Structure
GPCRs are composed of seven transmembrane (7-TM) α-helices connected by three
extracellular loops (ECL) and three intracellular loops (ICL). The ECLs, which includes the N-
terminus, form part of the binding pocket for certain receptor ligands but small molecules like
the catecholamines bind deep within the transmembrane bundle. Particularly, the ECL2 adopts
a β-hairpin conformation that keeps the binding pocket open, and thus is crucial for ligand
binding to GPCRs (White et al., 2012). On the other hand, the ICLs are responsible for G-
protein coupling and the binding of proteins such as the arrestins, both of which are responsible
for initiating discrete cell signaling cascades (Katritch et al., 2012). The ICLs also includes the
C-terminus and the cytoplasmic helix H8, which is important for receptor stabilization and its
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proper functioning (Maeda et al., 2010). The ECLs feature high structural diversity amongst
GPCRs in order to accommodate various types of ligands, while the G-protein-binding ICLs
are generally more conserved between GPCRs (Katritch et al., 2012). The α-helices of the 7-
TM core are frequently tilted due to the presence of helix-breakers amino acids such as proline,
serine, threonine and glycine residues (von Heijne, 1991; Senes et al., 2000; Deupi et al., 2004).
The degree of tilt and the rotation angles of these α-helices differ depending on the types of
GPCRs and their activation state (Yohannan et al., 2004; Meruelo et al., 2011; Latek et al.,
2012). Nevertheless, the 7-TM core contains well conserved motifs characteristic of GPCRs
including the “E/DRY” on TM3 (Rasmussen et al., 2011), “CwxP” on TM6 (Vaidehi et al.,
2014), and “nPxxy” on TM7 (Rosenbaum et al., 2009). The Arg residue of the “E/DRY” motif
interacts with a glutamic acid residue on TM6 to form an “ionic-lock” that was proposed to
stabilize the GPCR in the inactivated state (Rasmussen et al., 2011). In addition, a disulphide
bridge forms between ECL2 and TM3 to maintain structural stability (Hanson et al., 2012). The
crystal structures of several GPCR have been reported to date, including those for the A2A
adenosine receptor, β1 and β2 adrenergic receptors, CXCR4 chemokine receptor, D3 dopamine
receptor, H1 histamine receptor, M2 and M3 muscarinic receptors, µ, δ, and κ opioid receptors,
5HT1B serotonin receptor, rhodopsin, CRF1 corticotrophin receptor, and the glucagon receptor
(Reviewed in: Vaidehi et al. 2014). Of note, the recent release of high-resolution crystal
structures of the µ-opioid and the β1-adrenergic receptors demonstrated that GPCRs exist as
oligomers, or receptor complexes, via hydrophobic interactions between the TM domains
(Manglik et al., 2012; Huang et al., 2013). Specifically, an interface between TM1, TM2, and
helix H8 was found in both µ-opioid and β1-adrenergic receptor oligomers (Manglik et al.,
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2012; Huang et al., 2013), suggesting oligomerization of other GPCRs (Section 1.9) may occur
via a similar mechanism.
1.1.2 GPCR Activation
Under basal conditions, GPCRs exist in both active and inactive conformational states
that are in equilibrium, with a higher number of receptors in the inactive state when no ligand
is present (Niesen et al., 2011; Vaidehi and Bhattacharya, 2011). Stimulation of a GPCR by
ligand binding to the ECLs triggers a slight change in receptor conformation through the 7-TM
domain, which ultimately leads to structural alterations in the ICLs that permit receptor binding
to its cognate G-protein (Rasmussen et al., 2011). Upon binding to the GPCR, the G-protein
undergoes nucleotide exchange from GDP to GTP in its Gα subunit, thereby dissociating the
trimeric G-protein into Gα and Gβγ subunits, each capable of mediating its own signal
transduction pathways (De Lean et al., 1980; Leff, 1995; Freire, 1998). The C-terminus of a
stimulated GPCR is subjected to phosphorylation by G-protein receptor kinases (GRKs), which
then triggers the binding of arrestins to the GPCR that are responsible for the blockade of G-
protein activation and rapid homologous desensitization of the GPCR and its associated
signalling (Kohout and Lefkowitz, 2003; Reiter and Lefkowitz, 2006). The binding of arrestins
also results in clathrin-mediated endocytosis of the GPCR, which then may be subjected to
lysosomal degradation or resensitization and re-expression on the plasma membrane
(Palczewski et al., 1989; Freedman and Lefkowitz, 1996). Interestingly, arrestins were also
recently shown to induce their own non-canonical signalling cascades (Reviewed in: Maudsley
et al. 2013). In addition, the termination of the signalling can also be achieved via the intrinsic
4
GTPase activity of the Gα subunit that hyrdolyzes the GTP to GDP in the Gα subunit, resulting
in the re-association of Gα and Gβγ, and thus inactivation of the G-protein (Gilman, 1987).
During the process of GPCR activation, the intracellular portion of the TM6 tilts
outward towards TM5, and the Arg within the “E/DRY” motif on TM3 extends towards the
GPCR core to interact with the tyrosine residue of the “nPxxY” motif on TM7, thereby
breaking the “ionic lock” between TM3 and TM6 and turning the receptor into its active
conformation (Rasmussen et al., 2011).
1.1.3 G-proteins
The Gα subunit activates various types of signal transduction pathways, and can be
divided into four families, Gαs, Gαi/o, Gαq/11, and Gα12/13, based on the α subunit sequence
and functional characteristics. The Gαs and Gαi/o function to respectively, activate or inhibit
various types of adenylyl cyclases (AC) to modulate the intracellular concentration of cyclic
adenosine monophosphate (cAMP) (Wettschureck and Offermanns, 2005). The Gαq/11 protein
couples the GPCR to the β-isoform of phospholipase C (PLCβ) to increase intracellular
calcium levels (Wettschureck and Offermanns, 2005). In contrast, the functions of Gα12/13 are
still unclear due to the lack of specific inhibitors, although studies have shown that Gα12/13 are
activated by GPCRs that couple to Gαq/11 (Strathmann and Simon, 1990; Dhanasekaran and
Dermott, 1996). The Gα subunit has two structural domains: a nucleotide-binding, Ras-like
domain and an α-helical (AH) domain that partially occludes the bound nucleotide, and the two
domains are interconnected by two flexible linkers (Oldham et al., 2006). In addition, the Gα
5
subunit binds the ligand-bound GPCR via its C-terminus and associates with Gβγ through N-
terminal myristoylation (Resh, 1999).
1.1.4 GPCR Oligomerization
Classically, a GPCR was thought to exist and function as a monomeric entity that was
activated by a single ligand. However, studies using biophysical and molecular techniques
including co-immunoprecipitation (Co-IP), bioluminescence resonance energy transfer (BRET)
and fluorescent resonance energy transfer (FRET) have demonstrated that GPCRs can form
and function as oligomers both in vitro and in vivo (Reviewed in: Rivero-Müller et al. 2013).
Such findings have thus added another layer of complexity to our understanding of the
physiology, signalling and pharmacology of GPCRs.
Evidence has shown that GPCRs of the glutamate family exist as obligatory dimers, as
is the case for the GABAB receptor and the metabotropic glutamate receptors (mGluRs).
Specifically, the GABAB1 protomer is able to bind to its ligand but unable to transduce a signal,
whereas the GABAB2 protomer could couple to the G-protein but is incapable of ligand binding
(Kaupmann et al., 1998). Correct trafficking and signalling of the GABAB receptor only occurs
following the heterodimerization of the two protomers via coiled-coil interactions between
their C-termini (Comps-Agrar et al., 2011). Similarly, mGluRs were found to express in the
membrane as strict dimers via a disulphide bond linkage in the ECLs, and their proper
signalling requires intersubunit rearrangement between the two protomers (Tsuji et al., 2000;
El Moustaine et al., 2012). Furthermore, studies also suggest that GPCRs of the rhodopsin
family form homoligomers in the endoplasmic reticulum (ER) during protein synthesis and
6
maturation prior to cell surface delivery (Salahpour et al., 2004; Kong et al., 2006), although
the interactions between the protomers occur mainly via hydrophobic interactions between the
TM domains rather than the ECLs and ICLs like the glutamate family GPCRs. For instance,
homodimers of both the α1b-adrenergic receptor or dopamine D2 receptor are formed via
symmetrical interfaces between the TM1 and TM4 of the two protomers (Lee et al., 2003;
Lopez-Gimenez et al., 2007; Guo et al., 2008). In addition, different types of GPCRs can also
form heteroligomers primarily via electrostatic interactions between the ICLs (Navarro et al.,
2010). As an example, the heterodimer between the dopamine D2 and cannabinoid CB1
receptors is formed via electrostatic interactions between phosphorylated Thr321
–Ser322
in ICL3
of the CB1 receptor and an Arg-rich epitope in ICL3 of the D2 receptor.
At the physiological level, GPCR oligomerization could lead to the generation of novel
signalling cascades, the amplification or inhibition of an existing signalling, alteration in ligand
binding affinity, receptor delivery to cell surface, and heterologous receptor desensitization or
internalization (Rivero-Müller et al., 2013). To date, numerous GPCR homo- and
heteroligomers have been identified (Reviewed in: George et al. 2002; Moreno et al. 2013;
Gonzalez et al. 2014), many of which are implicated in various disease states ranging from
neuropsychiatric to cardiovascular disorders due to the ability of oligomerization to modulate
and diversify the signalling generated by GPCRs.
However, it should be noted that basic functioning of GPCRs may not require
oligomerization, since monomeric rhodopsin and β2-adrenergic receptor are capable of G-
protein coupling, GRK-mediated phosphorylation, and arrestin-binding when they are
reconstituted into high-density lipoprotein particles (Whorton et al., 2007, 2008). However, this
is an entirely artificial system for study and may not be physiologically relevant. In addition,
7
the crystal structure of an active state complex consisted of a ligand-bound, monomeric β2-
adrenergic receptor-T4-lysozyme fusion protein, a nucleotide-free Gs heterotrimer, and a
nanobody has also recently been solved (Rasmussen et al., 2011). Nevertheless, whether the
experimental setup of these studies reflects physiological conditions is still a matter of debate.
1.2 Dopamine Receptors
Dopamine (3-hydroxytyramine) is a catecholaminergic neurotransmitter that is critically
involved in the regulation of important physiological functions including voluntary movement,
feeding, affect, reward, sleep, attention, learning and memory (Missale et al., 1998; Sibley,
1999; Iversen and Iversen, 2007). Thus, disturbances in dopaminergic system in the brain have
been linked to a wide array of disorders including major depression and drug addiction, as well
as Parkinson’s disease (PD), Huntington’s disease, and schizophrenia, amongst others
(Ehringer and Hornykiewicz, 1960; Seeman et al., 1976; Jakel and Maragos, 2000; Koob and
Volkow, 2010a). Therefore, dopamine has been the target of active research since its discovery
more than 50 years ago.
1.2.1 The Genetic and Structural Properties of Dopamine Receptors
Dopamine exerts its physiological functions by binding to dopamine receptors, which
are GPCRs of the rhodopsin subfamily. Therefore, dopamine receptors possess the seven-
transmembrane-spanning structural phenotype that is iconic of GPCRs and signal through G-
proteins. There are five different subtypes of dopamine receptors, which are classified as either
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D1-like (D1R and D5R) or D2-like (D2R, D3R and D4R) on the basis of their structural,
biochemical and pharmacological properties (Spano et al., 1978; Kebabian and Calne, 1979;
Andersen et al., 1990; Tiberi et al., 1991). Members of the same dopamine receptor subfamily
share high sequence homology in their TM domain, with D1R being 80% homologous to the
D5R, and the D3R and the D4R being 75% and 53% homologous to the D2R, respectively
(Gingrich and Caron, 1993; Missale et al., 1998). The N-terminal domains of all dopamine
receptors are similar in length, while the C-termini of the D1-like receptors are seven times
longer than that for the D2-like receptors (Gingrich and Caron, 1993; Missale et al., 1998). To
date, the crystal structure has only been reported for the D3R (Chien et al., 2010).
The D1-like receptors couple to the Gαs/olf proteins to stimulate cAMP production by
AC, whereas the D2-like receptors activate the Gαi/o proteins to negatively modulate cAMP
production (Spano et al., 1978; Kebabian and Calne, 1979). In addition, evidence also suggests
that the D5R can couple to cAMP (Sunahara et al., 1991) and the Gαq protein to increase
intracellular calcium concentration via the activation of the IP3 receptor (Sahu et al., 2009; So
et al., 2009). The two dopamine receptor subfamilies are also distinct in their gene structures.
The coding regions of the D1R and D5R do not contain introns, whereas several introns have
been identified in the genes that encode the D2-like receptors, with six introns in the D2R, five
in the D3R and three in the D4R genes (Gingrich and Caron, 1993). As a result, genes of the
D2-like receptors can be alternatively spliced to produce receptor variants. For instance, there
are two D2R isoforms termed D2R-long (D2L) and D2R-short (D2S) that differ by 29 amino
acids in their third ICL (Usiello et al., 2000). Similarly, splice variants for the D3R and D4R
have also been described (Giros et al. 1991; Van Tol et al. 1992). For the D4R, its isoforms are
characterized by a 48-base-pair repeat sequence in the third ICL, the the number of repeats
9
varying from 2 up to 11 repeats (Van Tol et al. 1992). In general, each splice variant of the
D2-like receptors possesses distinct anatomical, physiological and pharmacological properties
(De Mei et al., 2009).
1.2.2 Dopamine Receptor Expressions
Dopaminergic neurons are much more prominent in the brain compared to peripheral
areas, and are subdivided into four major pathways in brain: the nigrostriatal, mesolimbic,
mesocortical, and tuberoinfundibular pathways (Anden et al., 1964; Dahlstroem and Fuxe,
1964). The D1-like receptors are expressed exclusively on the postsynaptic membrane, whereas
the D2R and D3R are found both pre- and postsynaptically (Sokoloff et al., 1992). The D2S
isoform is selectively expressed on the presynaptic terminals and is thought to be involved in
autoreceptor functions that provide negative feedback modulation on synaptic dopamine
release (Usiello et al., 2000; De Mei et al., 2009). In contrast, the D2L isoform is mostly found
in postsynaptic sites where it inhibits intracellular cAMP concentration via the coupling to
Gαi/o protein (Usiello et al., 2000; De Mei et al., 2009). In addition to their differences in the
expression sites, the D2S and D2L also differ in their affinity to dopamine receptor agonists,
with the D2S being activated at a lower dose of dopamine agonists than necessary to activate
postsynaptic receptors (De Mei et al., 2009).
In the brain, the highest density of the D1R is found in the caudate putamen (CP),
nucleus accumbens (NAc), ventral tegmental area (VTA), substantia nigra, olfactory bulb,
amygdala, and the frontal cortex. The D1R is also identified at a lower level in the
hippocampus, cerebellum, thalamic and hypothalamic areas (Levey et al., 1993; Missale et al.,
10
1998). The expression of the D2R largely overlaps with that of the D1R, which includes the CP,
NAc, VTA, substantia nigra, olfactory bulb, hypothalamus, frontal cortex, septum, amygdala
and hippocampus (Levey et al., 1993; Missale et al., 1998; Seeman, 2006). For the basal
ganglia, studies using transgenic mice with bacterial artificial chromosomes (BAC) that
express fluorescent proteins under the control of specific promoters have demonstrated distinct
segregation of D1R- and D2R-containing medium spiny neurons (MSNs) predominantly in the
CP, and to a lesser degree in the NAc (Shuen et al., 2008; Valjent et al., 2009). Specifically,
MSNs that project to the substantia nigra pars reticulata (SNr) and the entopeduncular nucleus
(EPN) comprise the direct striatonigral pathway that selectively expresses the D1R. In contrast,
the MSNs that project to the globus pallidus (GP) selectively expresses the D2R and are known
as the indirect striatopallidal pathway, since they indirectly reach the SNr/EPN via synaptic
relays in the GP and the subthalamic nucleus (STN) (Figure 1).
Nevertheless, recent studies have also identified a subpopulation of MSNs in the NAc
(17-30%) and CP (~6%) of BAC transgenic mice that co-express both the D1R and D2R
(Bertran-Gonzalez et al., 2008; Matamales et al., 2009; Gangarossa et al., 2013b). Using highly
validated selective antibodies for the D1R and the D2R, we have demonstrated the co-
expression of both receptors in a number of nuclei within the rat basal ganglia, including the
NAc core (~25% of D1R-expressing neurons), NAc shell (~35%), CP (~7%), ventral pallidum
(VP) (~30%), GP (~60%), and STN (~50%) (Perreault et al., 2010), suggesting that, in ventral
striatum, the direct and indirect pathways are not completely segregated, and there likely exists
a third neuronal pathway within the basal ganglia circuitry that co-express both the D1R and
the D2R (Perreault et al., 2011) (Figure 2). Additionally, we showed that D1R and D2R co-
expression were found in both the cell bodies and exclusively on the presynaptic, but not the
11
Figure 1: The basal ganglia circuitry in rats. A schematic depicting the connections between
the nuclei within the basal ganglia circuitry. The D1R and D2R-expressing medium spiny
neurons (MSNs) were thought to be largely segregated and respectively comprise the direct
striatonigral and indirect striatopallidal pathways.
SNc: substantia nigra pars compacta, SNr: substantia nigra pars reticulata, VTA: ventral
tegmental area, GP: globus pallidus, VP: ventral pallidum, STN: subthalamic nucleus, EPN:
entopeduncular nucleus
Cortex
Striatum
D2R D1R
SNc/VTA
GP/VP
STN
EPN/SNr
Thalamus
Functional
Output
Ind
irect d
irect
Inhibitory
Excitatory
12
postsynaptic, terminals, as indicated by the D1R and D2R co-expression with the presynaptic
marker synaptophysin, but not with the postsynaptic marker PSD95 (Perreault et al., 2010).
Since presynaptic dopamine receptors on MSN terminals have been shown to modulate both
the excitatory and inhibitory postsynaptic currents (Ford, 2014; Zhang et al., 2014), we
postulated that the neurons within the basal ganglia that co-express both the D1R and the D2R,
which terminate in regions within both the direct and indirect pathways, may function to
maintain homeostatic balance between the two neuronal pathways, perhaps via the regulation
of GABA and glutamate expression (Perreault et al., 2011, 2012a). Indeed, we have later
revealed that striatal D1R/D2R co-expressing neurons also exhibited a unique GABA/glutmate
co-expressing phenotype (Perreault et al., 2012a). In addition to the striatum, co-expression of
the D1R and D2R has also been observed in 20-25% of the pyramidal neurons in the PFC of
BAC transgenic mice, with almost all of the D1R-expressing neurons in the PFC also co-
expressing the D2R (Zhang et al., 2010), although the potential function of the D1R/D2R co-
expressing neurons in the PFC has yet to be elucidated.
The D3R has a relatively more limited distribution pattern in the brain, with the highest
expression being in the NAc shell, the olfactory tubercle, and the islands of Calleja (Sokoloff et
al., 1992). Amongst the dopamine receptor subtypes, the D4R has the lowest expression level
in the brain, which includes the frontal cortex, amygdala, hippocampus, hypothalamus, GP,
SNr and the thalamus (Missale et al., 1998). Lastly, the expression of the D5R has been
reported at high level in the pyramidal neurons of the PFC (Oda et al., 2010), and at lower level,
in the premotor cortex, the cingulated cortex, the entorhinal cortex, SN, hypothalamus,
hippocampus, the dentate gyrus, and in the interneurons of the striatum (Missale et al., 1998).
13
D1R, D2R Pathways
Striatum
D1R/D2R
SNc VTA
SNr
EPN VP GP
Putative D1R/D2R
Pathways
Figure 2: A potential third neuronal pathway within the basal ganglia. A schematic
depicting the known neuronal connections between the nuclei within the basal ganglia that
express exclusively the D1R or the D2R, and the putative D1R/D2R co-expressing pathways.
SNc: substantia nigra pars compacta, SNr: substantia nigra pars reticulata, VTA: ventral
tegmental area, GP: globus pallidus, VP: ventral pallidum, STN: subthalamic nucleus, EPN:
entopeduncular nucleus
14
1.2.3 Dopamine Receptor Signalling
The signalling initiation and modulation of dopamine receptors follows that of a typical
GPCR as described earlier in Section 1. In brief, the binding of dopamine induces
conformational changes in the dopamine receptor structure to promote G-protein coupling,
after which the guanine nucleotide exchange occurs on the Gα subunit, leading to the
dissociation of the trimeric G-protein into Gα and Gβγ, each capable of eliciting its own
signalling cascade (Gingrich and Caron, 1993). Activated dopamine receptors are then
subjected to phosphorylation by the GRKs, which in turn results in β-arrestin recruitment to the
receptor that induces receptor desensitization and internalization (Gainetdinov et al., 2003).
The RGS proteins also accelerate GTP-hydrolysis on the Gα subunit to terminate dopamine
receptor signalling (Dohlman and Thorner, 1997). This section will describe in detail both G-
protein-dependent and G-protein-independent signalling cascades that are mediated by
dopamine receptors in the brain.
1.2.3.1 The Gα-cAMP-PKA-DARPP-32 Pathway
As described in Section 2.1, D1-like receptors couple to the Gαs/olf protein to stimulate,
whereas D2-like receptors are coupled to the Gαi/o protein to inhibit, cAMP production and
PKA activity (Spano et al., 1978; Kebabian and Calne, 1979). Amongst the substrates for PKA,
the dopamine and cAMP-regulated protein phosphatase (DARPP-32) is one of the most
extensively studied effectors of dopaminergic signalling. DARPP-32 is highly expressed on the
MSNs in the striatum where it functions as an integrator of various cell signalling responses
(Svenningsson et al., 2004). PKA phosphorylates DARPP-32 at the Thr34 site, which promotes
15
its action as a potent inhibitor of protein phosphatase-1 (PP-1). It has been shown that DARPP-
32 is differentially phosphorylated at this site in the two subpopulations of MSNs in the
striatum following dopamine receptor stimulation, with increased Thr34 phosphorylation being
found selectively in the D1R-expressing neurons and reduced Thr34 phosphorylation in the
D2R-expressing neurons as a result of the opposing modulation of PKA activity by D1R and
D2R (Bateup et al., 2008). In addition, DARPP-32 Thr34 phosphorylation can be
dephosphorylated by the calmodulin-dependent protein phosphatase 2B (PP2B) following D2R
stimulation (Nishi et al., 1997). Another site of phosphorylation on DARPP-32 is Thr75 by
cyclin-dependent kinase 5 (Cdk5) which results in its action as an inhibitor of PKA, and
thereby releasing the inhibitory control of DARPP-32 on PP-1 activity (Bibb et al., 1999).
Furthermore, DARPP-32 is also phosphorylated at Ser137 by casein kinase 1 (CK1) and at
Ser97/102 by casein kinase 2 (CK2), which respectively reduces and enhances DARPP-32
Thr34 phosphorylation by PKA (Girault et al., 1989; Desdouits et al., 1995) (Figure 3). The
knockout of of CK2 selectively in D1R-expressing MSNs has recently been shown to alter the
biochemical and behavioural effects mediated by the D1R (Rebholz et al., 2013). Lastly, the
dephosphorylation of Ser97/102 by protein phosphatase 2A (PP2A) accumulates DARPP-32 in
the nucleus where it prevents dephosphorylaion of histone H3 by PP-1 to increase gene
expression in response to D1R stimulation (Stipanovich et al., 2008).
In the MSNs, the phosphorylation state of several PKA substrates such as AMPA and
NMDA glutamate receptors are governed by the equilibrium between DARPP-32-mediated
PP-1 and PKA activity (Snyder et al., 2000; Nishi et al., 2002, 2005). The inhibition of PP-1 by
DARPP-32 Thr34 phosphorylation shifts the equilibrium towards the phosphorylated state and
enhances the efficacy of D1R-PKA-mediated signalling. However, although DARPP-32
16
Figure 3: The regulation and function of DARPP-32 phophorylations. DARPP-32 can be
phosphorylated and dephosphorylated at four distinct sites respectitvely by different protein
kinases and protein phosphatases. Each phosphorylation carries out its own unique function.
PKA: protein kinase A, Cdk5: cyclin-dependent kinase 5, CK1&2: casein kinase 1&2,
PP2A/B/C: protein phosphatase 2A/B/C.
NH2 COOH Thr75 Ser97 Ser130 Thr34
P P P P
PKA
PP2B
PP2A
Cdk5
PP2A
CK2 CK1
PP2A PP2A
PP2C
Inhibits
PP-1
Inhibits
PKA
Enhances
PKA
Enhances
PP2B
17
functions as a crucial modulator or dopaminergic signalling, it is not the only effector that
regulates dopamine-mediated behaviours. For example, studies using DARPP-32 knockout
mice have demonstrated that behavioural responses to dopaminergic drugs such as cocaine and
apomorphine are only partially disrupted in the mutant mice, indicating that signalling
mechanisms other than the D1R-PKA-DARPP-32 cascade are involved in dopamine-related
behaviours (Fienberg et al., 1998; Nally et al., 2004). Nevertheless, the role of DARPP-32 in
various neuropsychiatric diseases involving a dysfunctional dopamine system has been
extensively studied, particularly in the drug addiction research (Reviewed in: Nairn et al. 2004;
Svenningsson et al. 2005; Yger and Girault 2011).
1.2.4.2 The MEK-ERK1/2 Pathway
Mitogen-activated protein (MAP) kinases have been shown to be involved in
dopamine-mediated signalling and behaviours (Berhow et al., 1996). Studies have
demonstrated that dopamine receptors can regulate ERK1 and ERK2 as administration of
indirect dopamine receptor agonists such as cocaine and amphetamine, as well as D2R
antagonist haloperidol have all been shown to enhance ERK1/2 phosphorylation and activation
(Valjent et al., 2000; Pozzi et al., 2003; Beaulieu et al., 2006). Specifically, genetic and
pharmacological manipulations determined that D1R is essential for striatal ERK1/2 activation,
whereas D2-like receptors, particularly the D3R, inhibit ERK1/2-mediated signalling in the
striatum (Zhang et al., 2004; Valjent et al., 2005). In addition, D1R-mediated ERK1/2
activation is also dependent on the NMDA glutamate receptor, the stimulation of which results
in a calcium influx that activates ERK kinase MEK via Ras-GRF1, a guanine nucleotide
18
exchange factor (Farnsworth et al., 1995; Valjent et al., 2005). In the absence of D1R
stimulation, ERK1/2 phosphorylation by MEK is counteracted by the activity of striatal-
enriched tyrosine phosphatase (STEP), which results in a zero-sum equilibrium where the
overall activity of ERK1/2 remain unchanged. Activation of the D1R-PKA-DARPP-32
pathway leads to the inhibition of PP-1 and the subsequent inactivation of STEP, which then
shifts the equilibrium between MEK and STEP activity in favour of the activation of ERK1/2
by MEK (Valjent et al., 2000, 2005) (Figure 4). This co-regulation of ERK1/2 activity by the
D1R and NMDA receptor suggest that ERK may be a signal integrator of dopaminergic and
glutamatergic neurotransmissions, particularly during the development of behavioural
responses to drugs of abuse. Indeed, inhibition of ERK1/2 activity by MEK inhibitor SL327
has been shown to antagonize the locomotor-stimulating effect of cocaine and amphetamine
(Beaulieu et al., 2006; Valjent et al., 2006b). Moreover, studies have suggested that ERK-
mediated signalling cascades are essential for the activation of transcription factors such as
cAMP response element binding protein (CREB), Zif268, and c-fos that are associated with
long-term changes in gene expression and the development of behavioural adaptations to drugs
of abuse (Brami-Cherrier et al., 2005; Valjent et al., 2006a).
1.2.4.3 Calcium Signalling via Gαq-coupling
In addition to their classic modulatory effect on cAMP/PKA signalling, some evidence
also suggests that specific subtypes of dopamine receptors can couple to the Gαq protein to
regulate PLC activity (Felder et al., 1989; Friedman et al., 1997; Lee et al., 2004; Sahu et al.,
2009).
19
Figure 4: The activation of the MEK-ERK pathway is dependent on the stimulation of
both the D1R and the NMDAR. D1R stimulation leads to the inhibition of PP-1 and STEP by
phosphorylating DARPP-32 at Thr34 to relieve the inhibition of NMDAR-mediated activation
of MEK-ERK pathway. PP-1: Protein Phosphatase 1, STEP: striatal-enriched tyrosine
phosphatase
AC
D1R NMDAR
Gs
D2R
Gi
cAMP
PKA
Ca2+
Ras
MEK
ERK
DARPP-32
Thr34
PP-1
STEP
CREB
Gene Expression c-Fos
Zif268
BDNF
Plasma Membrane
Nucleus
Cytoplasm
20
Activation of PLC leads to the production of inositol triphosphate (IP3) and diacylglycerol
(DAG), which in turn results in the activation of protein kinase C (PKC) by DAG and an
increased intracellular calcium release following IP3 receptor stimulation. It has thus been
delineated that the dopamine-induced PLC-Ca2+
signalling is mediated in part by D5R
stimulation, since the IP3 accumulation elicited by a D1-like receptor agonist SKF 83959 was
attenuated in striatal neurons derived from transgenic mice with D5R gene deletion (Sahu et al.,
2009). Similarly, it has been shown that the expression of D1R in human embryonic kidney
(HEK) cells did not induce intracellular calcium signalling following exposure to D1R-like
dopamine receptor agonist SKF 81297, whereas the stable expression of D5R in HEK cells
produced extensive calcium mobilization following SKF 81297 treatment (So et al., 2009). In
addition, the heterodimer between the D1R and D2R, known as the dopamine D1-D2 receptor
heteromer, can also regulate PLC-mediated calcium signalling both in vitro and in vivo (Lee et
al., 2004; Rashid et al., 2007; Hasbi et al., 2010), which will be discussed in detail in Section 3.
1.2.5 Dopamine Receptor Oligomerization
Similar to most GPCRs, it is now well accepted that dopamine receptors exist and
function as oligomers rather than monomers. For instance, both D1R and D2R require the
appropriate homomeric conformation for their proper signalling and cell surface trafficking
(Lee et al., 2003; Kong et al., 2006). In addition, dopamine receptors have been shown to form
heteromers between different dopamine receptor subtypes, as well as with other GPCRs and
ion channels (Reviewed in: Gomes et al. 2013). This thesis, however, will focus primarily on
one dopamine receptor heteromer, the dopamine D1-D2 receptor heteromer, a receptor
21
complex that has recently been identified, which may play a role in neuropsychiatric disorders
such as drug addiction and depression due to its unique signalling properties (Lee et al., 2004;
Rashid et al., 2007; Hasbi et al., 2009; Perreault et al., 2011).
1.3 The Dopamine D1-D2 Receptor Heteromer: A Novel Dopamine Receptor Complex
Coupled to Gq-PLC-Ca2+
In addition to the canonical cAMP/AC/PKA signalling mediated by the D1R and the
D2R, comprehensive behavioural studies conducted by Waddington et al using various
synthetic dopamine receptor agonists have revealed the existence of a “D1R-like dopamine
receptor” that was capable of AC-independent signalling (Deveney and Waddington, 1995;
Adachi et al., 1999; Tomiyama et al., 2002; O’Sullivan et al., 2005). Additional studies
performed by Friedman et al later revealed that this D1-like dopamine receptor signalled
through the PLCβ/IP3 cascade to release calcium from intracellular stores in the striatum and
the PFC in vivo (Undie et al., 1994; Wang et al., 1995; Jin et al., 2003; Zhen et al., 2004).
However, the exact nature of the D1R-like dopamine receptor that mediated such calcium
signaling in the brain remained elusive for many years. Although it had been demonstrated that
the D5R could couple to the Gαq protein to induce a PLC-mediated calcium signal in both the
striatum and the PFC (Sahu et al., 2009; Perreault et al., 2012b), its minimal expression in the
striatum necessitated the search for other possible mechanisms that could mediate the
production of a calcium signal by dopamine in this region.
Studies have shown that when the D1R alone was expressed in heterologous cell
systems such as Cos7, HEK293, CHO, BHK cells, intracellular calcium signalling in response
22
to dopamine treatment was not evident (Dearry et al., 1990; Pedersen et al., 1994; Lee et al.,
2004). This led to the idea that perhaps the calcium signal induced by dopamine may have
resulted from the co-activation of D1R with another GPCR also present in native striatal tissue
but not in cells. Amongst the possible GPCRs that could interact with the D1R, it was
postulated the D2R as a likely candidate as it was also highly expressed in the striatum
(Missale et al., 1998). Although the D1R and D2R were thought to be predominantly
segregated in striatum, many studies had reported that certain D1R- and D2R-mediated
biochemical, electrophysiological, and behavioural effects were synergistic upon the co-
activation of both receptors. For instance, coactivation of the D1R and D2R was shown to
potentiate immediate early gene expression, to synergistically inhibit NAc neuronal activity,
and to produce maximal locomotor behaviours, when compared to the stimulation of either
receptor alone (White et al., 1988; Hu et al., 1990; Keefe and Gerfen, 1995). There were three
possible explanations for these findings: 1) D1R and D2R on different neurons were interacting
through inter-neuronal circuitry communication, 2) D1R and D2R in the same neurons were
functionally interacting through their downstream signalling cascades, 3) D1R and D2R in
same neurons formed a receptor complex with unique signalling properties. However, the fact
that the D1R and D2R opposingly regulated the cAMP/PKA pathway (Spano et al., 1978;
Kebabian and Calne, 1979) suggested that these synergistic effects were not mediated simply
by the concurrent activation of individual Gαs-coupled D1R and Gαi/o-coupled D2R, but
rather may have been due to a novel dopamine receptor complex composed of D1R and D2R.
Indeed, our laboratory has thus far conducted thorough in vitro and in vivo analyses and
identified such a dopamine receptor complex, which was termed the dopamine D1-D2 receptor
heteromer.
23
1.3.1 The D1R and D2R form a Heteromeric Complex in vitro
Evidence for the existence of a heteromeric complex comprised of the D1R and the
D2R initially came from a study that showed the D1R and the D2R could be co-
immunoprecipitated from both primary-cultured striatal neurons and the rat striatum (Lee et al.,
2004). While such a finding suggested that the D1R and D2R were part of the same protein
complex, it did not indicate a direct physical interaction as the two receptors may have been
linked through scaffolding proteins. In later studies a direct physical interaction between the
two receptors was shown using a bioluminescence resonance energy transfer (BRET) technique
to assay D1R and D2R proximity in HEK cells stably expressing the D1R and D2R (Verma et
al., 2010; Hasbi et al., 2014). In these studies it was demonstrated that the D1R and D2R are in
physical contact with each other as indicated by the robust BRET signal elicited by the energy
transfer between D1R-Rluc and D2R-GFP. Furthermore, by tagging the D2R with a nuclear
translocation sequence (NLS) that induced D2R trafficking to the nucleus, we observed in
HEK cells the co-translocation of D1R with D2R to the nucleus, further demonstrating that the
D1R and D2R oligomerize into a heteromeric receptor complex (O’Dowd et al., 2005, 2012).
Additional studies using the NLS and BRET techniques from our laboratory subsequently
revealed that the D1R interacted with both the D2S and D2L isoforms, and that the D1-D2
heteromer was formed in the ER through the electrostatic interactions between two glutamic
acid residues in the C-terminal tail of D1R and two arginine residues in the ICL-3 of D2R
(O’Dowd et al., 2005, 2012; Hasbi et al., 2014) (Figure 5). Nevertheless, a separate study
reported that the D1R interacted with part of the additional 29 amino acids sequence that is
present in the D2L but not in the D2S isoform to form the D1-D2 heteromer (Pei et al., 2010),
however our laboratory and others (Chun et al., 2013) could not replicate this finding.
24
Fig
ure
5: T
he D
1R
an
d th
e D2
R in
tera
ct v
ia electro
static
intera
ction
s to fo
rm
the D
1-D
2 h
etero
mer. T
he electro
static
interactio
ns b
etween
the tw
o g
lutam
ic acid resid
ues (4
04-4
05
) in th
e C-term
inal o
f D1R
and th
e two arg
inin
e residues (2
74
-
275) in
the th
ird in
tracellular lo
op o
f D2R
interact fo
rm th
e D1
-D2 h
eterom
er.
25
1.3.2 Expression of the D1-D2 Heteromer in the Brain
Having confirmed the existence of the D1-D2 heteromer in vitro, we next wanted to
determine whether this receptor complex was present in vivo. Using highly specific
fluorophore-tagged antibodies against the D1R and the D2R (validated in D1-/- and D2-/- gene
deleted striatal tissue and in HEK cells expressing each of the 5 dopamine receptors
individually), the co-expression and co-localization of the two receptors was observed in adult
rat NAc core, NAc shell, CP, GP, VP, and the EPN to various degrees (Perreault et al., 2010).
Specifically, in both neonatal striatal neurons and in the dorsal and ventral striatum (CP and
NAc respectively), our antibodies were able to detect the co-localization of D1R and D2R with
each other, as well as with dynorphin (DYN) and enkephalin (ENK), well-known neuronal
markers for D1R- and D2R-expressing neurons respectively, thus validating the specificity of
our antibodies (Perreault et al., 2010). However, the co-localization of the D1R and D2R does
not necessarily mean the two receptors are physically interacting. Therefore, using the confocal
FRET microscopy in striatal slices in situ, which evaluates the proximity of the endogenous
D1R and D2R using fluorophore-tagged antibodies, we demonstrated a robust FRET efficiency
(~0.20) in the NAc core and shell, indicating that the two receptors were less than 100Ǻ apart,
with the majority of D1R/D2R co-expressing neurons (~90%) exhibiting D1-D2 heteromer
expression (Perreault et al., 2010). Since approximately 30% of D1R-expressing MSNs in the
NAc co-express the D2R, results from the FRET study indicated that roughly 27% of MSNs in
the NAc exhibited D1-D2 heteromer formation. Additionally, the expression of the D1-D2
heteromer in the NAc shell was subsequently found to increase with age, with the heteromer
expression being lower in juvenile rats compared to adults (Perreault et al., 2013, 2014). A
similarly high FRET efficiency (~0.22) was also observed between the D1R and the D2R in the
26
GP, suggesting a high degree of D1-D2 heteromer formation in this region as well, although
only a small number of neurons in the GP co-express both the D1R and the D2R (Perreault et
al., 2010, 2011). In contrast, while we were also able to detect a FRET signal between the D1R
and the D2R in the CP, the efficiency was much lower (~0.05), with only approximately 25%
of the D1R/D2R co-expressing neurons in the CP expressing the D1-D2 heteromer (Perreault et
al., 2010). Since only 4-6% of MSNs in the CP co-expressed the D1R and D2R, the FRET data
suggests merely 1-2% of total MSNs in the CP express the D1-D2 heteromer. Furthermore,
immunohistochemical analysis revealed localization of the D1-D2 heteromer selectively in cell
bodies and presynaptic terminals in the NAc and CP, indicating a possible role for the receptor
complex in neurotransmitter release from MSNs (Perreault et al., 2010).
The findings that demonstrated the abundant expression of the D1-D2 heteromer in the
NAc has thus been regarded as controversial, since it has traditionally been thought that the
expression of the D1R and D2R on striatal MSNs are largely segregated, with D1R residing
selectively on the direct striatonigral pathway and D2R localizing exclusively in the indirect
striatopallidal pathway (Gerfen and Surmeier, 2011). Nevertheless, several studies have also
identified the co-expression of D1R and D2R in striatal neuronal cultures and in native striatum,
thus supporting the existence of the D1-D2 heteromer in vivo (Bertran-Gonzalez et al., 2008;
Matamales et al., 2009; Perreault et al., 2010; Gangarossa et al., 2013b). For instance, using
BAC transgenic mice with eGFP-tagged to the promoter elements of D1R and D2R, a study by
Bertran-Gonzalez et al. (2008) showed that approximately 17% of NAc shell MSNs and 6% of
CP MSNs exhibit D1R/D2R co-localization. A separate study later validated the accuracy of
dopamine receptor expression and representation in the BAC transgenic mice by showing that
100% of MSNs in these mice expressed D1R, D2R or both, with the degree of co-localization
27
being similar to that was reported in the Bertran-Gonzalez study (Matamales et al., 2009).
Moreover, the subpopulation of MSNs with the highest co-expression of D1R and D2R was
found to be located specifically in the bundle-shaped area in the caudomedial NAc shell
(Gangarossa et al., 2013b). Combined with the FRET analyses, collectively these studies
strongly indicate that the D1R and D2R are co-expressed in a specific subset of MSNs in the
NAc and form the D1-D2 heteromer in these neurons.
1.3.3 A Unique Functional Role for the D1-D2 Heteromer
Using an in vitro cellular system, it was first demonstrated that the co-administration of
individual D1R and D2R selective agonists stimulated a calcium signal only in HEK293 cells
that stably co-expressed both the D1R and D2R, but not in cells that expressed the individual
D1R or D2R homoligomers alone (Lee et al., 2004). In addition, the calcium signal was
abolished by selective antagonists for the D1R or the D2R, as well as by inhibitors for Gαq,
PLC, and the IP3 receptor (Lee et al., 2004; Rashid et al., 2007; Hasbi et al., 2009), thus
indicating that the calcium signal was mediated by Gαq coupling, dependent on PLC activation,
resulted from an intracellular calcium source, and required the co-activation of both D1R and
D2R. In addition, the involvement of the Gαq protein in mediating this calcium signal was
further confirmed by the finding showing increased GTPγS incorporation into the Gαq protein
following D1R and D2R co-activation in HEK293 cells stably expressing both receptors
(Rashid et al., 2007).
A variety of dopamine receptor agonists were subsequently screened to determine their
effects at the D1-D2 heteromer. A synthetic benzazepine derivative known as SKF 83959 was
28
identified that could stimulate intracellular calcium mobilization in HEK293 cells and primary
cultured striatal neurons without affecting the D1R- or D2R-mediated AC activity (Rashid et
al., 2007; Hasbi et al., 2009; Verma et al., 2010). Additional analyses revealed that SKF 83959
increased the GTPγS incorporation into the Gαq protein in striatal tissue and generated a
calcium signal that was abolished by selective D1R or D2R antagonists, as well as by inhibitors
for Gαq, PLC, and the IP3 receptor in primary cultured striatal neurons (Rashid et al., 2007;
Hasbi et al., 2009). Additionally, SKF 83959-induced calcium signal was also absent in
primary cultured striatal neurons derived from transgenic mice gene-deleted for the D1R
presumably due to the lack of D1-D2 heteromers (Hasbi et al., 2009).
Further characterization of SKF 83959 demonstrated that within the D1-D2 heteromer
the compound acted as a full agonist at the D1R and as a partial agonist at the D2R (Rashid et
al., 2007). This was deduced from the findings showing SKF 83959 alone elicited a calcium
signal that was approximately 60-75% of that was generated by dopamine, but could be
enhanced upon the co-administration of D2R agonist quinpirole to the same extent as that was
induced by dopamine (Rashid et al., 2007; So et al., 2007). Moreover, SKF 83959 exhibited
high affinity binding to the D1R homoligomers and low affinity for the Gαi-coupled D2R
homoligomers, but did not activate either of these receptors (Andringa et al., 1999; Chun et al.,
2013). Nevertheless, the use of pertussis toxin (PTX) that renders the D2R to a state of low
affinity to agonist binding showed that SKF 83959 was able to bind to a PTX-insensitive D2R
with high affinity in rat striatum and in HEK293 cells stably expressing both the D1R and D2R,
but not in cells expressing either D1R or D2R alone (Rashid et al., 2007). This finding
suggested that SKF 83959 was able to bind to a specific D2R linked to the PTX-resistant Gαq
protein, such as the D2R within the D1-D2 heteromer, thus supporting the idea that SKF 83959
29
stimulated the D1-D2 heteromer by binding to both protomers within the receptor complex.
Collectively, these data suggested that SKF 83959 was an agonist for the D1-D2 heteromer,
and thus could be a useful tool to study the physiological role of the D1-D2 heteromer in vivo.
1.3.4 Regulation of the D1-D2 Heteromer Signalling
Similar to other GPCRs, the D1-D2 heteromer is subjected to signal desensitization and
receptor internalization following its activation. It was shown that D1-D2 heteromer-mediated
calcium signal was desensitized by prior exposure to dopamine, SKF 83959, the D1R-selective
agonist SKF 83822, or D2R-selective agonist quinpirole, albeit at different rates (So et al.,
2007; Verma et al., 2010), suggesting that agonist occupancy of either receptor within the D1-
D2 heteromer was sufficient to induce a conformation change that led to the desensitization of
the calcium signal even without heteromer stimulation. This desensitization occurred
independent of calcium storage depletion, exogenous calcium entry or receptor internalization,
and was specifically mediated by GRK2 via its RGS and catalytic domains (So et al., 2007;
Verma et al., 2010). Similarly, the D1-D2 heteromer was also found to internalize by the co-
activation of both D1R and D2R by dopamine or by the sole activation of either receptor within
the receptor complex (Verma et al., 2010). Since the D1-D2 heteromer was previously shown
to be formed in the ER (O’Dowd et al., 2005), the ability of agonist occupancy to either
constituent receptor to desensitize and internalize the D1-D2 heteromer indicates that the
integrity of the receptor complex was maintained throughout the typical GPCR life cycle from
the initial cell surface trafficking to the eventual signal termination.
30
1.3.5 Generation of a Novel Antagonist for the D1-D2 Heteromer: The TAT-D1 Peptide
Although SKF 83959 was shown to be an agonist for the D1-D2 heteromer, the
compound has been shown to have affinity for, or activate, a number of other receptors
including the D5R, the 5HT-2C receptor, and the α2c-adrenergic receptor (Andringa et al., 1999;
Sahu et al., 2009; Chun et al., 2013). In addition, there are also conflicting reports as to whether
SKF 83959 acts as an antagonist, partial agonist, or has no effect at the D1R (Andringa et al.,
1999; Jin et al., 2003; Rashid et al., 2007; Lee et al., 2014). The lack of specificity of SKF
83959 is a major caveat when the compound is used in studies that aim to address the
physiological effects of the D1-D2 heteromer, as systemic administration of SKF 83959 could
result in various off-target effects in addition to D1-D2 heteromer activation. In the past, SKF
83959 was used in combination with D1R and D2R antagonists or in gene-deleted mice to
validate D1-D2 heteromer-specific effects in vitro (Rashid et al., 2007; Hasbi et al., 2009;
Perreault et al., 2012b). However, such manipulations would also inevitably disrupt the
physiological function of the D1R and the D2R in vivo. It was therefore necessary to develop
an antagonist selective to the D1-D2 heteromer to establish specificity for SKF 83959-induced
effects.
Using serial deletions and point mutations of the D1R and the D2R, it was identified
that the two receptors heteroligomerize via electrostatic interactions between two glutamic acid
residues on the C-terminus of the D1R and two arginine residues on the ICL3 of the D2R
(O’Dowd et al., 2012; Hasbi et al., 2014). The loss of any of these four residues abolished the
BRET signals in HEK293 cells (Hasbi et al., 2014), and prevented the nuclear translocation of
D1R with D2R-NLS in HEK293 cells (O’Dowd et al., 2012). A short peptide was then
synthesized that was comprised of the same 16 amino acid sequence as the D1R C-terminus
31
that included the two Glu residues. The peptide was fused to a TAT sequence, a cell-
penetrating motif derived from human immunodeficiency virus, at its N-terminus to render it
cell permeable (Schwarze et al., 1999). The resulting TAT-D1 peptide was able to dose-
dependently reduce the BRET and FRET signals elicited by the D1-D2 heteromer when it was
incubated with HEK293 cells stably co-expressing both receptors and primary cultured striatal
neurons, respectively (Hasbi et al., 2014). In addition, the TAT-D1 peptide also reduced the co-
immunoprecipitation of D1R and D2R in cells as well as in native striatal tissues, and dose-
dependently attenuated SKF 83959-induced calcium mobilization in primary cultured striatal
neurons (Hasbi et al., 2014). The specificity of the TAT-D1 peptide towards the D1-D2
heteromer was confirmed by findings showing that the BRET signals elicited by D1R
homoligomer, D2R homoligomer, or D2-D5 heteromer were not affected by TAT-D1
administration (Hasbi et al., 2014). Furthermore, a TAT-scrambled control peptide that
contains the same amino acid residues as the TAT-D1 peptide but in random order had no
effect in the BRET, FRET, Co-IP and calcium mobilization experiments (Hasbi et al., 2014).
Collectively these findings indicate that the TAT-D1 peptide was able to selectively disrupt the
D1-D2 heteromer formation and antagonize its signalling. Therefore, the use of the TAT-D1
peptide would allow us to exclusively determine D1-D2 heteromer-specific physiological
effects.
1.3.6 Physiological Effects of the D1-D2 Heteromer
A physiological role for the D1-D2 heteromer in vivo was first demonstrated in rat
striatal neurons and tissue which showed increased phosphorylation and total expression of
32
calcium calmodulin kinase II (CaMKII), specifically in the NAc, as a result of increased
intracellular calcium release following acute D1-D2 heteromer stimulation (Lee et al., 2004;
Rashid et al., 2007; Hasbi et al., 2009; Ng et al., 2010). CaMKII has been highly implicated in
the regulation of synaptic plasticity through the modulation of glutamatergic transmission
(Anderson et al., 2008; Jenkins and Traynelis, 2012), and has also been shown to play a role in
the transcriptional regulation of brain-derived neurotrophic factor (BDNF) through
phosphorylation of the transcriptional repressor MeCP2 (Zhou et al., 2006). Indeed, it was
shown that subsequent to CaMKII phosphorylation following acute D1-D2 heteromer
stimulation by SKF 83959 also increased the expression of BDNF in both the NAc and the
VTA, and promoted morphological maturation and differentiation of primary cultured striatal
neurons (Hasbi et al., 2009; Perreault et al., 2012a). In contrast, a reduction in BDNF levels
and CaMKII phosphorylation was observed in the SN following acute D1-D2 heteromer
stimulation, whereas no effect was observed in the PFC (Perreault et al., 2012a, 2013). The
involvement of the D1-D2 heteromer in the modulation of CaMKII phosphorylation and BDNF
expression in the striatum was further confirmed using primary cultured striatal neurons
derived from D1R or D5R gene-deleted transgenic mice, which showed that the absence of
D1R, but not the D5R, abolished the effect of SKF 83959 on CaMKII phosphorylation and
BDNF expression in striatal neurons (Hasbi et al., 2009), likely due to the lack of D1-D2
heteromer formation in the D1R knockout mice.
As described in Section 1.3.2, the D1-D2 heteromer is expressed in a specific subset of
MSNs in the NAc that co-express both the D1R and D2R. Intriguingly, we further
demonstrated that these D1-D2 heteromer-expressing MSNs also co-express both GABA and
glutamate (Perreault et al., 2012a), thus potentially allowing these neurons to exert dual
33
modulation on the neuronal activity of specific regions in the brain via their efferent
projections. Indeed, we showed that acute D1-D2 heteromer stimulation by SKF 83959
increased the expression of a major GABA-producing enzyme, GAD67, in the NAc and VTA,
whereas a reduction in GAD67 expression was observed in the SN (Perreault et al., 2012a). On
the other hand, the expression of vesicular glutamate transporter-2 (VGLUT2), which regulates
glutamate release into the synapse, was increased in the CP, VTA and SN, whereas VGLUT1
expression was increased in the SN only, by acute D1-D2 heteromer stimulation (Perreault et
al., 2012a). In addition, selective stimulation of the D1-D2 heteromer in the NAc shell, where
the heteromer is predominantly expressed (Section 1.3.2), increased the expression of GAD67
in the VTA, and enhanced the expression of both GAD67 and VGLUT1/2 in the SN (Perreault
et al., 2012a). Subsequent quantification of GABA and glutamate levels following SKF 83959
treatment using dot blots found that the GABA to glutamate ratio was increased in the NAc,
but was reduced in the CP, in response to D1-D2 heteromer stimulation (Perreault et al., 2012a),
indicating differential modulation of the inhibitory GABAergic tone by the D1-D2 heteromer
in the two regions. Collectively, the ability of the D1-D2 heteromer to regulate GABA and
glutamate activity in a region-specific manner supports the hypothesis that one function of the
D1R/D2R co-expressing neurons, which extend efferent projections to nuclei in both the direct
and indirect pathways, may be to fine-tune neuronal activity and maintain homeostatic balance
between the direct and the indirect pathways (Perreault et al., 2011).
Behaviourally, acute D1-D2 heteromer stimulation by SKF 83959 enhanced basal
locomotion and induced robust grooming in rats (Perreault et al., 2010). This grooming
response elicited by SKF 83959 was further confirmed to involve the D1-D2 heteromer as the
behaviour was not observed following selective D1R stimulation by SKF 83822, was abolished
34
by D2R antagonist raclopride (Perreault et al., 2010) and the D1-D2 heteromer disrupting
peptide TAT-D1 (Hasbi et al., 2014), was absent in transgenic mice gene-deleted for D1R, but
not D5R (Perreault et al., 2012a), and was attenuated in juvenile rats possibly as a result of
lower D1-D2 heteromer expression compared to adult rats (Perreault et al., 2013).
Lastly, in contrast to that was observed with acute stimulation, repeated D1-D2
heteromer stimulation by daily SKF 83959 treatment for 7 days was found to reduce total
CaMKII expression in the NAc without affecting its phosphorylation (Perreault et al., 2010).
Since CaMKII mediates the Ser831 phosphorylation of AMPA glutamate receptor subunit
GluA1 (Snyder et al., 2000), repeated D1-D2 heteromer stimulation concurrently resulted in
reduced GluA1 Ser831 phosphorylation in the NAc (Perreault et al., 2010). However, the exact
mechanism responsible for the opposing effects between acute and repeated D1-D2 heteromer
stimulation on CaMKII phosphorylation remain to be elucidated.
In summary, we have identified a novel dopamine receptor complex in the brain with a
novel signalling properties and a unique expression pattern. Furthermore, D1-D2 heteromer
stimulation in vivo modulated the expression of various proteins in the brain that have been
critically implicated both depression and drug addiction, such as BDNF and GAD67, which
will be discussed in detail in Section 1.5.
1.4 The Role of Dopamine in the Brain Reward System
A reward is a stimulus that, when given to humans or animals, will alter their behaviour
to reinforce them to actively acquire such stimulus. The brain reward system is critical for the
reward function in response to various external stimuli such as food, water, and sex, and is
35
therefore evolutionarily crucial for survival (Wise, 2004; Grace et al., 2007). A dysfunctional
brain reward system has been critically implicated in the pathophysiology of neuropsychiatric
disorders such as depression and drug addiction (Koob and Volkow, 2010b; Russo and Nestler,
2013). The primary reward pathway in the brain consists of the dopaminergic projection from
the VTA to the NAc, where the limbic information is translated into goal-directed behaviour
via the projections from the NAc to various nuclei of the basal ganglia motor circuitry (Sesack
and Grace, 2010). As the D1-D2 heteromer is predominantly expressed in the NAc, and since
its stimulation was shown to modulate the expression of various proteins in both the NAc and
the VTA, it possible that the D1-D2 heteromer may play an important role in the modulation of
brain reward function.
1.4.1 The Neurocircuitry of the Brain Reward System
Although the primary component of reward circuit is comprised of dopaminergic
projections from the VTA to the NAc, also known as the mesolimbic circuitry, the activity of
this pathway can be in turn modulated by inputs from nuclei of the cortical and limbic regions,
including the PFC, basolateral amygdala (BLA), the ventral subiculum (vSub) of the
hippocampus for the NAc, and PFC, pedunculopontine tegmentum (PPTg), laterodorsal
tegmentum (LDTg), rostromedial tegmental nucleus (RMTg), and the lateral habenula (Lhb)
for the VTA. Collectively, interconnections between these nuclei and the mesolimbic pathway
comprise the brain reward system (Figure 6).
36
Figure 6: The neurocircuitry of the brain reward system. The NAc receives dopaminergic
projections from the VTA and glutamatergic projections from the mPFC, BLA, and vSub. The
NAc itself sends out GABAergic projections to the VTA and the lateral hypothalamus. The
LDTg and RMTg respectively send glutamatergic and GABAergic projections to the VTA to
control dopamine release in the NAc. The RMTg in turn receives glutamatergic projections
from the Lhb.
NAc: Nucleus Accumbens, VTA: Ventral Tegemental Area, BLA: Basolateral Amygdala,
vSub: Ventral Subiculum, LDTg: Laterodorsal Tegmentum, RMTg: rostromedial tegmental
nucleus, LHb: Lateral Habenula. (Figure cited from Russo and Nestler 2013, Figure 1).
37
1.4.1.1 The Nucleus Accumbens
The NAc is critical for the regulation of reward-seeking behaviours (Mogenson et al.,
1980; Groenewegen et al., 1996). The NAc receives afferent projections from limbic regions
such as the VTA, BLA, and the vSub (Kelley and Domesick, 1982; Kelley et al., 1982;
Groenewegen et al., 1987), and sends efferent projections to nuclei of the basal ganglia
circuitry including the VP, STN, SNr, and the thalamus (Heimer et al., 1991; Usuda et al., 1998;
Dallvechia-Adams et al., 2001). Therefore, anatomically the NAc serves as an interface
between the limbic and motor regions to convert learned associations of motivational
significance into goal-directed behaviours (Sesack and Grace, 2010). The NAc is subdivided
into two anatomically and functionally distinct regions, the NAc shell and NAc core.
Traditionally it has been thought that the NAc shell is linked to the limbic system (Rodd-
Henricks et al., 2002; Sellings and Clarke, 2003), whereas the NAc core is part of the basal
ganglia motor circuitry (Nicola, 2007). Furthermore, the NAc shell is primarily implicated in
the regulation of reward- or drug-seeking behaviour by spatial or contextual information,
whereas the NAc core is in control over such behaviours by discrete cues (Robbins et al., 2008).
Approximately 90% of the neurons in the NAc are GABAergic medium spiny neurons
(MSNs), with the remaining neurons being cholinergic and GABAergic interneurons
(Kawaguchi et al., 1995; Meredith, 1999). In addition to the dopaminergic projections from the
VTA, the NAc also receives excitatory glutamatergic projections from the medial and lateral
PFC, the BLA, and the vSub that modulates goal-directed behaviours generated by different
sources (Ishikawa et al., 2008; Ito et al., 2008; Gruber et al., 2009). Specifically, inputs from
the PFC are thought to supply executive control, BLA to communicate information regarding
conditioned associations and affective drive, and the vSub to provide spatial and contextual
38
information (Ishikawa et al., 2008; Ito et al., 2008; Gruber et al., 2009). Interestingly, it has
been shown that the dopaminergic and glutamatergic inputs converge onto the same spines of
the MSNs in the NAc, forming a “synaptic triad” that allows dopamine to modulate the
plasticity of glutamatergic transmissions into the NAc, a process that is crucial for the
establishment of stable reward-seeking behaviours (Dehaene and Changeux, 2000; Wolf et al.,
2004; Moss and Bolam, 2008). In addition, the NAc also receives the reciprocal inhibitory
GABAergic input from the VP and modulatory dopaminergic input from the substantia nigra
pars compacta (SNc) that function to provide negative feedback control and to modulate motor
function, respectively (Brog et al., 1993; Ikemoto, 2007).
The targets of efferent MSNs from the NAc include the VTA, VP, and the SNr (Haber
et al., 1990; Heimer et al., 1991; Usuda et al., 1998). The projections from the NAc shell to the
VTA have been shown to influence the dopaminergic neurons that project back to the NAc
core, thereby creating a medial to lateral series of spirals that allow limbic associated structures
to progressively influence transmission in more motor-related parts of basal ganglia (Nauta et
al., 1978; Zahm and Heimer, 1993; Haber et al., 2000). Through this spiral of connections,
motivational limbic information can be processed into appropriate motor responses to obtain
reward. The projections from the NAc to the SNr of the basal ganglia are connected directly via
the striatonigral and indirectly via the striatopallidal pathways, where the D1R and the D2R are
respectively expressed (Gerfen and Surmeier, 2011) (see Section 1.2.2 & 1.3.2). For the NAc
core, cortical activation of the direct pathway to the EPN/SNr complex leads to the
disinhibition of appropriate action plans that facilitate reward acquisition (Redgrave et al.,
1999). On the other hand, cortical activation of the indirect pathway from the NAc core to the
GP and STN before reaching the SNr is thought to inhibit actions that are maladaptive for
39
obtaining reward, or for the purpose of avoiding punishment (Redgrave et al., 1999). In
contrast to the NAc core, the direct and indirect pathways from the NAc shell are less well-
defined since the shell subregion is a hybrid structure that is part basal ganglia and part limbic
(Heimer et al., 1991). One theory suggests that both direct and indirect pathways from the NAc
shell first project the GP, where a subset of neurons extend efferent projections to the
mediodorsal thalamus to form the direct pathway, and another subset to the STN to form the
indirect pathway (O’Donnell et al., 1997; Nicola et al., 2000). Another theory postulates that
the direct and indirect pathways from the NAc shell converge onto the VTA instead of the SNr,
with the direct pathway involving MSNs from the NAc shell to the VTA, and the indirect
pathway consisting of MSNs from the NAc shell to the ventromedial VP before reaching the
VTA (Sesack and Grace, 2010). In addition, a subset of striatal MSNs that co-express
DYN/ENK-D1R/D2R were shown to project to nuclei within both the direct and indirect
pathways, specifically to the GP, EPN, and the VP (Perreault et al., 2011) (Figure 2 in Section
1.2.2). Unpublished data from our laboratory also indicated a putative projection of the
D1R/D2R co-expressing striatal MSNs from the NAc shell to the PFC. The function of these
DYN/ENK-D1R/D2R co-expressing striatal MSNs remain to be elucidated, although it has
been postulated that they function to maintain homeostatic balance between the direct and the
indirect pathways due to their efferent projections in both pathways (Perreault et al., 2011).
1.4.1.2 The Ventral Tegmental Area
The VTA is located in the midbrain in close proximity with the SN, and is the major
source of dopaminergic input into the NAc. The VTA-NAc, mesolimbic dopamine circuitry is
40
consistently recruited by both drugs of abuse and natural rewards, making it an essential
component of the brain reward system (Wise, 2004). Approximately 60-65% of neurons in the
VTA are dopaminergic, and they are highly heterogeneous in their morphological
characteristics, afferent influences, efferent projection targets, and firing properties (Nair-
Roberts et al., 2008). The non-dopaminergic neurons in the VTA are primarily GABAergic
(30-35%), either as interneurons or as projection neurons that function to provide local
inhibition of dopaminergic neurons and long-range inhibition of projection regions (Nair-
Roberts et al., 2008). The remaining neurons in the VTA were found to be glutamatergic (2-
3%), with the ability to co-release dopamine (Yamaguchi et al., 2007).
The projection targets of the VTA vary depending on the location and the types of
neurons that originate from the VTA. Neurons from the medial VTA project to the NAc, VP,
STN, olfactory tubercle, and the amygdala (Lammel et al., 2008). The medial most rostral
linear VTA sends projections to VP, olfactory tubercle, preoptic and lateral hypothalamic areas,
Lhb, mediodorsal thalamus, and also to a lesser extent to the PFC, BLA, and dorsal raphe (Del-
Fava et al., 2007). The caudomedial and ventromedial VTA innervate the NAc, the bed nucleus
of the stria terminalis, the pallidum, the central amygdaloid nucleus (CeA), and the BLA (Del-
Fava et al., 2007). The mesostriatal (VTA-NAc) projections can be further subdivided, with the
posterior VTA projecting to the medial NAc shell, and the lateral VTA innervating the NAc
core and the lateral shell (Ikemoto, 2007). Furthermore, the dopaminergic, GABAergic and
glutamatergic neurons from the VTA each extend efferents to different destinations. The
dopaminergic neurons from the VTA project primarily to the NAc, mPFC, and the amygdala
(Ungless and Grace, 2012), while the GABAergic neurons extensively innervate the VP, lateral
hypothalamus, and the Lhb (Taylor et al., 2014). Lastly, the glutamatergic neurons from the
41
VTA were shown to project to the NAc shell, PFC, VP, amygdala, and the Lhb (Hnasko et al.,
2012). Recent studies using optogenetics to selectively stimulate specific projection neurons
from the VTA delineated that each subset of neurons influence reward-seeking behaviour in
different manners depending on the projection targets and neuronal subtypes (Reviewed in:
Lammel et al. 2014).
The VTA receives excitatory glutamatergic projections from a wide array of structures;
each provides a modest input to the VTA but also innervates other regions that also project to
the VTA. Therefore, VTA neuronal activity is modulated by an integrated network of inputs
rather than by a discrete set of brain structures (Geisler and Zahm, 2005). The major source of
excitatory cortical input originates from the PFC, involving predominantly the prelimbic and
infralimbic cortices, and to a lesser extent the cingulate and orbital divisions (Geisler and Zahm,
2005). The PFC projections synapse onto dopaminergic neurons in the VTA that project back
to the PFC to create a microcircuit that allows DA to modulate glutamatergic input from the
PFC (Carr and Sesack, 2000). Neurons from the PFC also innervate GABAergic neurons
within the VTA that in turn project to the NAc that allows the PFC to indirectly inhibit NAc
neuronal activity (Carr and Sesack, 2000). Projections from the LDTg and PPTg are another
source of excitatory input to the VTA. Stimulation of the LDTg/PPTg complex has been shown
to promote burst-firing of dopaminergic neurons to enhance dopamine release from the VTA to
the NAc lateral shell that ultimately led to reward-seeking behaviours (Lammel et al., 2012).
Other sources of excitatory input to the VTA include the lateral hypothalamus, VP, Lhb,
periaqueductal grey, and reticular formation (Geisler et al., 2007). In particular, the inputs from
the lateral hypothalamus, VP, and the Lhb to the VTA have also recently been implicated in the
42
regulation of reward-related behaviours (Aston-Jones et al., 2010; Lammel et al., 2012; Mahler
et al., 2014).
The major extrinsic inhibitory inputs into the VTA arise from the GABAergic neurons
of the NAc shell and the VP, which function as a negative feedback and a gating mechanism,
respectively, for dopamine release in response to rewarding stimuli (Heimer et al., 1991;
Geisler and Zahm, 2005). Recently, the RMTg has been identified to be another important
source of inhibitory input to the VTA. The RMTg is located just caudal to the VTA and sends
extensive GABAergic projections to the VTA (Jhou et al., 2009b). The activity of the RMTg in
turn is under the influence of excitatory glutamatergic input from the Lhb (Jhou et al., 2009b).
Optogenetic stimulation of the Lhb was found to enhance RMTg GABAergic activity that
selectively inhibits the dopaminergic neurons from the VTA to the NAc lateral shell to produce
aversive conditioning (Lammel et al., 2012). Furthermore, Lhb activity, in turn, can be
modulated by the GABAergic and glutamatergic neurons from the VTA. Selective stimulation
of VTA GABAergic neurons that project to the Lhb was shown to inhibit Lhb neuronal firing
to promote reward-seeking behaviour (Stamatakis et al., 2013), whereas the stimulation of
VTA glutamatergic neurons to the Lhb had the opposite effect (Root et al., 2014). Therefore,
the VTA is able to modulate its own neuronal activity via its efferent projections to the Lhb. In
addition to the extrinsic GABAergic projections, the effects of local GABAergic interneurons
on VTA neuronal activity have also been recently elucidated. Selective optogenetic stimulation
of GABAergic interneurons within the VTA was shown to reduce the spontaneous firing rate
of VTA dopaminergic neurons that in turn led to the production of conditioned place aversion
and the disruption of reward consumption (Tan et al., 2012; Van Zessen et al., 2012).
43
1.4.2 The Role of Mesolimbic Dopamine in Depression and Drug Addiction
Given the ability of the mesolimbic dopamine system to modulate both reward and
aversion, numerous studies have investigated the role of the mesolimbic dopamine system in
neuropsychiatric disorders that are characterized by dysfunctional brain reward function,
mainly depression and drug addiction (Nestler and Carlezon, 2006a; Koob and Volkow, 2010a;
Baik, 2013; Russo and Nestler, 2013).
One of the major symptoms of depression is the emergence of anhedonia, which is the
inability to perceive natural reward (Der-Avakian and Markou, 2012). Therefore, it has been
postulated that the mesolimbic dopamine system may be critically involved in the
pathophysiology of depression (Russo and Nestler, 2013). Studies have shown that chronic
restraint stress and repeated social defeat stress, which are rodent models for depression,
increased spontaneous and burst/phasic firing of VTA dopaminergic neurons both in vivo and
ex vivo in brain slices (Anstrom and Woodward, 2005; Chaudhury et al., 2013). More
importantly, chronic administration of the antidepressant fluoxetine was able to reverse the
enhancement of VTA dopaminergic neuron activity induced by both chronic stress models for
depression (Anstrom and Woodward, 2005; Chaudhury et al., 2013). Nevertheless, the precise
involvement of the mesolimbic dopamine system in depression is complicated by two recent
optogenetic studies that yielded opposite conclusions. In the study by Chaudhury et al (2013),
optogenetically induced burst/phasic firing of VTA dopaminergic neurons was shown to
produce a susceptible, depression-related behavioural phenotype in mice that underwent
subthreshold social defeat stress, as well as in previously resilient animals that underwent
repeated social defeat stress. They further showed that the pro-depressive effect of VTA
dopaminergic neuron burst firing was mediated by dopamine release into the NAc but not the
44
mPFC. In contrast, the study by Tye et al (2013) demonstrated that optogenetic phasic
stimulation of VTA dopaminergic neurons reversed the depressive behavioural phenotypes in
animals that were subjected to chronic mild stress, and that the antidepressant-like activity of
VTA dopaminergic stimulation was dependent on dopamine receptor activation in the NAc. In
addition to the differences in the stress models used, a possible explanation for the
discrepancies between the two studies would be that each study may have targeted different
subpopulations of VTA dopaminergic neurons that project to the NAc (Walsh and Han, 2014).
Indeed, as mentioned earlier, two distinct subpopulations of VTA dopaminergic neurons that
innervate the NAc were identified that differ drastically in their electrophysiological properties
(Hnasko et al., 2012). Therefore, it is likely that the optogenetic stimulation of each
subpopulation may result in different behavioural outcomes. Nevertheless, both studies
confirmed the potential involvement of the mesolimbic dopamine system in depression-related
behaviours despite the opposite direction of modulation.
In addition to major depression, numerous studies have conclusively demonstrated that
the mesolimbic dopamine system plays a central role in the pathogenesis of drug addiction
(Wolf et al., 2004; Thomas et al., 2008; Koob and Volkow, 2010a; Baik, 2013). Specifically, it
has been postulated that drugs of abuse hijack the brain’s reward system to produce abnormal
neuroadaptations in the mesolimbic circuitry, thus leading to heightened responses to drug-
associated stimuli and persistent drug-seeking behaviours despite negative consequences
(Lüscher and Malenka, 2011). All drugs of abuse, regardless of their respective mechanisms of
actions, ultimately result in increased dopamine release from the VTA to the NAc and an
AMPA glutamate receptor-mediated LTP of the VTA dopaminergic neurons (Lüscher and
Malenka, 2011). Moreover, although natural rewards such as food also induced LTP of VTA
45
dopaminergic neurons, its effect was transient, while the LTP caused by cocaine self-
administration was shown to last for more than 3 months (Chen et al., 2008). However, the
heterogeneity of VTA dopaminergic neurons necessitates further studies to identify the
selective neurons that are modified by drugs of abuse. To address this issue, a recent study by
Lammel et al. (2012) found that cocaine potentiated the excitatory synapse of VTA
dopaminergic neurons that project to the NAc medial and lateral shell, but not the synapse of
neurons that innervate mPFC, thus establishing the critical involvement of VTA-NAc shell
projection in mediating the behavioural effect of drugs of abuse.
1.5 The D1-D2 Heteromer as a Modulator of Brain Reward Function: Relevance to
Addiction and Depression
As described earlier in Section 1.3.6, D1-D2 heteromer stimulation modulated the
expression of several proteins in the NAc, including CaMKII, BDNF, and GAD67 (Hasbi et al.,
2009; Perreault et al., 2012a). Interestingly, each of these proteins has been previously
implicated in reward-related behaviours in the context of drug addiction and depression (Xi et
al., 2003; Fuchs et al., 2008; Russo and Nestler, 2013; Robison, 2014; Li and Wolf, 2015).
Thus, the D1-D2 heteromer may act as a modulator of brain reward function via its ability to
modulate the expression of CaMKII, BDNF, and GAD67 in the NAc.
46
1.5.1 CaMKII
CaMKII is a multifunctional Ser/Thr protein kinase that is critically involved in
learning and memory, with the α and β isoforms being the most abundant in the brain (Hudmon
and Schulman, 2002). A rise in intracellular calcium level initiates the binding of CaM to
CaMKII to relieve autoinhibition and allow the phosphorylation of its substrates following
autophosphorylation at Thr286 (Coultrap et al., 2012). Therefore, the calcium signalling
elicited by acute D1-D2 heteromer stimulation was able to increase the activation and total
expression of CaMKII in the NAc (Hasbi et al., 2009). However, repeated D1-D2 heteromer
stimulation was shown to, unexpectedly, reduce total CaMKII expression in the NAc (Perreault
et al., 2010).
Recent studies have implicated CaMKII function in the NAc in reward-related
neuropsychiatric disorders such as drug addiction and major depression due to its ability to
modulate synaptic plasticity in the mesolimbic reward system, mainly via the phosphorylation
of AMPA and NMDA glutamate receptors (Reviewed in: Robison 2014). Specifically, repeated
exposure to psychostimulants such as cocaine and amphetamine was shown to increase
CaMKII expression in the NAc (Loweth et al., 2010; Robison et al., 2013), which in turn
resulted in the accumulation of transcription factor ΔFosB to lead to increased expression of
several genes that could mediate the subsequent drug-seeking behaviours (McClung and
Nestler, 2003; Robison et al., 2013). Indeed, herpes simplex viral (HSV) vector-mediated
overexpression of CaMKII in the NAc was found to enhance amphetamine self-administration
(SA) (Loweth et al., 2010), whereas pharmacological inhibition of CaMKII in the NAc shell
was shown to attenuate the cocaine-induced reinstatement of cocaine-seeking behaviours, a
behavioural paradigm that models relapse to drug-taking in humans (Anderson et al., 2008).
47
With respect to depression, it has been shown that HSV vector-mediated inhibition of CaMKII
activity in the NAc prevented the manifestation of depressive phenotype as measured by
sucrose preference test in rats that underwent chronic social defeat stress, a commonly used
paradigm to induce a depressive state in rodents (Robison et al., 2014). Conversely, chronic
social defeat stress alone enhanced the binding of ΔFosB to CaMKII promoter to increase its
transcription, an effect that was abolished by choric treatment of antidepressant fluoxetine
(Robison et al., 2014). Collectively, these findings clearly implicated a role for CaMKII in
mediating reward-seeking and depression-related behaviours. Therefore, as acute and chronic
D1-D2 heteromer stimulation were able to differentially affect CaMKII expression in the NAc
(Hasbi et al., 2009; Perreault et al., 2010), this receptor complex may thus be potentially
involved in the etiology of drug addiction and major depression.
1.5.2 BDNF
BDNF is a neurotrophic factor that is important for neuronal cell growth and survival
(Lessmann et al., 2003). BDNF is synthesized as a pro-peptide (32kDa) in the ER and is
proteolytically processed either intracellularly or extracellularly into its mature form (14kDa).
Although bdnf mRNA is present in the NAc, BDNF in the NAc was thought to originate
predominantly from the PFC and VTA through anterograde axonal transport rather than from
local synthesis (Lessmann et al., 2003). However, we have demonstrated that D1-D2 heteromer
stimulation resulted in increased BDNF expression in striatal neurons and in the NAc (Hasbi et
al., 2009), thus providing a novel mechanism through which BDNF can be synthesized locally
in the NAc. The binding of mature BDNF to its receptor TrkB, initiates TrkB dimerization and
48
autophosphorylation of intracellular tyrosine residues, which would lead to the activation of
signalling cascades including Ras/ERK, phosphoinositide 3-kinase (PI3K)/Akt, and
phospholipase Cγ (PLC-γ) /Ca2+
pathways (Corominas et al., 2007).
Several studies have implicated BDNF in the NAc as an important mediator of drug-
seeking and depression-related behaviours. For instance, the development of stable cocaine SA
behaviour was associated with increased BDNF expression in the NAc (Grimm et al., 2003),
and the infusion of BDNF or BDNF antibody into the NAc was shown to respectively enhance
or reduce cocaine SA (Graham et al., 2007a). Similarly, increased BDNF expression was also
observed in the NAc of animals that underwent chronic social defeat stress (Berton et al., 2006).
Moreover, animals that exhibited reduced immobility in the forced swim test, another a
commonly used animal model for depressive behaviour, exhibited reduced BDNF expression
in the NAc (Sequeira-Cordero et al., 2014), whereas animals displaying anhedonic behavior in
the sucrose preference test showed increased NAc levels of BDNF mRNA (Bessa et al., 2009).
Consequently, as D1-D2 heteromer stimulation is able to modulate BDNF expression in the
NAc, it is possible that this receptor complex may similarly modulate the drug-seeking and
depression related behaviours that are, in part, mediated by BDNF in the NAc.
1.5.3 GAD67
Glutamic acid decarboxylase (GAD) is an enzyme that is responsible for the
decarboxylation of glutamate to produce GABA (Kaufman et al., 1991). Two distinct isoforms
of GAD exists, namely GAD65 and GAD67, that differ in their subcellular localizations,
functions, regulatory properties and co-factor interactions (Kaufman et al., 1991). Due to its
49
function as a major GABA producer, the level of GAD in the brain closely reflects the
inhibitory GABAergic activity in regions where it is expressed. Particularly, the expression of
GAD67 was shown to be increased in the NAc following D1-D2 heteromer stimulation
(Perreault et al., 2012a), suggesting that the D1-D2 heteromer may exert negative modulation
on the neuronal activity in this region. Indeed, further analysis showed that D1-D2 heteromer
stimulation also enhanced GABA to glutamate ratio in the NAc (Perreault et al., 2012a).
The dysregulation of GABAergic activity within the NAc has been shown to be
associated with the etiology of drug addiction and depression. For instance, the development of
cocaine SA was accompanied by a reduction in GABA level in the NAc, whereas the
extinction of such cocaine-seeking behaviour was associated with increased GABA level in the
same region (Wydra et al., 2013). The microinjection of a GABA agonist, baclofen, into the
NAc also attenuated context-induced reinstatement of cocaine SA (Fuchs et al., 2008). On the
other hand, increased GABA level was observed in the NAc of animals that underwent
psychostimulant withdrawal (Xi et al., 2003), a paradigm that also induces a depressive state in
animals (Barr and Markou, 2005a). Along the same lines, the neuronal activity of the NAc was
also found to be reduced in depressed patients (Mayberg et al., 2000). Together these findings
suggest that enhanced GABAergic activity in the NAc would not only suppress reward-seeking
behaviour, but also further promote a depressive state, likely due to reduced dopamine release
in the same region (Shirayama and Chaki, 2006). Consequently, it is possible that D1-D2
heteromer stimulation, which indirectly increased GABAergic activity in the NAc by
increasing GAD67 expression, may exert negative modulation on reward-seeking behaviours,
and if excessive, may also induce a depressive state.
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6. Research Rationale and Objectives
Emerging evidence has suggested that abnormal dopaminergic neurotransmission in the
mesolimbic circuitry contributes to the pathogenesis of neuropsychiatric disorders involving a
dysfunctional brain reward system, such as depression and drug addiction. However, despite
our growing knowledge of the mesolimbic dopamine system, currently available interventions
that target the dopamine system show little and variable efficacies. As a result, a novel
treatment target is desirable or novel strategy is warranted to better manage the conditions.
Our laboratory has identified and thoroughly characterized a novel dopamine receptor
complex, the dopamine D1-D2 receptor heteromer. We have demonstrated that the D1-D2
heteromer is predominantly expressed in the NAc, a region that is critically involved in brain
reward function. In addition, D1-D2 heteromer stimulation by agonist SKF 83959 leads to
Gαq-mediated, PLC-dependent calcium signalling that subsequently increases the expression
of proteins that have been strongly implicated in drug addiction and depression in the NAc, and
thus it is possible that the D1-D2 heteromer may also be involved in the pathophysiology of
these neuropsychiatric disorders. Therefore, the goal of my thesis is to characterize the function
of the dopamine D1-D2 receptor heteromer in reward-related behaviours, specifically in the
context of depression and drug addiction. I investigated the effects of D1-D2 heteromer
stimulation by SKF 83959 and its inactivation by the TAT-D1 peptide on depression-related
and reward-seeking behaviours using well-established animal models for aspects of depression
and drug addiction, respectively. In addition, I also examined whether and how D1-D2
heteromer stimulation and inactivation modulate the neuronal activity of specific nuclei within
the brain reward circuitry using immunohistological staining for c-Fos, a commonly used
marker for neuronal activity.
51
OVERALL HYPOTHESIS: D1-D2 heteromer stimulation and inactivation will differentially
modulate reward-related behaviours in rodents.
The specific hypotheses were,
Hypothesis 1: D1-D2 heteromer stimulation and inactivation will bidirectionally modulate
psychostimulant-induced behaviours.
Hypothesis 2: D1-D2 heteromer stimulation will induce a depression- and anxiety-like
behavioural phenotype, while D1-D2 heteromer inactivation will exert antidepressant-like and
anxiolytic activities.
Hypothesis 3: D1-D2 heteromer stimulation and inactivation will differentially modulate the c-
Fos expression in specific nuclei of the brain reward circuitry.
52
2. The role of the dopamine D1-D2 receptor heteromer in cocaine addiction.
Abstract
The dopamine D1-D2 receptor heteromer is a novel dopamine receptor complex that is
predominantly expressed in the NAc, and its physiological function has yet to be elucidated.
Upon stimulation by agonist SKF 83959, the D1-D2 heteromer elicits a Gαq-mediated, PLC-
dependent calcium signal that subsequently increased expression of CaMKII and BDNF in the
NAc. As both of these proteins have been critically implicated in the etiology of cocaine
addiction, the aim of the current study was to investigate the effects of D1-D2 heteromer
stimulation and inactivation on cocaine-induced behaviours using animal models including the
conditioned place preference (CPP), locomotor sensitization, and intravenous self-
administration (SA) models. The results showed that D1-D2 heteromer stimulation by SKF
83959 induced conditioned place aversion (CPA), abolished the acquisition and expression of
cocaine CPP, prevented the expression of locomotor sensitization to cocaine, attenuated the
maintenance of cocaine SA, and obviated cocaine- and cue-induced reinstatement of cocaine
SA. In contrast, selective D1-D2 heteromer inactivation by the heteromer-disrupting TAT-D1
peptide consistently produced an opposite effect in all three behavioural models examined,
thereby affirming the role of the D1-D2 heteromer in these cocaine-induced behaviours. More
importantly, the sole treatment of the TAT-D1 peptide induced CPP, indicating that the D1-D2
heteromer exerts tonic inhibitory modulation on brain reward function. Collectively, these
findings indicate that the D1-D2 heteromer inhibits cocaine-induced behaviours, and thus may
potentially be utilized as a novel therapeutic target for cocaine addiction.
53
1. Introduction
Cocaine addiction is a chronic relapsing neuropsychiatric disorder that is characterized
by the persistent use of cocaine despite the adverse psychosocial and physiological
consequences. The rewarding effect of cocaine is attributed to its ability to enhance dopamine
level in the mesolimbic circuitry by blocking the reuptake of endogenously released dopamine
via the inhibition of the dopamine reuptake tranportors (DAT) (Gawin, 1991). Approximately
0.3 million individuals suffered from cocaine addiction in Canada in 2011 (Statistics Canada,
2011), and there is currently no definitive therapeutic intervention available for the treatment of
the condition.
The three most commonly used animal tests for studying aspects of cocaine addiction
include conditioned place preference (CPP), locomotor sensitization, and drug self-
administration (SA). The conditioned place preference paradigm is a commonly used animal
model that examines the rewarding or aversive effects of drugs (Tzschentke, 1998). After
repeatedly pairing the hedonic value of a drug with a specific environment, the animal’s
preference to that environment is then assessed in comparison to a saline-associated neutral
environment in a free-choice, drug-naive trial. Conditioned place preference (CPP) and
conditioned place aversion (CPA) are defined respectively by the animal spending more or less
time in the drug-paired context compared to the saline-paired context (Tzschentke, 1998).
Cocaine exhibits a classic biphasic effect in the CPP model, where low to moderate doses of
cocaine (5-20mg/kg) induce CPP and a high dose of cocaine (30mg/kg) produces CPA
(Reviewed in: Aguilar et al. 2009).
54
Locomotor sensitization to cocaine is the progressive augmentation of locomotor
responsiveness to the drug following repeated administration of the same dose in the same
environmental context (Robinson and Berridge, 1993). This phenomenon consists of two
phases: the initiation and the expression. The initiation refers to the successive enhancement of
locomotor activity following daily cocaine injections that is associated with an increase in
extracellular dopamine release in the NAc as a result of reduced D2R autoreceptor inhibition of
VTA dopaminergic neurons (Henry et al., 1989). The expression of sensitization is
characterized by the persistent hyper-responsiveness to the priming injection of cocaine
following a period of withdrawal, which is associated with long-term neuroadaptations within
the mesolimbic circuitry that may underlie the compulsive drug cravings in humans.
Amongst the three animals tests used in this study to assess aspects of cocaine addiction,
the intravenous self-administration paradigm (SA) has the best face validity since it directly
measures drug-taking behaviour as the number of active lever presses and total cocaine
infusions (Woolverton, 1992; Mello and Negus, 1996). In addition, the reinstatement phase of
cocaine SA also models the persistent relapse to drug-taking in humans that frequently occurs
even after a long period of abstinence.
Numerous studies to date have strongly implicated calcium calmodulin kinase II
(CaMKII), dopamine and cAMP-regulated protein phosphatase (DARPP-32), and brain-
derived neurotrophic factor (BDNF) in the NAc in mediating these cocaine-induced behaviours
(Svenningsson et al., 2005; Anderson et al., 2008; Li and Wolf, 2015), and the expressions of
these proteins specifically in the D1R- or the D2R-expressing MSNs were shown to have
differential behavioural effects (Bateup et al., 2008; Lobo et al., 2010a; Robison et al., 2013).
55
More recently, evidence has also emerged that indicated the existence of a MSN
population that co-expressed both the D1R and the D2R (~17% of all MSNs) in the NAc of
mice (Bertran-Gonzalez et al., 2008; Matamales et al., 2009) and rats (Perreault et al., 2010),
within which a novel dopamine receptor complex known as the dopamine D1-D2 receptor
heteromer is expressed. The potential physiological relevance of this unique subset of MSNs
with regards to cocaine addiction remains to be elucidated.
The heteromeric complex formation between the D1R and the D2R was initially
identified in HEK293 cells stably co-expressing both receptors by elucidation of a calcium
signal upon the coactivation of both receptors (Lee et al., 2004). The physical interaction
between the D1R and the D2R was later confirmed in HEK293 cells by bioluminescence
resonance energy transfer (BRET) (So et al., 2005), in primary cultured striatal neurons by
fluorescence resonance energy transfer (FRET) (Hasbi et al., 2009), and in native rat striatum
by co-immunoprecipitation and FRET (Lee et al., 2004; Perreault et al., 2010). The D1-D2
heteromer is predominantly expressed in the NAc on approximately 27% of D1R-expressing
MSNs as FRET analysis indicated that 90% of D1R/D2R co-expressing neurons in the NAc
exhibited heteromer formation (Perreault et al., 2010). The expression of the D1-D2 heteromer
was found to be much less in the CP (6-7% of D1R/D2R co-expressing neurons) (Perreault et
al., 2010). Stimulation of the D1-D2 heteromer required agonist occupancy of both constituent
receptors, and generated a Gαq-mediated, PLC-dependent, intracellular calcium release that
subsequently activated CaMKII in the NAc (Rashid et al., 2007; Hasbi et al., 2009; Ng et al.,
2010). The screening of a series of synthetic benzazepines identified a D1R-like agonist, SKF
83959, that can stimulate a calcium signal without affecting the canonical cAMP pathway
mediated by homomeric D1R and the D2R (Rashid et al., 2007). Using SKF 83959, it was
56
subsequently shown that D1-D2 heteromer stimulation modulated the expression of DARPP-32
(unpublished finding, manuscript in preparation), BDNF (Hasbi et al., 2009), and ΔFosB
(unpublished finding, manuscript in preparation) in the NAc in a manner that was opposite to
that was induced by cocaine administrations. It is likely that such opposing effects of D1-D2
heteromer stimulation on cocaine-induced actions at the molecular level may also be reflected
behaviourally in animal models for cocaine addiction.
Therefore, the purpose of the present study was to examine the physiological effects of
the D1-D2 heteromer stimulation by agonist SKF 83959 in the context of cocaine addiction
using behavioural models including CPP, locomotor sensitization, and SA paradigms. In
addition, as SKF 83959 was reported to have affinity for, or activate, a number of other
receptors, such as the dopamine D5 receptor, the α-adrenergic receptor 2C, and the serotonin
5HT-2C receptor (Sahu et al., 2009; Perreault et al., 2012b; Chun et al., 2013), we have
synthesized a peptide that can selectively disrupt and antagonize the D1-D2 heteromer by
preventing the two glutamic acid residues in the C-terminus of the D1R from electrostatically
interacting with the two arginine residues in the third intracellular loop of the D2R (O’Dowd et
al., 2012; Hasbi et al., 2014). The behavioural effect of D1-D2 heteromer inactivation by this
highly selective disrupting TAT-D1 peptide on cocaine-induced behaviours was also examined.
2. Materials and Methods
2.1 Animals
Adult male Sprague-Dawley rats (Charles River, Canada), weighing 300-350 g at the
start of the experiment, were used. Rats were housed in polyethylene cages in a temperature-
57
controlled colony room, maintained on a 12-h light-dark cycle (lights on at 0700h), with ad
libitum access to food and water. Rats were handled daily for 5 days before the start of the
experiment. All treatments were performed during the light phase of the day-night cycle.
Animals were housed and tested in compliance with the guidelines described in the Guide to
the Care and the Use of Experimental Animals (Canadian Council on Animal Care), and were
approved by the Animal Care Ethics Committee of the University of Toronto. Experimentally
naïve animals were used for each behavioural study.
2.2 Drugs
SKF 83959 hydrobromide (Tocris Bioscience) was dissolved in physiological saline
containing 5% DMSO, and was administered subcutaneously (s.c.). Amphetamine
hydrochloride (Sigma-Aldrich) was dissolved in physiological saline (0.9% NaCl), and was
administered intraperitoneally (i.p.). The TAT-D1 disrupting peptide was dissolved in saline
and administered into the intracerebroventricular (i.c.v.) space 15 minutes prior to vehicle, SKF
83959, or amphetamine injection. For non-drug injections, an equivalent volume of saline or
vehicle was administered. All systemic injections were given at a volume of 1.0 ml/kg just prior
to behavioural testing.
2.3 Surgery
Rats were anesthetized with isoflurane (5%), administered analgesic ketoprofen (5
mg/kg, s.c.) and secured in a stereotaxic frame. A cannula (22-gauge, Plastics One) was placed
58
unilaterally into the intracerebroventricular space close to the midline according to the
following stereotaxic coordinates: AP -0.8mm, ML + 1.3mm, DV – 3.7mm, and was secured by
dental cement anchored with four stainless steel screws (Plastics One) fixed on the dorsal
surface of the skull. AP and ML coordinates were taken from bregma, DV coordinate from the
dura (Paxinos and Watson, 1998). The animals were allowed to recover in their home cage for a
minimum of five days before the experiments were performed. Cannulae placement was
visually validated postmortem in brain slices. For the self-administration studies, a catheter
constructed of silastic tubing was surgically implanted in the right jugular vein. The terminal
end of the catheter consisted of a 22-gauge guide cannula (Plastics One) and was anchored
subcutaneously between the scapulae with a small piece of Marlex mesh.
2.4 Apparatus
2.4.1 Place Preference Chamber
The place preference chamber (Harvard Apparatus, UK) consisted of two equally sized
compartments (45cm in height, 34cm in width, and 40cm in length) interconnected by a
transparent rectangular corridor. The compartments were differentiated by the motifs painted
on the walls (dots or stripes) and the colour (different shade of grey tones, light or dark) and
texture (smooth or rough) of the floor.
59
2.4.2 Locomotor Activity Chamber
The locomotor activity chambers were 20cm in height, 25cm in width, and 40cm in
length. Two arrays of 16 infrared photocells were attached along the longer sides of the
polyethylene cages. The activity chambers were interfaced to a computer that provided
automated recording of horizontal locomotor activity when both top and bottom infrared
photocells were triggered. Ventilated polyethylene lids were used to cover the activity
chambers to prevent animals from escaping.
2.4.3 Operant Chamber
The operant chambers were 21cm in height, 21 cm in width, and 28 cm in length (Med.
Associates Inc., USA). Each chamber contained two response levers, with a Sonalert and a
stimulus light located above each lever. A syringe mounted on a motor driven pump (Razel)
delivered cocaine infusions to the rat’s jagular catheter via Tygon tubing attached to a swivel
located above the test chamber. Each chamber was illuminated by a house light and housed in a
sound-attenuating box equipped with a ventilating fan. All boxes were controlled by a PC
running Med-PC-IV. All programs were written in-house.
2.5 Experimental Procedures
2.5.1 Conditioned Place Preference
The animals were first habituated to the CPP chambers for 2 days, followed by the
measurement of their baseline preference for each of the two chambers. The baseline
60
preference determined which side of the chamber the drug would be paired for a balanced
experimental design. To examine the effects of the D1-D2 heteromer on cocaine CPP
acquisition, the rats underwent 6 days of conditioning sessions, during which they received 3
drug treatments (vehicle, 1.0mL/kg; SKF 83959, 1.0mg/kg, s.c.; cocaine, 10mg/kg, i.p.; TAT-
D1 peptide, 300pmol, i.c.v.; TAT-Sc peptide, 300pmol, i.c.v.) and 3 saline treatments in
alternating order. Immediately after the injection the rats were confined to the assigned
chamber for 30 minutes. The TAT-D1 peptide was given 15 minutes prior to the start of each
conditioning session. On the test day, the rats were allowed to freely explore the two chambers,
and the time the animals spent in each chamber was recorded. To examine the effects of the
D1-D2 heteromer on the expression of cocaine CPP, animals first underwent 6 days of
conditioning sessions with cocaine (10mg/kg, i.p.) and saline in alternating order. On the test
day, the animals received an injection of SKF 83959 (2.5mg/kg, s.c.) or vehicle, and the time
they spent in each chamber was recorded. Place preference or aversion was established if the
animal spent significantly more or less time in drug-paired over saline-paired chamber, and
thus each rat acted as its own control. Animals that spent more than 40% of total time in the
middle compartment were excluded.
2.5.2 Locomotor Sensitization
The animals were first habituated to the locomotor chamber for 3 days following which
they received the assigned drug treatment daily for 7 days (saline, 1.0 ml/kg, i.p.; SKF 83959,
0.4 mg/kg, s.c.; TAT-D1 peptide, 300pmol, i.c.v.; cocaine, 10 mg/kg, i.p.; cocaine + SKF
83959, cocaine + TAT-D1 peptide). The dose of SKF 83959 was previously shown to attenuate
61
amphetamine-induced locomotor sensitization without desensitizing the D1-D2 heteromer over
repeated injections (Shen et al., 2014b), while the dose of cocaine was the same as described by
Robison et al (2014). The locomotor activity of the animals was measured for a total of 60
minutes daily, 30 minutes prior and 30 minutes following the assigned treatment over 7
injection days (Referred to as the development of locomotor sensitization). All animals received
a single injection of priming cocaine (5.0 mg/kg, i.p.) on day 8 following a 24 hour withdrawal,
and their locomotor activity was again evaluated (Referred to as the expression of locomotor
sensitization).
2.5.3 Intravenous Self-Administration
Adult male Sprague-Dawley rats under a restricted diet were first trained to lever-press
for food under an FR1 schedule. Rats were allowed a total of 100 food pellets during each 30
minute training session. The rats that consumed 100 pellets for 3 consecutive days they were
considered lever-trained. Trained animals then underwent surgery for intravenous catheter
implantation to allow for IV cocaine infusion. The animals were allowed to recover from
surgery for a week, and then were first trained for cocaine SA under the FR1, FR3, and then
eventually FR5 schedules of reinforcement. Cocaine infusions were delivered only when the
left (active) lever was pressed, and the delivery was associated with a 5 second light cue
located directly above the left lever. Pressing of the right (inactive) lever had no functional
consequence. For each infusion the animals received 0.25 mg cocaine/0.1mL/5.5s. Once stable
responding was achieved, the dose response relationship of D1-D2 heteromer stimulation (SKF
83959, 0.05, 0.5, 1.5mg/kg, s.c.) and cocaine SA under the FR5 schedule was examined. Next,
62
the animals underwent extinction training, during which no cocaine was infused following
active lever presses. Extinction was achieved when responding on the active lever had reached
a stable level of less than 15 responses over 2h. Once the extinction training was completed,
the animals underwent surgery for intracerebroventricular cannula implantation for TAT-D1
peptide infusions. Following a week of recovery, the effects of D1-D2 heteromer stimulation
(SKF 83959, 0.5, 1.5mg/kg, s.c.) and inactivation (TAT-D1 peptide, 300pmol, i.c.v.) on
cocaine- (5 or 10mg/kg, i.p.) and cue-induced reinstatement of cocaine SA was then
investigated
2.6 Statistical Analysis
All data are reported as mean ± s.e.m, and was analyzed for normality prior to
ANOVAs using the Shapiro-Wilk test. Post-hoc analysis following ANOVAs was performed
using Duncan’s Multiple Range test or Student’s t-test for planned comparisons. Computations
were performed using the SPSS statistical program. Statistical criteria for significant
differences were set at p<0.05.
2.6.1 Conditioned Place Preference
The time spent in the drug-paired chamber versus the saline-paired chamber for each
drug treatment was compared using paired Student’s t-test.
63
2.6.2 Locomotor Sensitization
For the development of locomotor sensitization, the total horizontal activity was
analyzed using repeated measures of ANOVA with Injection (Injections 1 to 7) as the within
subject factor and Treatment (Veh, Cocaine, SKF 83959, TAT-D1 peptide, Cocaine+SKF
83959, Cocaine+TAT-D1 peptide) as the between subject factor. For the expression of
locomotor sensitization, the total horizontal activity was analyzed using one-way ANOVA with
Treatment (Veh, Cocaine, SKF 83959, TAT-D1 peptide, Cocaine+SKF 83959, Cocaine+TAT-
D1 peptide) as the between subject factor. Planned comparisons between groups were done
using Student’s t-test. Planned comparions were conducted in Figure I-5, Expression of cocaine
sensitization, between the following groups: Saline vs. Cocaine; Cocaine vs. SKF+Cocaine;
Cocaine vs. TAT-D1+Cocaine.
2.6.3 Intravenous Self-Administration
For the dose response study that examined the effect of D1-D2 heteromer stimulation
by SKF 83959 on the maintenance of cocaine SA under the FR5 schedule of reinforcement, the
active lever response was analyzed using repeated measures of ANOVA with Dose (0, 0.05,
0.5, 1.5mg/kg SKF 83959) as the within-subject factor. For the reinstatement studies, the active
lever response was analyzed using repeated measures of ANOVA with Treatment (Cocaine,
Cue, SKF 83959, TAT-D1 peptide, Cocaine+SKF 83959, Cue+SKF 83959, Cocaine+TAT-D1
peptide) as the within-subject factor. Planned comparisons between groups were done using
paired Student’s t-test. Planned comparions were conducted in Figure I-8C, Effect of TAT-D1
on cocaine-induced reinstatement, between the following groups: Saline vs. 10mg/kg Cocaine;
64
5mg/kg Cocaine vs. 5mg/kg Cocaine+TAT-D1; 10mg/kg Cocaine vs. 10mg/kg Cocaine+TAT-
D1.
3. Results
3.1 Conditioned Place Preference
We first investigated the effects of D1-D2 receptor heteromer activation and disruption
on basal CPP behaviour. Saline treated animals received saline in both chambers and did not
exhibit preference towards a specific chamber on average (Figure I-1A). SKF 83959 induced
place aversion as the animals spent significantly less time in the drug-paired chamber
compared to the saline-paired chamber on the test day (Figure I-1A, 432.11±80.83s vs.
1055.22±92.26s, t(9)=3.72, p<0.01). In contrast, disruption of the D1-D2 receptor heteromer
by the TAT-D1 peptide alone, but not the scrambled peptide, resulted in place preference
(Figure I-1B, 812.00±59.54s vs. 514.67±27.38s, t(9)=3.87, p<0.01). To further confirm the role
of the D1-D2 receptor heteromer in the induction of place aversion by SKF 83959 treatment,
the animals were co-treated with SKF 83959 plus the TAT-D1 peptide or the scrambled
peptide during the conditioning sessions (Figure I-1C). The place aversion induced by SKF
83959 was abolished by the TAT-D1 peptide but not the scrambled peptide, suggesting that the
aversive effect was mediated by D1-D2 receptor heteromer activation.
The effects of D1-D2 receptor heteromer activation and disruption on the acquisition
and expression of cocaine-induced CPP were next examined. Repeated cocaine treatment
successfully induced a place preference as expected, as the animals spent significantly more
A) B)
65
Figure I-1: The effects of D1-D2 heteromer stimulation and inactivation on basal
conditioned place preference. A) Vehicle-conditioned animals did not exhibit a preference
towards a particular chamber. D1-D2 heteromer stimulation by SKF 83959 induced a
conditioned place aversion as the animals spent significantly less time in the drug paired
chamber, whereas B) its inactivation by TAT-D1 resulted in a conditioned place preference as
the animals spent significantly more time in the drug paired chamber. C) The CPA induced by
SKF 83959 was abolished by the pre-treatment of the TAT-D1 but not the TAT-Sc peptide.
Data represent means ± s.e.m. of n=8-10 rats/group. (*p<0.05, **p<0.01: compared to saline-
paired).
Veh
icle
SKF 8
3959
0
250
500
750
1000
1250
Saline PairedDrug Paired
**
Tim
e (
s)
TAT-D
1-SKF
TAT-S
c-SKF
0
250
500
750
1000
1250
Saline PairedDrug Paired
*
Tim
e (
s)
TAT-D
1
TAT-S
c0
250
500
750
1000
1250
Saline PairedDrug Paired
**
Tim
e (
s)
A) B) C)
66
Figure I-2: The effects of D1-D2 heteromer stimulation and inactivation on cocaine-
induced CPP. A) Vehicle-conditioned animals did not exhibit a preference towards a
particular chamber. Cocaine-conditioned animals exhibited a conditioned place preference
(CPP) as they spent significantly more time in the drug paired chamber. The acquisition
cocaine CPP was respectively abolished and enhanced by SKF 83959 and the TAT-D1 peptide.
B) A single injection of vehicle or SKF 83959 did not affect the chamber preference of vehicle-
conditioned animals. A single injection of vehicle also did not affect the CPP of cocaine-
conditioned animals. However, a single injection of SKF 83959 abolished the expression of
cocaine CPP in cocaine-conditioned animals. Data represent means ± s.e.m. of n=8-10
rats/group. (*p<0.05, **p<0.01: compared to saline-paired).
Veh
icle
Coca
ine
SKF-C
oc
TAT-D
1-Coc
0
250
500
750
1000
1250
Saline PairedDrug Paired
* **
Tim
e (
s)
Veh
+Veh
SKF+V
eh
Coc+
Veh
Coc+
SKF
0
250
500
750
1000
1250
Saline PairedDrug Paired
**
Tim
e (
s)
A) B)
67
Figure I-3: The effect of Cdk5 inhibitor roscovitine on SKF 83959-induced CPA. Animals
conditioned with SKF 83959 exhibited conditioned place aversion (CPA) as the spent
significantly less time in the drug paired chamber. The CPA induced by D1-D2 heteromer
stimulation was abolished by roscovitine pre-treatment. Data represent means ± s.e.m. of n=8-
10 rats/group. (*p<0.05: compared to saline-paired).
Veh
icle
Rosc
o0
250
500
750
1000
1250
Saline Paired83959 Paired
*
Tim
e (
s)
68
time in the cocaine-paired chamber compared to the saline-paired chamber (Figure I-2A,
1050.11±101.53s vs. 581.67±88.54s, t(9)=2.52, p<0.05). Co-treatment of cocaine with SKF
83959 during the conditioning sessions abolished the acquisition of cocaine CPP (Figure I-2A,
613.88±87.85s vs. 815.38±122.23s, p>0.05). Co-treatment of cocaine with the TAT-D1 peptide
during the conditioning sessions had no apparent effect on cocaine CPP, but did increase in
statistical significance (Figure I-2A, 1028.14±122.06s vs. 379.86±75.76s, t(7)=3.28, p<0.01).
The expression of cocaine CPP was similarly abolished by a single injection of SKF 83959 on
the test day (2.5mg/kg, s.c.) in animals that were conditioned with cocaine (Figure I-2B,
758.44±147.53s vs. 894.89±131.51s, t(9)=3.55, p<0.01). The single injection of SKF 83959 on
the test day in saline treated animals had no effect, which was an expected outcome given the
aversive effect of D1-D2 receptor heteromer activation was not associated with a particular
environment.
Lastly, the effect of cdk5 inhibitor roscovitine on CPA induced by D1-D2 receptor
heteromer activation was investigated. (Figure I-3) Since it has previously been shown that the
activation of the D1-D2 receptor heteromer led to increased DARPP-32 Thr75 phosphorylation,
which is regulated by cdk5, it was postulated that cdk5 would be a downstream effector
following D1-D2 receptor heteromer activation. Co-treatment of SKF 83959 plus saline or
roscovitine (25-30nmol/0.5µL) during the conditioning sessions demonstrated that the
inhibition of cdk5 successfully blocked the CPA induced by SKF 83959 (696.17±68.12s vs.
667.33±87.46s, p>0.05), suggesting that cdk5 activation may be responsible for the aversive
effects of the D1-D2 receptor heteromer.
69
Figure I-4: The effects of D1-D2 heteromer stimulation and inactivation on locomotion
induced by acute and repeated cocaine treatment. A) Acute treatment of the TAT-Sc, SKF
83959, and TAT-D1 peptide did not affect basal locomotion. Acute cocaine treatment
significantly increased basal locomotion, which was further significantly enhanced by the
TAT-D1 peptide. An increase in statistical significance was found in animals treated acutely
with cocaine plus SKF 83959 compared to the saline control. B) Repeated treatment of saline,
TAT-Sc, SKF 83959, and the TAT-D1 peptide did not affect basal locomotion over the 7-day
injection period. The locomotor activity of animals treated with repeated cocaine increased
steadily over injections, indicating sensitization. Although the locomotor-stimulating effect of
cocaine was retained in animals co-treated with cocaine plus SKF 83959, the sensitization in
locomotor activity was abolished by SKF 83959 co-treatment. Animals treated with cocaine
plus TAT-D1 exhibited significantly higher locomotor activity compared to cocaine-treated
animals across injections, and the sensitization in locomotor activity occurred at an earlier time
point. Data represent means ± s.e.m. of n=8-10 rats/group. (*p<0.05, ***p<0.001: compared to
Saline; #p<0.05, ##p<0.01, ###p<0.001: compared to Cocaine).
Sal
ine
TAT-S
c
SKF 8
3959
TAT-D
1
Coca
ine
SKF+C
oc
TAT+C
oc0
1000
2000
3000
4000
5000
****
***#
Ho
rizo
nta
l A
ctiv
ity
(b
eam
bre
aks)
A)
1 2 3 4 5 6 70
1000
2000
3000
4000
5000
6000
7000
SalineTAT-ScTAT-D1
SKF+CocCocaine
***
TAT+Coc
*
##
### ### ##
#
**
** *** *SKF 83959
Injection
Ho
rizo
nta
l A
cti
vit
y
(b
ea
m b
rea
ks)
B)
70
3.2 Locomotor Sensitization
The total horizontal activity in response to the assigned treatment for 30 minutes was
reported. One-way ANOVA of the results of acute locomotor activity showed a significant
effect of Treatment {F(6,57)=19.37, p<0.001}. As shown in Figure I-4A, the animals’ basal
locomotion was significantly elevated by acute cocaine treatment (10mg/kg, i.p.), which was
not affected by SKF 83959 co-treatment (0.5mg/kg, s.c.) but was further significantly enhanced
by TAT-D1 pre-treatment (300pmol, i.c.v., p<0.05). Repeated measures of ANOVA on
locomotion across the development of sensitization phase revealed significant effects of
Injection {F(6,57)=3.08, p<0.01}, Treatment {F(6,57)=31.81, p<0.001}, and Injection x
Treatment interaction {F(36,57)=4.21, p<0.001}. Across the 7 injections, the locomotor
activity of saline-treated controls remained low, while SKF 83959, TAT-D1 and TAT-
Scrambled peptide treatment each produced a slight and non-significant increase in locomotion
compared to the saline control (Figure I-4B). In contrast, all cocaine-treated groups (cocaine
alone, cocaine plus SKF 83959, and cocaine plus TAT-D1) had significantly higher locomotor
activity compared to the saline control over the 7 injections (Figure I-4B, p<0.001 vs. Saline
group for all 3 groups). Furthermore, animals treated solely with repeated cocaine exhibited a
steady increase in locomotor activity across injections that became significant on Injection 6
and 7 (p<0.01 and p<0.001, vs. Injection 1, respectively), indicating successful development of
locomotor sensitization. SKF 83959 co-treatment abolished the development of locomotor
sensitization to cocaine but did not alter the locomotor-stimulating effect of cocaine, as the
locomotor activity for the SKF 83959 co-treated group remained elevated and unperturbed
throughout the 7 injections. In contrast, TAT-D1 peptide pre-treatment significantly augmented
cocaine-induced locomotor activity at all injections except for Injection 3. Repeated measures
71
Figure I-5: The effects of D1-D2 heteromer stimulation and inactivation on the expression
of cocaine-induced locomotor sensitization. An injection of a subthreshold dose of cocaine
did not affect the basal locomotor activity of animals previously treated with repeated saline,
TAT-Sc, and SKF 83959, but significantly increased the locomotor acitivity of cocaine-treated
animals, indicating expression of sensitization. In response to the cocaine injection at a
subthreshold dose, a sensitized locomotor phenotype was observed in animals previously
treated with repeated TAT-D1 peptide. The expression of locomotor sensitization was
respectively abolished and enhanced by SKF 83959 and the TAT-D1 peptide. Data represent
means ± s.e.m. of n=8-10 rats/group. (*p<0.05, ***p<0.001: compared to Saline; ###p<0.001:
compared to Cocaine).
Salin
e
TAT-S
c
SKF 8
3959
TAT-D
1
Coca
ine
SKF+C
oc
TAT+C
oc0
1000
2000
3000
4000
5000
****
***###
Ho
rizo
nta
l A
ctiv
ity
(b
eam
bre
aks)
5mg/kg Cocaine
Chronic
Pre-treatment
Priming
72
of ANOVA also showed that the TAT-D1 pre-treated group had significantly higher locomotor
activity than the cocaine-alone group across the 7 injections (p<0.001 vs. Cocaine group).
Moreover, the sensitization of locomotor response to cocaine was not only retained in TAT-D1
peptide pre-treated animals, but it occurred at an earlier time point compared to the cocaine
alone group (Injection 4 vs. Injection 6) and remained elevated throughout the rest of the
injection period.
24 hours following the 7th
injection, animals from all treatment groups received a
cocaine challenge (5mg/kg, i.p.) and their locomotor activity was measured (Figure I-5). One-
way ANOVA revealed a significant effect of Treatment {F(6,56)=20.92, p<0.001}. Repeated
treatment of the TAT-Scrambled peptide or SKF 83959 had no effect on the locomotor activity
induced by cocaine challenge compared to the saline-treated animals. Animals with prior
repeated exposure to the TAT-D1 peptide alone and cocaine alone both exhibited significantly
higher locomotor activity in response to cocaine challenge compared to the saline control,
which is indicative of successful expression of locomotor sensitization to cocaine (p<0.05 and
p<0.001, vs. Saline group, respectively). Interestingly, prior SKF 83959 co-treatment with
cocaine abolished, while prior TAT-D1 peptide pre-treatment with cocaine significantly
enhanced (p<0.001 vs. Cocaine group), the expression of locomotor sensitization to cocaine.
3.3 Intravenous Self-Administration
With successive cocaine SA training, the animals exhibited a steady increase in the
number of active lever presses and the number of infusions, with the latter plateaued at
approximately 25 infusions per 2 hour session (Figure I-6A). Once stable responding in the
73
Veh
icle
0.05
mg/k
g
0.5m
g/kg
1.5m
g/kg
0
50
100
150
200
0
50
100
150
200
***
**
InfusionsPresses
Activ
e L
ev
er
Pre
ss
Infu
sio
ns
Figure I-6: The effect of D1-D2 heteromer stimulation on cocaine self-administration
under the FR5 schedule. A) The animals exhibited steady cocaine self-administration
behaviour over the 2 hour session following training. B) SKF 83959 dose-dependently reduced
the number of active lever press and total cocaine infusions. Data represent means ± s.e.m. of
n=15-16 rats/group. (**p<0.01, ***p<0.001: compared to Vehicle).
1 2 3 4 5 6 7 8 90
25
50
75
100
125
150
175
200
0
10
20
30
40
50
60
70
80PressesInfusions
Training Day
Ac
tive
Le
ve
r P
ress
Infu
sio
ns
A) B)
A) B)
74
number of active lever pressed and cocaine infusions were achieved, three different doses of
SKF 83959 were tested (0.05, 0.5, and 1.5 mg/kg) to examine the dose-response relationship
between D1-D2 heteromer stimulation and cocaine self-administration behaviour under the
FR5 schedule of reinforcement (Figure I-6B). Repeated measures of ANOVA revealed a
significant effect of Dose on the number of active lever responses {F(3,16)=9.85, p<0.001} and
the number of cocaine infusions {F(3,16)=12.14, p<0.001}. Post-hoc comparisons further
showed that the pre-treatment with SKF 83959 dose-dependently reduced the number of active
lever press and the number of infusions that was significant at the 0.5mg/kg (p<0.05) and
1.5mg/kg (p<0.01) doses.
The rats then underwent extinction training, during which the number of active lever
presses steadily decreased and was eventually stabilized at 15 presses per 2 hour session
(Figure I-7). Following extinction training, two separate groups of animals were used to
examine the effect of D1-D2 stimulation on cocaine and cue-induced reinstatement using SKF
83959 at the 0.5mg/kg and 1.5mg/kg dose. A third group of animals was used to examine the
effect of D1-D2 heteromer inactivation on cocaine-induced reinstatement. Repeated measures
of ANOVA revealed a significant effect of Treatment on the number of active lever press
(0.5mg/kg SKF 83959: {F(3,8)=11.26, p<0.0001}; 1.5mg/kg SKF 83959: {F(3,10)=22.66,
p<0.0001}; TAT-D1: {F(5,15)=10.15, p<0.0001}). A priming injection of cocaine at 10mg/kg
successfully reinstated the cocaine SA behaviour as indicated by a significant increase in the
number of active lever presses in cocaine-primed animals compared to the vehicle-treated
controls (Figure 8: t(7)=3.44, p<0.05; Figure 3.4b: t(9)=5.14, p<0.01). SKF 83959 co-treatment
at 0.5mg/kg and 1.5mg/kg (Figure I-8A&B) abolished cocaine-induced reinstatement of
cocaine SA behaviour, while SKF 83959 by itself (without cocaine priming) had no effect. In
75
Figure I-7: The lever-pressing behaviour during extinction training. The number of active
lever press steadily declined over the extinction training, which was eventually maintained at
approximately 15 presses per 2 hour session.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 260
10
20
30
40
50
60
70
80
90
100
110
120
Ac
tiv
e L
ev
er
Pre
ss
Training Day
76
Figure I-8: The effects of D1-D2 heteromer stimulation and inactivation on cocaine-
induced reinstatement. A&B) A single injection of saline or SKF 83959 did not reinstate the
SA behaviour. A priming cocaine injection reinstated the SA behaviour, which was abolished
by SKF 83959 at the 0.5 mg/kg and 1.5 mg/kg dose. C) A single injection of TAT-Sc, TAT-D1
or cocaine at a subthreshold dose (5mg/kg) did not reinstate the SA behaviour, whereas the SA
behaviour was reinstated by a priming dose of cocaine (10mg/kg). Pre-treatment of the TAT-
D1 peptide facilitated the reinstatement of SA behaviour induced by a subthreshold dose of
cocaine and further enhanced the reinstatement induced by a priming dose of cocaine. Data
represent means ± s.e.m. of n=10-12 rats/group. (*p<0.05, **p<0.01, ***p<0.001: compared to
Veh and TAT-Sc; #p<0.05: compared to Cocaine).
Veh
1.5m
g/kg S
KF
Coc
1.5
SKF+C
oc0
25
50
75
100
125
150
**
InactiveActive
Lev
er
Pre
sses
TAT-S
c
TAT-D
1
5mg C
oc
TAT-D
1+5o
c
10m
g Coc
TAT-D
1+10
coc
0
25
50
75
100
125#
***
****#
Inactive
Active
Lev
er
Pre
sses
Veh
0.5m
g/kg S
KF
Coc
0.5
SKF+C
oc0
25
50
75
100
125
150
*
InactiveActive
Lev
er
Pre
sses
A) B)
C)
0
25
50
75
100
125
5mg C
oc
TAT-D
1+Coc
Activ
e L
ev
er
Pre
sses
0
50
100
150
200
10m
g Coc
TAT-D
1+Coc
Activ
e L
ev
er
Pre
sses
77
contrast, the co-treatment of the TAT-D1 peptide with the priming cocaine injection at a
subthreshold dose, 5mg/kg (Figure I-8C, t(14)=2.82, p<0.05), and the usual priming dose,
10mg/kg (Figure 8C, t(14)=2.26, p<0.05), further enhanced the number of active lever presses
induced by the cocaine injections (Figure I-8C, t(14)=3.54, p<0.01). Similar to cocaine priming,
the presentation of the light cue that was associated with cocaine delivery during cocaine SA
training was also able to reinstate cocaine SA behaviour (Figure I-9A: t(10)=3.29, p<0.01;
Figure I-9B: t(9)=3.70, p<0.01), an effect that was abolished by SKF 83959 treatment at the
1.5mg/kg dose (Figure I-9B) but not the 0.5mg/kg dose (Figure I-9A). The inactive lever
presses were not affected by any of the drug treatments.
It should be noted that SKF 83959-treated animals exhibited otherwise normal
behaviours such as sniffing and exploring that were comparable to vehicle-treated animals, and
thus the reduction in active lever responses following SKF 83959 administration was not due to
motor impairment. On the contrary, acute SKF 83959 treatment actually enhanced basal
locomotion (Perreault et al., 2010; Shen et al., 2014b).
4. Discussion
In the present study, we assessed the effects of D1-D2 heteromer stimulation by SKF
83959, and its selective inactivation by a disrupting TAT-D1 peptide, on cocaine conditioned
place preference (CPP), locomotor sensitization, and intravenous self-administration (SA),
three most commonly used behavioural models for cocaine addiction. The results consistently
showed that D1-D2 heteromer stimulation attenuated or abolished, whereas its inactivation
78
Figure I-9: The effect of D1-D2 heteromer stimulation on cue-induced reinstatement. The
presentation of a light cue that was previously associated with cocaine infusions was able to
reinstate the SA behaviour. Cue-induced reinstatement of SA behaviour was A) not affected by
0.5mg/kg SKF 83959, but B) was abolished by 1.5mg/kg SKF 83959. Data represent means ±
s.e.m. of n=10-12 rats/group. (**p<0.01: compared to No Cue).
No C
ue
Cue
Alo
ne
Cue+
0.5
SKF
0
25
50
75
100
125
150
****
InactiveActive
Lev
er
Pre
sses
A)
No C
ue
Cue
Alo
ne
Cue+
1.5
SKF
0
20
40
60
80
100
**
InactiveActive
Lev
er
Pre
sses
B)
79
enhanced, cocaine-induced behaviours in all three behavioural paradigms. Specific findings
from each experiment will be discussed in detail in the following sections.
4.1 The Dopamine D1-D2 Receptor Heteromer Modulates Basal and Cocaine Reward
Repeated stimulation of the D1-D2 heteromer by SKF 83959 during the conditioning
sessions produced CPA, indicating that the D1-D2 heteromer stimulation induced an aversion.
Moreover, the SKF 83959-induced CPA was abolished by TAT-D1 peptide-mediated D1-D2
heteromer inactivation, thus validating that the CPA was a D1-D2 heteromer specific effect. In
striking contrast, repeated inactivation of the D1-D2 heteromer by the TAT-D1 peptide during
the conditioning sessions resulted in CPP, indicating that the D1-D2 heteromer is tonically
active and its inhibition was rewarding. Having established that D1-D2 heteromer stimulation
and inactivation were able to bidirectionally modulate basal reward function, we next examined
the role of the D1-D2 heteromer in cocaine reward. Interestingly, stimulation of the D1-D2
heteromer simultaneously with cocaine conditioning abolished the acquisition of cocaine-
induced CPP. Moreover, acute stimulation of the D1-D2 heteromer on the test day eliminated
the expression of cocaine-induced CPP. Taken together, these data suggest that the D1-D2
heteromer possibly exerts a tonic suppression of the brain reward system. Thus, D1-D2
heteromer stimulation augments such suppression to induce a state of aversion, whereas D1-D2
heteromer inactivation removes such suppression to produce a net rewarding effect.
Furthermore, D1-D2 heteromer stimulation not only abolishes cocaine reward per se, but can
also abrogate the associative reward of a reinforcing environmental context.
80
Numerous studied have shown the critical involvement of the D1R or the D2R, as well
as D1R- or D2R-expressing MSNs, in the NAc in the modulation of cocaine CPP. For instance,
systemic or intra-NAc selective antagonism of the D1R by SCH 23390 abolished the
acquisition of cocaine CPP (Cervo and Samanin, 1995; Baker et al., 1996). In contrast,
selective D2R antagonism in the NAc failed to influence the acquisition or expression of
cocaine CPP (Cervo and Samanin, 1995; Shippenberg et al., 1996), although mice lacking pre-
synaptic D2R autoreceptors selectively were shown to exhibit enhanced expression of cocaine
CPP (Bello et al., 2011), potentially due to the lack of presynaptic inhibition of dopamine
release that in turn increased the stimulation of postsynaptic D1R. On the other hand,
optogenetic stimulation of D1R- or D2R-expressing MSNs were shown to respectively enhance
or attenuate the expression of cocaine CPP (Lobo and Nestler, 2011). Similarly, inhibition of
the D1R-expressing MSNs by tetanus toxin attenuated cocaine CPP expression whereas the
abrogation of D2R-expressing MSNs had no such effect (Hikida et al., 2010). Collectively,
these studies clearly demonstrated that cocaine CPP acquisition is critically dependent on D1R
stimulation in the NAc, and its expression is differentially modulated by D1R- and D2R-
expressing MSNs.
Nevertheless, despite the classical understanding that the expression of the D1R and the
D2R in neurons is largely segregated in the NAc (Gerfen et al., 1990; Harrison et al., 1990; Le
Moine and Bloch, 1995; Surmeier et al., 2007), recent evidence has also identified a population
of NAc MSNs in mice and rats that co-express both the D1R and the D2R (Bertran-Gonzalez et
al., 2008, 2010; Matamales et al., 2009; Perreault et al., 2010; Gangarossa et al., 2013a), within
which the D1-D2 heteromer is expressed (Lee et al., 2004; Rashid et al., 2007; Hasbi et al.,
2009, 2014; Perreault et al., 2010, 2011). This unique set of D1R/D2R co-expressing MSNs
81
was shown to have representations in regions associated with the D1R-expressing direct and
D2R-expressing indirect pathways (Deng et al., 2006; Wang et al., 2006, 2007; Perreault et al.,
2011). Therefore, it was hypothesized that this putative third neuronal pathway within the basal
ganglia circuitry may influence the activity of both the direct and indirect pathways (Perreault
et al., 2011), which suggests these neurons may have the capabilities to regulate cocaine CPP.
Indeed, this idea fits with the current findings that showed D1-D2 heteromer stimulation and
inactivation bidirectionally modulated the acquisition of cocaine CPP. Therefore, it was
demonstrated for the first time the potential physiological relevance of the D1R/D2R co-
expressing MSNs in the NAc in the negative modulation of cocaine reward.
There are a number of mechanisms by which activation of the D1-D2 heteromer in
D1R/D2R-coexpressing MSNs may have regulated cocaine CPP. These D1R/D2R co-
expressing MSNs are also unique in that they exhibit a dual GABA/glutamate co-expressing
phenotype (Perreault et al., 2012a), potentially allowing them to modulate the neuronal activity
of their efferent targets or locally in the NAc. Indeed, D1-D2 heteromer stimulation was shown
to enhance the expression of a major GABA-producing enzyme, GAD67, and to increase the
GABA to glutamate expression ratio in the NAc (Perreault et al., 2012a). A recent study has
demonstrated that a reduction in GABA-mediated inhibition of MSNs in the NAc promoted the
expression of cocaine CPP (Maguire et al., 2014), suggesting that increased GABA activity in
the NAc, as was observed following acute D1-D2 heteromer stimulation, may function to
suppress cocaine CPP, which is consistent with the current finding.
Another possibility is that the effect of the D1-D2 heteromer on cocaine CPP was
mediated, at least in part, by D1-D2 heteromer-induced changes in BDNF expression and
release (Hasbi et al., 2009). Calcium signalling elicited by D1-D2 heteromer stimulation was
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shown to increase the phosphorylation and activation of CaMKII, which in turn enhanced
BDNF expression in the NAc (Hasbi et al., 2009), potentially via a transcriptional mechanism
(Zheng et al., 2009). Although increased BDNF in the NAc has been associated with enhanced
cocaine CPP (Bahi et al., 2008), which would potentially occlude a role for BDNF in D1-D2
heteromer-induced inhibition of cocaine CPP, a study also demonstrated that BDNF-mediated
signalling via its receptor TrkB exerted differential effects on cocaine CPP in D1R- versus
D2R-expressing MSNs (Lobo et al., 2010b). Specifically, TrkB knockout in the D1R-
expressing MSNs enhanced cocaine CPP whereas an opposite effect was observed when TrkB
was knocked-out in the D2R-expressing MSNs. This would suggest that BDNF signalling in
the D1R- and D2R-expressing MSNs may respectively function to inhibit and promote cocaine
CPP. Therefore, it is possible that D1-D2 heteromer-induced BDNF release in the NAc may
have preferentially impacted the D1R-expressing MSNs. In addition, a negative role for
mesolimbic BDNF expression in morphine reward lends further credence to the idea that
BDNF can exert positive or negative effects on reward depending on its expression in distinct
neuroanatomical subregions of the NAc (Koo et al., 2012, 2014). Furthermore, our findings
demonstrated a significant role for Cdk5 in mediating the effects of SKF 83959 on cocaine
CPP, a protein kinase that exhibits enhanced activity in the NAc following TrkB activation and
the associated PI3K/Akt signalling (Bogush et al., 2007). Interestingly, the conditional
knockout of Cdk5 in the NAc has been shown to enhance cocaine CPP (Benavides et al., 2007),
which supports our findings showing an inhibitory role for the kinase in this behaviour.
The link between D1-D2 heteromer stimulation and Cdk5 signalling additionally
suggests the potential regulation of DARPP-32 by the D1-D2 heteromer, as Cdk5 has been
shown to phosphorylate DARPP-32 at Thr75 (Bibb et al., 1999). DARPP-32, expressed in all
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MSNs, is an important mediator of the biochemical, electrophysiological, transcriptional, and
behavioural effects of dopamine (Nishi et al., 1997; Fienberg et al., 1998; Nestler, 1999; Yan et
al., 1999; Calabresi et al., 2000; Flores-Hernandez et al., 2000; Zachariou et al., 2006). As
cocaine enhances dopaminergic signalling in the mesolimbic circuitry by inhibiting the
dopamine reuptake transporter, studies have also established a critical role of DARPP-32 in
mediating cocaine actions (Nestler, 1999; Zachariou et al., 2002, 2006; Zhang et al., 2006).
Particularly, the expression of cocaine CPP was shown to be associated with increased
DARPP-32 phosphorylation at Thr34, a D1R/cAMP/PKA-mediated effect, together with a
reduction in Thr75 phosphorylation, in the NAc (Tropea et al., 2008). In addition, mice with a
Thr34-Ala mutation that prevented phosphorylation at this site exhibited diminished cocaine
CPP expression (Zachariou et al., 2002), whereas Cdk5 conditional knockout in the NAc,
which would indirectly reduce Thr75 phosphorylation (Bibb et al., 1999), promoted the
expression of cocaine CPP (Benavides et al., 2007). Collectively, these findings indicate a
positive role for NAc DARPP-32 Thr34 phosphorylation, and a negative role for Thr75
phosphorylation, in the modulation of cocaine CPP. Interestingly, we have observed an
increase in DARPP-32 Thr75 phosphorylation in native rat NAc following acute stimulation of
the D1-D2 heteromer by SKF 83959 (Hasbi et al., unpublished finding), which occurred via a
Cdk5-dependent mechanism. Moreover, we further determined that the D1-D2 heteromer-
mediated increase in DARPP-32 Thr75 phosphorylation occurred specifically in the D1R/D2R
co-expressing MSNs in primary cultured striatal neurons and in rat NAc, with a concomitant
reduction in the Thr34 phosphorylation in the same neuronal population (Hasbi et al.,
unpublished finding). Therefore, it is possible that D1-D2 heteromer stimulation may have
abolished the acquisition and expression of cocaine CPP by enhancing DARPP-32 Thr75
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phosphorylation in the NAc, which in turn would prevent the increase in Thr34
phosphorylation that is associated with cocaine CPP via the inhibition of PKA (Bibb et al.,
1999; Tropea et al., 2008). Taken together, D1-D2 heteromer stimulation increased BDNF
release in the NAc (Hasbi et al., 2009), which in turn could enhance Cdk5 activity via TrkB-
mediated PI3K/Akt signalling in the same region (Bogush et al., 2007), and ultimately result in
increased DARPP-32 Thr75 phosphorylation in the NAc to prevent the acquisition and
expression of cocaine CPP.
Lastly, as the acquisition of cocaine CPP is critically dependent on the stimulation of
D1R in the NAc (Cervo and Samanin, 1995; Baker et al., 1996), the possibility that SKF 83959
may antagonize the D1R (Downes and Waddington, 1993; Cools et al., 2002; Jin et al., 2003)
would confound the interpretation of the current findings. However, we argue that SKF 83959-
mediated abolishment of cocaine CPP acquisition and expression occurred through the D1-D2
heteromer as firstly, SKF 83959-induced aversion was abolished by the highly selective
antagonist TAT-D1 and secondly, place conditioning with the selective D1R antagonist SCH
23390 alone did not produce CPA (Meririnne et al., 2001, 2005) and thirdly, D1R antagonism
by SCH 23390 administered solely on the test day does not affect the expression of cocaine
CPP (Liao et al., 1998) whereas a similar administration of SKF 83959 abolished it.
4.2 The Dopamine D1-D2 Receptor Heteromer Modulates Behavioural Sensitization to
Cocaine
In addition to its inhibitory effects on the acquisition and expression of cocaine CPP, it
was further demonstrated that the D1-D2 heteromer also exerts a negative modulatory role in
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both the development and expression of locomotor sensitization to cocaine. More importantly,
repeated D1-D2 heteromer inactivation during the initiation phase by the TAT-D1 peptide
alone was sufficient to induce a sensitized locomotor phenotype in response to cocaine priming
after a 24-hour withdrawal, indicating that the D1-D2 heteromer exerts a tonic inhibition to the
neurobiological processes underlying the development and expression of locomotor
sensitization to cocaine.
Despite its lack of clear face validity, locomotor sensitization to cocaine has been
associated with long-term neuroadaptations in the mesolimbic system that are critically
involved in the regulation of reward and motivation (Robinson and Becker, 1986; Henry and
White, 1991; Kantor et al., 1999), and which have been proposed to enhance the incentive
salience, or “wanting”, of the drug itself, as well as drug-related stimuli such as the
environmental context associated with drug use, leading to compulsive and persistent drug
seeking behaviours that are typically seen in addicted individuals (Robinson and Berridge,
2000). Supporting this notion, the progressive augmentation in locomotor activity in response
to repeated non-contingent cocaine administration mirrors the escalation of cocaine intake as
observed in animals that underwent extended access of cocaine self-administration (Ahmed and
Koob, 1998, 1999; Berridge, 2007). As both cocaine-induced locomotion and SA are critically
dependent on D1R stimulation in the NAc, it is possible that repeated exposure to cocaine, be it
contingent or non-contingent, may result in the same neurobiological alterations in the NAc
that ultimately augments the desire for cocaine. Indeed, not only have cocaine sensitized
animals been shown to exhibit enhanced cocaine CPP and SA (Lett, 1989; Vezina, 2004), but
the neurocircuitry of cocaine-induced locomotor sensitization was also shown to largely
overlap with that of the reinstatement of cocaine SA (Steketee and Kalivas, 2011), suggesting
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that the persistent neuroadaptations associated with cocaine locomotor sensitization may also
contribute to eventual drug relapse. Thus, the ability of D1-D2 heteromer stimulation to
prevent specifically the sensitization response to cocaine, while leaving the locomotor-
stimulating effect of cocaine intact, strongly implicates this receptor complex as a negative
regulator of the physiological processes associated with cocaine addiction.
Studies to date have demonstrated that locomotor sensitization to cocaine is a complex
behaviour involving the close interplay between various neurotransmitter systems in regions of
the mesolimbic circuitry. The initiation of locomotor sensitization cocaine is thought to depend
on enhanced VTA dopaminergic neuron excitability in the form of long-term potentiation (LTP)
(Borgland et al., 2004), which occurs as a consequence of reduced D2 auto-receptor inhibition
(White and Wang, 1984) and reduced inhibitory GABAergic control in the VTA (Liu et al.,
2005) following repeated non-contingent cocaine administration. This subsequently results in
increased dopamine release in the NAc in response to cocaine priming that acts on
supersensitized D1 receptors (Henry and White, 1991), leading to the expression of cocaine-
induced locomotor sensitization. Therefore, a potential mechanism by which the D1-D2
heteromer prevents cocaine-induced locomotor sensitization may be through its ability to
enhance the expression of GAD67 in the VTA following acute stimulation (Perreault et al.,
2012a). This in turn could enhance GABAergic control in the VTA and prevent the LTP of
VTA dopaminergic neurons that are known to be associated with the initiation of cocaine-
induced locomotor sensitization.
Similar to that observed with the CPP findings, evidence suggests a potential role for
Cdk5 in the regulation of cocaine-induced locomotor sensitization by the D1-D2 heteromer. It
has been shown that rats treated with repeated intra-NAc infusions of Cdk5 inhibitor
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roscovitine alone during the initiation phase produced a sensitized locomotor phenotype in
response to cocaine priming to a magnitude that was comparable to those exhibited by cocaine-
sensitized animals (Taylor et al., 2007). Similarly, concomitant intra-NAc infusion of
roscovitine with cocaine during the initiation phase further augmented the subsequent
expression of locomotor sensitization to cocaine (Taylor et al., 2007). These effects of Cdk5
inhibition in the NAc on cocaine-induced locomotor sensitization remarkably mirrors the
effects of selective D1-D2 heteromer inactivation as observed in the current study. Together
with the CPP data, these findings suggest that Cdk5 in the NAc may play a crucial role in the
inhibition of the development and expression of locomotor sensitization to cocaine by the D1-
D2 heteromer.
It is noteworthy that, although locomotor sensitization to cocaine was opposingly
modulated by D1-D2 heteromer stimulation and inactivation, the acute locomotor-stimulating
effect of cocaine was enhanced by both SKF 83959 and the TAT-D1 peptide, an apparent
contradictory finding as the two pharmacological agents were shown to exert opposite actions
at the D1-D2 heteromer (Rashid et al., 2007; Hasbi et al., 2014). However, the findings also
showed that while acute SKF 83959 treatment augmented basal locomotion, this effect was not
abolished by TAT-D1 peptide treatment, indicating that the locomotor stimulation induced by
SKF 83959 was not mediated through the D1-D2 heteromer (Shen et al., 2014b). Indeed,
although PLCβ-mediated signalling in the NAc has been shown to promote basal locomotion,
such an effect was dependent selectively on the D1R but not the D2R (Medvedev et al., 2013),
thus excluding the possible involvement of the D1-D2 heteromer in regulating basal
locomotion. A possible mechanism by which SKF 83959 enhanced basal locomotion may be
through the stimulation of D5R (Perreault et al., 2012b), as mice with D5R gene-deletion
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exhibited reduced locomotor response to a D1R/D5R agonist SKF 81297 (Holmes et al., 2001),
which suggests a locomotor-stimulating action of the D5R. On the other hand, in contrast to its
stimulating effect on cocaine-induced locomotion, basal locomotion was not affected by acute
D1-D2 heteromer inactivation (Shen et al., 2014b), and thus the potentiating effect of D1-D2
heteromer inactivation on acute cocaine-induced locomotion was likely a sensitization response
to cocaine, possibly via a Cdk5-dependent mechanism as described in the previous section.
4.3 The Dopamine D1-D2 Receptor Heteromer Modulates Cocaine-seeking Behaviours
The present finding demonstrated that the D1-D2 heteromer exerts negative modulation
on cocaine CPP and locomotor sensitization, and a similar inhibitory effect was also observed
in the cocaine SA paradigm. Specifically, D1-D2 heteromer stimulation dose-dependently
reduced total cocaine intake during an SA session as reflected by reduced total number of
active lever presses and cocaine infusions. In addition, cocaine-induced reinstatement of SA
was similarly abolished by D1-D2 heteromer stimulation, whereas D1-D2 heteromer
inactivation further enhanced this behaviour. However, the reinstatement of cocaine SA by a
discriminative light cue that was previously associated with each cocaine infusion during FR5
training was only abolished by D1-D2 heteromer stimulation when a higher dose of SKF 83959
was used (1.5mg/kg but not 0.5 mg/kg), although both doses completely prevented cocaine-
induced reinstatement.
There are two possible interpretations for the reduction in cocaine SA under the FR5
schedule of reinforcement that was observed following D1-D2 heteromer stimulation. First,
such an effect may suggest that the D1-D2 heteromer enhanced the rewarding effect of each
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cocaine infusion so that a fewer number of total infusions were required to achieve the same
euphoric state (Richardson and Roberts, 1996). Indeed, a higher dose of cocaine was shown to
result in fewer total infusions compared to a lower dose of cocaine when used in SA under the
FR5 schedule (Depoortere et al., 1993). However, as D1-D2 heteromer stimulation reduced
cocaine reward in the CPP model, it is highly unlikely that D1-D2 heteromer stimulation
enhanced cocaine reward in this paradigm. A more likely explanation is that the observed
reduction in cocaine SA reflected a loss of motivation to acquire cocaine infusions. This
interpretation is supported by the fact that D1-D2 heteromer stimulation and inactivation
respectively prevented and enhanced priming cocaine-induced reinstatement of cocaine SA
Although stimulation of the D1-D2 heteromer by SKF 83959 at the two doses examined
both prevented priming cocaine-induced reinstatement of cocaine SA, the lower dose of SKF
83959 did not affect reinstatement of SA induced by a discriminative cue. This discrepancy
may be explained by the distinct neurocircuitry involved in priming cocaine- versus cue-
induced reinstatement of SA (Shaham et al., 2003). Thus, whereas cocaine priming-induced
reinstatement was critically dependent on D1R and D2R receptor stimulation in the NAc shell
(Schmidt et al., 2006), reinstatement by discriminative cues was shown to require D1R
stimulation in the BLA but not the NAc (Ciccocioppo et al., 2001). Therefore, since it has been
shown that the expression of the D1-D2 heteromer is most prominent in the NAc shell
(Perreault et al., 2010), it is possible that D1-D2 heteromer stimulation by a lower dose of SKF
83959 will affect a behaviour that is more dependent on dopaminergic activity in the NAc shell
rather than the BLA. Furthermore, no evidence yet exists that indicate D1-D2 heteromer
expression is present in the BLA or that D1R/D2R neuron projections to the BLA exists, and
thus we cannot predict the impact of this region on our observed effects.
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The D1-D2 heteromer may have attenuated cocaine SA through several mechanisms.
One mechanism may occur through the increase in GAD67 elicited by D1-D2 heteromer
stimulation and enhanced GABA/glutamate ratio in NAc (Perreault et al., 2012a), since
GABAergic activity in the NAc has been implicated in the attenuation of cocaine SA. The
administration of GABAB receptor agonists into NAc and VTA were shown to attenuate the
maintenance of cocaine SA under both the FR and PR schedules (Shoaib et al., 1998; Brebner
et al., 2000; Di Ciano and Everitt, 2003), as well as cocaine- and context-induced reinstatement
(Fuchs et al., 2008; Peng et al., 2008). Moreover, rats that underwent extinction of cocaine SA
were shown to have increased basal GABA levels in the NAc (Wydra et al., 2013). Together
these findings suggest that increased GABAergic activity in the NAc attenuates cocaine-
seeking behaviours, and may be an underlying neurobiological alteration that is associated with
the extinction of cocaine-taking. Therefore, the D1-D2 heteromer may have attenuated and
prevented the maintenance and reinstatement of cocaine SA respectively by enhancing
GABAergic activity in the NAc.
Another mechanism may be through the ability of the D1-D2 heteromer to regulate
DARPP-32 phosphorylation in NAc, which may also allow it to exert an inhibitory modulation
on the maintenance of cocaine SA. The establishment of stable cocaine SA was associated with
increased DARPP-32 phosphorylation at Thr34 in the NAc (Lynch et al., 2007), a site that is
phosphorylated by D1R/cAMP/PKA signalling (Hemmings et al. 1984). In addition, mice with
an inactive Thr34 phosphorylation site required a longer training period to achieve stable
cocaine SA (Zhang et al., 2006). Therefore, similar to its effects on other cocaine-mediated
behaviours (Scheggi et al., 2007; Tropea et al., 2008), DARPP-32 Thr34 phosphorylation in the
NAc seems to be an important mediator for the maintenance of cocaine SA. In contrast,
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DARPP-32 phosphorylation at Thr75, an effect mediated by Cdk5, turns it into a potent
inhibitor of PKA, and thus the phosphorylation at this site may attenuate cocaine SA by
reducing DARPP-32 Thr34 phosphoryation. Since intra-NAc infusion of a Cdk5 inhibitor
enhanced cocaine SA under the PR schedule (Taylor et al., 2007), a negative modulatory role
for DARPP-32 Thr75 phosphorylation in cocaine SA has been demonstrated. Therefore, since
D1-D2 heteromer stimulation resulted in an increase in DARPP-32 Thr75 phosphorylation in
the NAc via a Cdk5-dependent mechanism (unpublished finding), the D1-D2 heteromer may
therefore have exerted its inhibitory modulation on the maintenance of cocaine SA by reducing
Thr34 phosphorylation in the NAc.
In addition to the mesolimbic circuit, the corticostriatal circuit (PFC-NAc) has also
been implicated in the reinstatement of cocaine SA. The PFC is anatomically divided into
various subregions, including the infralimbic cortex (IFC), prelimbic cortex (PLC), and the
orbitofrontal cortex (OFC), and each was shown to play a role in the modulation of the
reinstatement of SA behaviour induced by cocaine priming (Capriles et al., 2003; Fuchs et al.,
2004; Peters et al., 2008; Martín-García et al., 2014; Shen et al., 2014a) or cocaine-associated
cues (McLaughlin and See, 2003; Fuchs et al., 2004; Lasseter et al., 2009; LaLumiere et al.,
2012). Interestingly, acute D1-D2 heteromer inactivation by the TAT-D1 peptide was shown to
enhance neuronal activity in the IFC, PLC, and OFC as indicated by increased c-Fos
expression in these regions (Perreault et al., 2015), providing yet another potential mechanism
through which the D1-D2 heteromer could regulate cue- and cocaine-induced reinstatement of
SA behaviour. However, the use of c-Fos as the marker for neuronal activity cannot
differentiate which subpopulation of neurons in these prefrontal subregions were activated by
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D1-D2 heteromer inactivation, and thus we cannot surmise at this point exactly how this may
contribute to the observed effects on the reinstatement of cocaine SA.
4.4 Potential Limitations
A potential limitation of the current study is the use of SKF 83959 to examine the
effects of D1-D2 heteromer stimulation on cocaine-seeking behaviours. To date, no selective
agonist for the D1-D2 heteromer has been identified, and while SKF 83959 has been shown to
definitively induce the Gq-PLC-linked calcium signalling mediated by the D1-D2 heteromer,
its pharmacology has been shown to extend to include a few other receptors including the
dopamine D5 receptor and the 5-HT2C serotonin receptor (Sahu et al., 2009; Perreault et al.,
2012b; Chun et al., 2013; Guo et al., 2013). Since the involvement of both the D5R and the 5-
HT2CR in the modulation of cocaine-seeking behaviours have been previously implicated (Filip
et al., 2000; Rocha et al., 2002; Fletcher et al., 2004; Perreault et al., 2012b), it is possible that
systemic injection of SKF 83959 may have elicited off-target effects that may also contribute
to its inhibitory modulation on cocaine-seeking behaviours as observed in the current study.
For instance, a D5R-mediated increase in BDNF expression in the mPFC has been shown and
5-HT2CR stimulation in the VTA was shown to attenuate cocaine SA (Fletcher et al., 2004;
Berglind et al., 2007; Perreault et al., 2012b). To overcome this specificity issue, we
synthesized a highly selective TAT-D1 peptide that occludes the interaction site between the
two receptors (O'Dowd et al., 2012), thus inhibiting D1-D2 heteromer expression and function
and abolishing the physiological effects of D1-D2 heteromer activation by SKF 83959 without
affecting other receptor oligomers such as D1-D1 homomers, D2-D5 heteromers, or other
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heteromers (Hasbi et al., 2014). The use of the TAT-D1 peptide allowed us to conclusively
define the effects of D1-D2 heteromer inactivation on cocaine-seeking behaviours. However, it
is highly noteworthy that we did not directly show the abolition of SKF 83959-induced
regulation of cocaine effects by TAT-D1 in many instances as we were concerned about the
confounding effects of the repeated administration of more than two agents simultaneously to
overwhelm the in vivo system, as both SKF 83959 and cocaine affect various neurotransmitter
systems via multiple mechanisms of action (Rothman et al., 2001; Perreault et al., 2012b; Chun
et al., 2013; Guo et al., 2013). Nonetheless, the ability of SKF 83959 and the highly specific
TAT-D1 peptide to consistently exert opposite effects in all three behavioural models
examined argues for a pivotal role for the D1-D2 heteromer in mediating cocaine-seeking
behaviours.
4.5 Significance and Conclusion
In summary, I have demonstrated that D1-D2 heteromer stimulation and inactivation
consistently exerted bidirectional modulation of cocaine-induced behaviours in the three most
commonly used animal experimental paradigms that model aspects of cocaine addiction.
Specifically, D1-D2 heteromer stimulation by the agonist SKF 83959 abolished the acquisition
and expression of cocaine CPP, prevented the development and expression of cocaine-induced
locomotor sensitization, attenuated the maintenance of cocaine SA, and blocked priming
cocaine- and cue-induced reinstatement of SA. In contrast, selective D1-D2 heteromer
inactivation increased the acquisition of cocaine CPP, augmented cocaine-induced locomotor
sensitization, and enhanced priming cocaine-induced reinstatement of SA. Numerous studies to
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date have clearly implicated the critical involvement of D1R and D2R, as well as the D1R- and
D2R-expressing MSNs, in the NAc in cocaine-induced behaviours (Thomas et al., 2008; Baik,
2013), and the present findings show for the first time a highly significant role for the
dopamine D1-D2 receptor heteromer and the unique subset of D1R/D2R co-expressing MSNs
in the NAc may also play an important role in the modulation of cocaine-induced behaviours.
Indeed, the findings of the current study indicate that the D1-D2 heteromer exerts tonic
inhibition on the neurobiological processes underlying cocaine-induced behaviours, potentially
via its signalling through Cdk5 and DARPP-32 in the NAc. Thus, the stimulation and
inactivation of this dopamine receptor complex will respectively enhance and remove such
inhibitory modulation to reduce and promote cocaine-induced behaviours.
Despite constant research efforts, no effective therapeutic intervention for cocaine
addiction has yet emerged, and a novel therapeutic target is thus warranted. Therefore, the
identification of the dopamine D1-D2 receptor heteromer as a specific negative modulator of
cocaine-induced behaviours may have great implications for the understanding and treatment
of cocaine addiction. Furthermore, given the established involvement of the D1-D2 heteromer
in amphetamine-induced locomotor sensitization, it is possible that these therapeutic effects
may be translated across other psychoactive substances.
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3. Rapid anti-depressant and anxiolytic actions following dopamine D1-D2 receptor
heteromer inactivation
Abstract
A role for the mesolimbic dopaminergic system in the pathophysiology of depression
has become increasingly evident. Specifically, brain-derived neurotrophic factor (BDNF) has
been shown to be elevated in the nucleus accumbens of depressed patients and to positively
contribute to depression-like behaviour in rodents. The dopamine D1-D2 receptor heteromer
exhibits significant expression in NAc and has also been shown to enhance BDNF expression
and signalling in this region. We therefore examined the effects of D1-D2 heteromer
stimulation in rats by SKF 83959, or its inactivation by a selective heteromer-disrupting TAT-
D1 peptide on depression- and anxiety-like behaviours in non-stressed animals and in animals
exposed to chronic unpredictable stress. SKF 83959 treatment significantly enhanced the
latency to immobility in the forced swim test, increased the latency to drink condensed milk
and reduced total milk consumption in the novelty-induced hypophagia test, and additionally
reduced the total time spent in the open arms in the elevated plus maze test. These pro-
depressant and anxiogenic effects of SKF 83959 were consistently abolished or attenuated by
TAT-D1 peptide pre-treatment, signifying the behaviours were mediated by the D1-D2
heteromer. More importantly, in animals exposed to chronic unpredictable stress (CUS), TAT-
D1 peptide treatment alone induced significant and rapid anxiolytic and antidepressant-like
effects in two tests for CUS-induced anhedonia-like reactivity and in the novelty-suppressed
feeding test. Together these findings indicate a positive role for the D1-D2 heteromer in
mediating depression- and anxiety-like behaviours and suggest its possible value as a novel
therapeutic target.
The work on the chronic unpredictable stress in this study was done by Dr. Fancis
Bambico.
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1. Introduction
Major depression is a chronic psychiatric disorder that is characterized by low mood,
despair, and the inability to experience pleasure from normally rewarding stimuli that severely
reduce the quality of life of patients. Roughly half of the affected individuals do not respond to
available antidepressants (Culpepper, 2010) and reports indicate that greater than 50 percent of
depressed patients also suffer from anxiety (Carter et al., 2001), which can result in increased
symptom severity and delayed recovery (Hirschfeld, 2001). Interestingly, since virtually all
antidepressants are also anxiolytic, it has been suggested that the neurobiology of anxiety and
depression may share certain common molecular substrates (Conway et al., 2006).
The hippocampus and the frontal cortex have been the most widely studied brain
regions in relation to depression, however it is unlikely that these regions account for all the
symptoms of the disorder (Russo and Nestler, 2013). The mesolimbic dopamine system for
example, comprised of the dopaminergic projection from the ventral tegmental area (VTA) to
the nucleus accumbens (NAc), plays a critical role in reward and has been most widely studied
for its role in addiction. Studies are now demonstrating, however, a major role for the
mesolimbic pathway in the regulation of depression (Nestler and Carlezon, 2006b; Krishnan et
al., 2007; Russo and Nestler, 2013; Tye et al., 2013; Friedman et al., 2014). The activity of the
NAc is reduced in depressed patients (Mayberg et al., 2000) and animal studies suggest that
this may result from increased GABAergic activity in the region leading to heightened
inhibitory tone on dopamine neurons, which could manifest as a loss of pleasurable feelings
and the development of depression-like behaviours with anhedonia (Nestler and Carlezon,
2006b; Tye et al., 2013). One key player in the mediation of depression-like responses is brain-
97
derived neurotrophic factor (BDNF) (Nestler and Carlezon, 2006b; Russo and Nestler, 2013).
Interestingly in the NAc, many of the proteins identified to be involved in depression are
downstream of BDNF signaling. These findings suggest that the mechanisms which regulate
the expression of BDNF in the NAc may represent novel therapeutic targets in depression. The
effects of BDNF appear to be region-specific, being pro-depressant in the mesolimbic system
but anti-depressant in hippocampus and frontal cortex (reviewed in: Duman, 2014).
The dopamine D1-D2 receptor heteromer is a dopamine receptor complex that is
predominantly expressed in the NAc (Perreault et al., 2010), and which has been shown to
increase the expression of a major GABA producing enzyme GAD67 in this region, thus
potentially enhancing inhibitory GABAergic tone of neurons in the NAc (Perreault et al.,
2012a). In addition, D1-D2 heteromer stimulation produced a Gq-mediated, phospholipase C
(PLC)-dependent calcium signal (Lee et al., 2004; Rashid et al., 2007; Hasbi et al., 2009, 2014;
Verma et al., 2010) that was shown to contribute to increased expression of BDNF and its
signaling in the NAc through its receptor tropomyosin receptor kinase B (TrkB) (Hasbi et al.,
2009; Perreault et al., 2012b, 2013). Given these findings, we postulated that the D1-D2
heteromer could positively contribute to the pathogenesis of depression, a notion supported by
recent studies that demonstrated the ability of D1-D2 heteromer disrupting peptides to exert
antidepressant-like activity in rats (Pei et al., 2010; Hasbi et al., 2014).
The physiological role of D1-D2 heteromer stimulation in mediating depression- and
anxiety-like behaviours in rodents can be delineated through the use of the agonist SKF 83959
(Rashid et al. 2007). However, recent reports have indicated that the pharmacology of SKF
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83959 also involves several other receptors, such as the dopamine D5 receptor, in addition to
the D1-D2 heteromer (Sahu et al., 2009; Perreault et al., 2012b; Chun et al., 2013). Thus, we
sought to validate whether the pro-depressive effects of SKF 83959 can be attributed to the D1-
D2 heteromer using the recently developed TAT-D1 disrupting peptide (Hasbi et al., 2014).
The highly selective TAT-D1 peptide, which occludes the interaction site between the two
receptors (O’Dowd et al., 2012), disrupts D1-D2 heteromer formation and abolishes the
physiological effects of D1-D2 heteromer activation and signalling without affecting other
receptor oligomers such as D1-D1 homomers or D2-D5 heteromers (Hasbi et al., 2014).
Several lines of evidence have supported the functional existence of the D1-D2
heteromer in the brain despite the concerns raised by a recent report (Frederick et al., 2015).
We and other groups have consistently observed the co-expression of the D1R and the D2R in
a unique subset of medium spiny neurons (MSNs) in rodent striatum (Matamales et al., 2009;
Bertran-Gonzalez et al., 2010; Perreault et al., 2010; Gangarossa et al., 2013b), and that the two
receptors physically interact in the rat NAc and human striatal tissues as evidenced by in situ
confocal fluorescent resonance energy transfer (FRET) and FRET photobleaching (Hasbi et al.,
2009; Perreault et al., 2010), co-immunoprecipitation (Lee et al., 2004; Pei et al., 2010),
glutathione-S-transferase (GST) pulldown (Pei et al., 2010), and proximity ligation assays (R.
Franco, personal communication). Moreover, the calcium signalling associated with the D1-
D2 heteromer was shown to require the co-activation of both the D1R and the D2R in the
striatum (Lee et al., 2004; Rashid et al., 2007; Hasbi et al., 2014), and was dose-dependently
reduced by the TAT-D1 peptide in primary cultured striatal neurons (Hasbi et al., 2014).
Collectively, these findings clearly supported the functional existence of the D1-D2 heteromer
99
in the brain, and here we propose to further characterize the physiological role for the receptor
complex in depression and anxiety. Using a number of behavioural paradigms we were able to
demonstrate in rats that D1-D2 heteromer activation by SKF 83959 promoted both depression-
and anxiety-like behaviours. The pro-depressive and anxiogenic effects of SKF 83959 were
attenuated, or in some cases reversed, by TAT-D1 peptide pre-treatment, thereby confirming
the involvement of the D1-D2 heteromer in mediating the behavioural effects of SKF 83959. In
addition, acute TAT-D1 peptide administration also induced significant and rapid anxiolytic
and antidepressant-like properties in animals exposed to chronic unpredictable stress (CUS),
which models chronic depressive-like states and has been shown to better predict the
therapeutic onset of antidepressants (Willner et al., 1987a).
2. Materials and Methods
2.1 Animals
Adult male Sprague-Dawley and Fischer 344 rats weighing 300-400g at the beginning
of the experiments (Charles River, Canada) were used. The mesolimbic dopamine system of
Fischer 344 rats are highly responsive to CUS, which were thus used in the CUS protocol
(Ortiz et al., 1996). Animals were housed in polyethylene cages in a temperature-controlled
colony room, maintained on a 12-h light-dark cycle (lights on at 0700), with ad libitum access
to food and water. Animals were handled daily for 3 days before the start of the experiments.
All treatments were performed during the light phase of the day-night cycle. Animals were
housed and tested in compliance with the guidelines described in the Guide to the Care and the
Use of Experimental Animals (Canadian Council on Animal Care), and protocols were
approved by the Animal Care Ethics Committee of the University of Toronto.
100
2.2 Drugs
SKF 83959 hydrobromide (Tocris Bioscience) was dissolved in physiological saline
containing 5% DMSO, and was administered subcutaneously (s.c.). All systemic injections
were given at a volume of 1.0 mL/kg and administered 5 minutes prior to behavioural testing.
The TAT-D1 disrupting peptide or an inactive TAT-scrambled peptide control (TAT-Sc) was
dissolved in physiological saline containing a protease inhibitor cocktail (1:1000) and
administered into the intracerebroventricular space (300 pmol/4µL, i.c.v.) 15 minutes prior to
SKF 83959 injection. The amino acid sequence of the TAT-D1 peptide derived from the D1R
carboxyl-tail is a unique sequence not appearing in any other protein (BLAST search). For non-
drug injections, an equivalent volume of saline or vehicle was administered.
2.3 Surgery
Rats were anaesthetized with isoflurane (5%), administered analgesic ketoprofen
(5mg/kg) and secured in a stereotaxic frame. A cannula (22-gauge, Plastics One) was placed
unilaterally into the intracerebroventricular space close to the midline according to the
following stereotaxic anterior-posterior (AP), mediolateral (ML), and dorsoventral (DV)
coordinates: AP -0.8mm, ML + 1.3mm, DV – 3.7mm, and was secured by dental cement
anchored with four stainless steel screws (Plastics One) fixed on the dorsal surface of the skull.
AP and ML coordinates were taken from bregma, DV coordinate from the dura (Paxinos and
Watson, 1998). The animals were allowed to recover in their home cage for five days before
the experiments were initiated. Cannulae placement was visually verified in post-mortem brain
slices.
101
2.4 Apparatus and Procedures
All animals received a minimum of five days of handling (2 minutes/animal/day) prior
to behavioural testing. The dose of the TAT-D1 peptide (300pmol, i.c.v.) was previously
shown to disrupt D1-D2 heteromer formation in vivo as indicated by the loss of co-
immunoprecipitation of D1R with D2R in rat NAc tissue (Hasbi et al., 2014).
2.4.1 Forced Swim Test
The forced swim test (FST) was conducted as previously described (Hasbi et al., 2014)
in a non-colony room isolated from external noise. During the pre-test, animals were placed in
a glass container with room temperature water filled to a height of approximately 40 cm. The
animals remained in the water for 15 minutes after which they were towel-dried and placed in a
cage under a heat lamp until completely dry. 24 hours following the pre-test, animals were
administered their designated treatment, TAT-D1 peptide (0, 300 pmol, i.c.v; given 15 minutes
prior to SKF 83959 treatment) and SKF 83959 (0 or 2.5 mg/kg, s.c), and placed again in the
water-filled container for 5 minutes. The following parameters were measured at 5-second
intervals: immobility (passively floating in water), swimming (moving across the water
surface), or climbing (front paws touching the glass wall while attempting to jump out of
water). Behavioural testing took place 5 minutes following SKF 83959 injection. To avoid
experimenter bias, behavioural measurements were done by an individual who was blind to the
treatment assigned to each animal.
2.4.2 Elevated Plus Maze
102
Behavioural testing was conducted as previously described (Rogóż and Skuza, 2011) in
an elevated plus maze (EPM) (Harvard Apparatus) situated in a non-colony room isolated from
external noise. The EPM was constructed of black plexiglass and consisted of a central square
with two sets of opposing open and closed arms each with dimensions 50cm x 10cm. Closed
arms were enclosed by 40cm high black plexiglass walls along the longitudinal edges, with the
roof and ends open. The entire maze was suspended 50cm off the ground. Following the
assigned drug treatment, TAT-D1 (0, 300 pmol, i.c.v; given 15 minutes prior to SKF 83959
treatment) and SKF 83959 (0, 0.5, 2.5 mg/kg, s.c.), rats were placed in the center of the maze
and behaviour was recorded for 5 minutes. Behavioural scoring of the videos occurred after the
testing was complete and the following parameters were measured: the number of open arm
and closed arm entries, time spent in the open and closed arms, and time spent per entry in
open and closed arms. Entry to or exit from an arm was defined by both front paws crossing the
arm boundaries. Behavioural testing took place 5 minutes following SKF 83959 injection. To
avoid experimenter bias, behavioural measurements were done by an individual who was blind
to the treatment assigned to each animal.
2.4.3 Novelty-induced Hypophagia
Behavioural testing was conducted as previously described (Dulawa and Hen, 2005).
Animals were trained to consume sweetened condensed milk (1:3 ; milk:water) in the home
cage under dim light for 2 hours daily over 3 days to establish stable preference to milk over
water. Milk and water were presented in plastic bottles closed with rubber stoppers and
positioned through meshed cage lids. Upon the establishment of stable milk preference (day 4),
the latency to drink from milk bottle and total milk consumed in the training environment was
103
measured. The few rats that failed to establish milk preference (~2-3%) were eliminated from
the test. On the test day (day 5), after receiving the assigned drug treatment, TAT-D1 (0, 300
pmol, i.c.v; given 15 minutes prior to SKF 83959 treatment) and SKF 83959 (0, 0.5 mg/kg,
s.c.), rats were placed in novel cages without bedding and under bright light to induce anxiety.
The latency to drink from the milk bottle and total milk consumed over the 2 hour period in the
novel environment was measured. Behavioural testing began 5 minutes following SKF 83959
injection.
2.4.4 Chronic Unpredictable Stress (CUS)
The CUS protocol employed was modified after Bambico et al. (2009) and Willner et al.
(1987). All animals were handled during one week of acclimation. They were then exposed to
sucrose solution (1% w/v) for 3 days, and then allowed to discriminate between the sucrose
solution and water (sucrose preference test) for 1 hour every day (at 10:00) after overnight
water deprivation for 16-18 hours. The position of the bottles on the cage top cover was
switched midway to minimize a possible egocentric orientation bias. Sucrose preference was
calculated as the ratio between sucrose solution intake and total fluid intake. During this task,
the animals were individually caged, tested within the same housing room under normal
lighting condition. After 4 days of initial sucrose preference tests, the last sucrose preference
score was used to assign animals to 4 groups such that no significant between-groups
difference was obtained. Two CUS groups were exposed to stress for 5 weeks. Two control
groups were not exposed to any of the stressors and were housed in a different vivarium.
Sucrose preference testing was performed for all animals after each week. Overtly stable
anhedonia-like reduction of sucrose preference scores in CUS animals typically ensues after a
104
few weeks of exposure. The CUS regimen consisted of various non-debilitating, inescapable
and uncontrollable physical, psychological and circadian stressors with the following elements:
modifications of the light/dark cycle and of the lighting characteristics (light-dark cycle
reversal, intermittent on-off lighting and stroboscopic lights), in food and water availability
(12-18 hours of deprivation), housing conditions (isolation, damp bedding, novel room
environment, cage tilt), and various ecological challenges (restraint, cold room, high frequency
sound or static noise, predator odor). Stress schedules across 5 weeks were not identical.
Within each one week block, stressors could occur at any time of the day (or night), and were
each applied for a period of 15 min to 18 hours. Sequence was at random and combinations
varied, in order to be completely unpredictable to the animal. However, more severe stressors
were not combined. The combinatory and cumulative effect of these stressors but not any one
of the individual stressors is sufficient and necessary to produce depressive-like reactivity.
During the post-CUS test day, animals were administered with TAT-D1 or TAT-Sc
(300pmol, i.c.v.) and were subjected to two tests for anhedonia (Sucrose Preference Test and
Fruit Loops Test) and two tests for anxiety-related reactivity (Novelty-Suppressed Feeding test
and Elevated Plus Maze) in the following order: Sucrose Preference Test, Novelty-Suppressed
Feeding, Elevated Plus Maze, and Fruit Loops Test. Procedures for the Sucrose Preference Test
and Elevated Plus Maze were as described earlier.
2.4.5 Fruit Loop Test
5 pieces of fruit loops (sweetened fruit-flavored cereal) were placed at the center of the
home cage. 15 minutes following the treatment with the TAT-D1 or the TAT-Sc peptide
105
(300pmol, i.c.v.), the animals were placed back into the home cage, and the latency to and total
fruit loop consumption over 10 minutes were recorded. Animals were pre-exposed to this
procedure for 5 days before the CUS exposure and before the TAT-D1 or TAT-Sc treatment to
eliminate a possible effect of novelty.
2.4.6 Novelty Suppressed Feeding Test
Animals were food-deprived for 36 hours, then placed in a novel environment (80x80x15
cm Plexiglas chamber) where the latency to approach and feed from 12 food pellets placed at
the center was measured. Once the animals approached the pellets, they were immediately
removed and placed in a familiar environment (Home Cage), where the latency to approach
and feed from 12 food pellets placed at the center was again measured. The cut-off time was
600 seconds. Novelty-induced suppression of feeding typically occurs in the novel but not in
the familiar environment, indicating an anxious-depressive behavioural phenotype that is
induced by the exposure to a novel environment. This delay in feeding has been shown to be
reversed by chronic but not acute treatment with standard antidepressants (Bodnoff et al.,
1989).
2.5 Statistical Analyses
Values are reported as mean ± s.e.m. All data was analyzed for normality prior to
ANOVAs using the Shapiro-Wilk test. Means were analyzed using the appropriate ANOVA
with Treatment as the between subjects factors for the forced swim test, elevated plus maze,
and novelty-induced hypophagia tests. Additionally for the novelty-induced hypophagia test,
data comparisons between the home cage and novel cage were performed by Student’s t test.
106
Sucrose preference scores during CUS exposure were analyzed with Stress Exposure as the
between subject factor and Weeks as the within subject factor. Post-CUS behavioural tests
were analyzed with Stress Exposure and Treatment as between subject factors. Duncan’s
multiple range test was used for post-hoc comparisons following appropriate ANOVAs.
Statistical significance was set at P<0.05 and computations were performed using the
SPSS/PC+ statistical package.
3. Results
3.1 Forced Swim Test
We first examined the effects SKF 83959 on passive coping or behavioural despair in
rats in the forced swim test, and determined whether SKF 83959-induced behaviour could be
reversed by the TAT-D1 peptide. The ANOVA revealed a significant main effect of Treatment
on the latency to {F(3,48)=13.5, p<0.0001} and total duration of {F(3,48)=26.4, p<0.0001}
immobility. Post-hoc analysis showed that SKF 83959 administration significantly reduced the
latency to develop immobility compared to the vehicle control, an effect that was abolished by
TAT-D1 peptide pretreatment (Figure II-1A, p<0.001). TAT-D1 peptide treatment alone did
not affect the latency to develop immobility. SKF 83959 did not alter the total immobility time
over the 5 minute testing period, whereas TAT-D1 administration alone or with SKF 83959
produced an antidepressant-like activity as indicated by significantly reduced total immobility
time (Figure II-1B, p<0.001). Time course analysis of the effects of SKF 83959 and TAT-D1
peptide on total immobility time during each minute of testing (Figure II-1C) revealed
significant effects of Time {F(4,120)=239.1, p<0.0001}, Treatment {F(3,43)=40.4, p<0.0001}
107
Figure II-1: The effects of D1-D2 heteromer stimulation or inactivation on the latency to
and total immobility time in the forced swim test in rats. A) SKF 83959 significantly
reduced the latency to immobility, and effect that was reversed by TAT-D1 peptide pre-
treatment. B) The total immobility was not affected by SKF 83959, but was significantly
reduced by TAT-D1 peptide alone or with SKF 83959. C&D) The time course of the total
immobility for each treatment over the 5 minute testing period. Data represent means ± s.e.m.
of n=10-14 rats/group. (***p<0.001, compared to vehicle-treated rats).
0
50
100
150
200
La
ten
cy
(se
c)
***
Veh
icle
SKF
8395
9
TAT-D
1
TAT-D
1+SKF
0
50
100
150
200
250
Imm
ob
ilit
y (
se
c)
***
Veh
icle
SKF
8395
9
TAT-D
1
TAT-D
1+SKF
***
-10
0
10
20
30
40
VehicleSKF 83959
TAT-D1TAT-D1+SKF
0 15 30 45 60
***
Time (sec)
Imm
ob
ilit
y (
se
c)
10
20
30
40
50
60
VehicleSKF 83959
TAT-D1TAT-D1+SKF
1 2 3 4 5
***
Time (min)
Imm
ob
ilit
y (
se
c)
A) B)
C) D)
108
and a Time x Treatment interaction {F(12,120)=15.6, p<0.0001}. SKF 83959 induced a
significant increase in immobility within the first minute of testing compared to controls
(Figure II-1D, p<0.001), an effect that was abolished by TAT-D1 pre-treatment.
3.2 Elevated Plus Maze
To examine the role of the D1-D2 heteromer in mediating anxiety, the effects of SKF
83959 and the TAT-D1 peptide were assessed in rats in the elevated plus maze. One-way
ANOVA revealed significant effects of Drug Treatment on the total time spent in the open
arms of the maze {Treatment: F(4,42)=4.6, p<0.01}, total time spent in the closed arms
{Treatment: F(4,42)=7.0, p<0.001}, total open arm entries {Treatment: F(4,42)=4.1, p<0.01},
and time spent in open arms per entry {Treatment: F(4,42)=16.8, p<0.001}. SKF 83959
administration resulted in a dose-dependent reduction in the total time spent in the open arms
that became significant at the 2.5 mg/kg dose, an effect that was abolished by TAT-D1 peptide
pre-treatment (Figure II-2A, p<0.05). TAT-D1 alone, or in combination with SKF 83959,
resulted in a small and insignificant increase in the total time spent in open arms (Figure II-2A).
Accordingly, the total time spent in the closed arms was significantly higher in animals treated
with 2.5mg/kg of SKF 83959 (Figure II-2B, p<0.05). In addition, the number of total open arm
entries was significantly reduced by SKF 83959 at the 2.5mg/kg dose (p<0.01), as well as by
TAT-D1 treatment alone (p<0.05) or with SKF 83959 (p<0.01) (Figure II-2C). Combined with
the total time spent in the open arm, the reduced number of total open arm entries for TAT-D1
peptide-treated animals reflected the significantly more time spent in the open arms with each
entry compared to the vehicle control, (p<0.01, Figure II-2D) indicating that D1-D2 heteromer
disruption is anxiolytic in the elevated plus maze. In contrast, the time spent in the open arm
109
Figure II-2: D1-D2 heteromer stimulation or inactivation had bidirectional effects on
anxiety-like behaviours in the elevated plus maze. A) SKF 83959 dose-dependently reduced
the total time spent in the open arms and B) increased the total time spent in the closed arms C)
SKF 83959 at 2.5mg/kg or the TAT-D1 peptide treatment alone significantly reduced total
open arm entries. D) SKF 83959 at 2.5mg/kg significantly reduced the time spent in open arms
per entry, whereas TAT-D1 peptide treatment alone or with SKF 83959 had the opposite effect.
Data represent means ± s.e.m. of n=8-12 rats/group. (*p<0.05, **p<0.01, ***p<0.001,
compared to vehicle-treated rats)
0
20
40
60
To
tal
Tim
e S
pe
nt
in
Op
en
Arm
(se
c)
*
Veh
icle
0.5_
SKF
2.5_
SKF
TAT-D
1
TAT-D
1+SKF
0
2
4
6
8
10
To
tal
Op
en
Arm
En
trie
s
** ***
Veh
icle
0.5_
SKF
2.5_
SKF
TAT-D
1
TAT-D
1+SKF
0
100
200
300
To
tal
Tim
e S
pe
nt
in
Clo
se
d A
rms (
se
c)
*
Veh
icle
0.5_
SKF
2.5_
SKF
TAT-D
1
TAT-D
1+SKF
0
5
10
15
Tim
e S
pe
nt
in O
pe
n
Arm
s P
er
En
try
(se
c)
Veh
icle
0.5_
SKF
2.5_
SKF
TAT-D
1
TAT-D
1+SKF
**
***
*
A) B)
C) D)
110
with each entry was significantly reduced by SKF 83959 treatment at the 2.5mg/kg dose, an
effect that was abolished by the pre-treatment with TAT-D1 (Figure II-2D, p<0.05).
3.3 Novelty-induced Hypophagia
To further assess the effects of the D1-D2 heteromer on anxiety responses we next
utilized the novelty-induced hypophagia paradigm, a behavioural model that examines anxiety
induced by the stress of a novel environment, and which assesses an animal’s willingness to
approach and consume food or drink in a novel environment. There were significant effects of
Drug Treatment in the total sweetened milk consumed {Treatment: F(4,42)=7.612, p<0.001}
and the latency to drink milk {Treatment: F(4,42)=672.219, p<0.001}. As expected, the
anxiogenic effect of the novel testing environment prolonged the latency to drink milk in
control animals (Figure II-3A, p<0.01), whereas it did not affect the total milk consumption
(Figure II-3B). For every rat tested, SKF 83959 treatment completely prevented any drinking
of milk over the 2 hour testing period (Figure II-3A&3B), whereas TAT-D1 treatment
significantly reduced the latency to drink milk in the novel environment (Figure II-3A, p<0.05)
without affecting total milk consumption (Figure II-3B). It should be noted that the SKF
83959-treated animals were able to move around normally in the novel cage. Thus the loss of
milk consumption was due to the animals’ choice rather than locomotor impairment. TAT-D1
pre-treatment robustly attenuated the enhanced latency to drink milk induced by SKF 83959
(Figure II-3B, p<0.001) and reinstated total milk consumption (Figure II-3B, p<0.001).
Together these findings indicate that D1-D2 heteromer activation has pro-depressive and
anxiogenic effects in rodents that in many instances are tonically active, which can be obviated
by disruption of the receptor complex.
111
Figure II-3: D1-D2 heteromer stimulation or inactivation had bidirectional effects on
anxiety-like behaviours in the novelty-induced hypophagia test. A) Exposure to novel
environment and SKF 83959 treatment significantly increased the latency to drink from milk
bottle, an effect that was significantly reduced by TAT-D1. B) The total milk consumption was
not affected by exposure to novel environment or TAT-D1, but was significantly reduced by
SKF 83959, an effect normalized by TAT-D1 pre-treatment. Data represent means ± s.e.m. of
n=8-12 rats/group. (*p<0.05, **p<0.01, ***p<0.001, compared to vehicle-treated group in
novel cage; ###p<0.001, compared to SKF 83959-treated group)
10
10
20
30
To
tal
Mil
k
Co
nsu
me
d (
mL
)
Pre
-Tes
t
Veh
icle
TAT-D
1
SKF
8395
9
TAT-D
1+SKF
###
***
Pre
-Tes
t
Veh
icle
TAT-D
1
SKF 8
3959
TAT-D
1+SK
F0
50
100
150
200
2000
4000
6000
80007200 ***
###
*
**
La
ten
cy
(se
c)
A) B)
112
3.4 CUS-induced Anhedonic Response
Having shown that D1-D2 heteromer stimulation by SKF 83959 induced a pro-
depressive and anxiogenic behavioural phenotype, we next wanted to determine whether D1-
D2 heteromer inactivation by the TAT-D1 peptide could alleviate the anhedonic response
exhibited by animals that underwent 5-week exposure to CUS. Two-way repeated measures
ANOVA on sucrose preference scores over the 5-week exposure showed significant effects of
Weeks {F(4,31)=11.08, p<0.001}, Stress Exposure {F(1,31)=40.34, p<0.001} and Weeks x
Stress Exposure interactions {F(4,31)=5.99, p<0.001}. Post-hoc comparisons revealed that
sucrose preference scores of CUS-exposed animals were significantly lower than control
animals at the end of the 5-week CUS exposure (Figure II-4A, p<0.01). On the test day, both
the control and CUS-exposed animals were administered TAT-D1 peptide or its inactive
control TAT-Sc. Two-way ANOVA revealed a significant effect of Stress Exposure
{F(1,31)=7.01, p<0.05}. Thus, TAT-Sc peptide-treated CUS animals had significantly lower
sucrose preference score compared to the control animals (Figure II-4B, p<0.05). Interestingly,
a single infusion of TAT-D1 peptide (300pmol, i.c.v.) partially reversed this CUS-induced
reduction of sucrose preference scores as no significant difference between scores of the
controls and those of the TAT-D1-treated CUS animals was observed (Figure II-4B).
In the fruit loops test, two-way ANOVA on the latency to consume fruit loops yielded a
significant main effect of Stress Exposure {F(1,27)=5.831, p<0.05} and Treatment
{F(1,27)=7.646, p<0.05}, with a non-significant Stress Exposure x Treatment interaction
{F(1,27)=3.192, p=0.085}(Figure II-4C). In non-CUS exposed animals, TAT-Sc and TAT-D1
113
Figure II-4. The effects of TAT-D1 peptide treatment on anhedonia-like reactivity
induced by chronic unpredictable stress. A) In the SPT, CUS-exposed animals (black)
exhibited a progressive and significant decrease in sucrose preference scores (% of total
volume consumed) in comparison to non-stressed controls (CTR, white) across 5 weeks of
exposure. B) A single administration of the TAT-D1 peptide (300pmol, i.c.v.) partially
reversed the CUS-induced reduction in sucrose preference score. C) In the FLT, CUS exposure
significantly increased the latency to consume fruit loops, an effect that was abolished by a
single TAT-D1 peptide infusion (300pmol, i.c.v.) D) CUS exposure had no effect on total fruit
loops consumption, whereas TAT-D1 peptide treatment (300pmol, i.c.v.) significantly
increased consumption in both the CTR and CUS-exposed animals. Data represent means ±
50
60
70
80
90
100
Su
cro
se
Co
nsu
mp
tio
n
(% o
f T
ota
l V
olu
me
)
TAT-D
1
TAT-S
c
TAT-D
1
TAT-S
c
CTRCUS
*
0
75
150
225
300
375
450
525
La
ten
cy
to
Co
su
me
Fru
it L
oo
ps (
se
c)
TAT-D
1
TAT-S
c
TAT-D
1
TAT-S
c
CTRCUS
**
0
1
2
3
4
5
6
To
tal
Fru
it L
oo
ps
Co
nsu
me
d
TAT-D
1
TAT-S
c
TAT-D
1
TAT-S
c
CTRCUS
**##
20
40
50
60
70
80
90
100
CTR CUS
1 2 3 4 5
Su
cro
se
Co
nsu
mp
tio
n
(% o
f T
ota
l V
olu
me
)
***
Weeks
A) B)
C) D)
114
peptide did not affect the latency to consume fruit loops (Figure II-4C). For TAT-Sc treated
animals, CUS exposure led to a significant increase in the latency to consume fruit loops
(Figure II-4C, p<0.01). The TAT-D1 peptide completely reversed CUS-induced increase in
latency to the level of TAT-Sc-treated control animals, with no significant effect in the control
animals (Figure II-4C). On the other hand, two-way ANOVA on the total fruit loops
consumption yielded a significant main effect of Treatment {F(1,22)=13.3, p<0.01}(Figure II-
4D), with TAT-D1 significantly increasing the number of fruit loops consumed in both the
control (p<0.05) and CUS-exposed rats (p<0.01), whereas CUS exposure did not affect total
fruit loop consumption compared to the control animals (Figure II-4D).
3.5 CUS-induced Anxiety-like Behaviours
In the novelty suppressed feeding test, exposure to the novel environment consistently
prolonged the feeding latency in all four groups as expected, regardless of stress exposure or
peptide treatment (Figure II-5A). Two-way ANOVA on the feeding latency in the novel
environment resulted in a significant Stress Exposure x Treatment interaction {F(1,27)=6.729,
p<0.05}(Figure II-5A). Post-hoc comparisons revealed that CUS exposure further significantly
prolonged the feeding latency in the novel environment (p<0.01), an effect that was completely
nullified by TAT-D1 peptide treatment (Figure II-5A). However, the TAT-D1 peptide did not
significantly reduce novelty-induced increase in feeding latency in the control animals (Figure
II-5A). On the other hand, despite the robust anxiogenic effect of CUS in the novelty-
suppressed feeding test, the total time spent in the open arms in the elevated plus maze was not
affected by CUS exposure or TAT-D1 peptide treatment (Figure II-5B).
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Figure II-5. The effects of TAT-D1 peptide treatment on anxiety-like reactivity induced
by chronic unpredictable stress. A) In the NSFT, exposure to the novel environment
significantly increased the feeding latency in control (CTR) rats, an effect that was further
enhanced by exposure to CUS. The effect of CUS on novelty-induced increase in feeding
latency was completely abrogated by the TAT-D1 peptide (300pmol, i.c.v.). B) In the EPM,
neither CUS nor TAT-D1 treatment influenced total time spent in the open arms. Data
represent means ± s.e.m. of n=7-8/group. (**p<0.01, compared to CTR/TAT-Sc; CUS:
Chronic Unpredictable Stress, NSFT: Novelty Suppressed Feeding Test, EPM: Elevated Plus
Maze)
0
50
100
150
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4. Discussion
In the present study we demonstrated that stimulation of the dopamine D1-D2 receptor
heteromer induced depression- and anxiety-like behaviours. In the forced swim test, used as a
measure of passive coping or behavioural despair, D1-D2 heteromer stimulation by SKF 83959
significantly reduced the latency to immobility. When anxiety responses were tested in the
elevated plus maze, D1-D2 heteromer stimulation resulted in less time spent in the open arms
of the maze. In the novelty-induced hypophagia test, which measures anxiety induced by the
stress of a novel environment, D1-D2 heteromer stimulation abolished the eagerness of trained
animals to approach and consume highly palatable sweetened milk. More importantly, these
effects were consistently attenuated, or reversed, by pre-exposure to the selective TAT-D1
disrupting peptide prior to D1-D2 heteromer stimulation by SKF 83959, thus confirming the
contribution of the D1-D2 heteromer in these depression- and anxiety-related behavioural
phenotypes.
In addition, TAT-D1 peptide administration on its own exhibited rapid antidepressant-
like and anxiolytic properties in animals exhibiting depressive and anxiety-like phenotypes. In
CUS-exposed animals, TAT-D1 peptide partially reversed anhedonia-like reductions in sucrose
preference after just a single administration, suggesting that it could have a rapid antidepressant
action. Indeed, such an effect requires at least 2 weeks of daily treatment with conventional
antidepressants, during which improvements in sucrose preference scores start to ensue
(Willner et al., 1987a). While the improvement we observed with a single dose of TAT-D1
peptide was partial, it is likely that a few additional administrations could completely
normalize sucrose preference scores. Further investigations are warranted to assess the effects
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of long-term TAT-D1 peptide treatments. Nevertheless, in our hands, TAT-D1 peptide
treatment significantly reversed CUS-evoked novelty-induced suppression of feeding to control
levels, thereby further supporting a potentially rapid-acting antidepressant-like activity induced
by D1-D2 heteromer inactivation. Such effect typically also requires chronic daily treatment
with conventional antidepressants to become apparent (Bodnoff et al., 1989; Bessa et al., 2009).
Moreover, TAT-D1 peptide also increased both the latency to and total consumption of fruit
loops in CUS-exposed animals, which further suggests an anti-anhedonic (or hedonic) property
that is associated with D1-D2 heteromer inactivation.
However, it is worth noting that TAT-D1 treatment by itself did not influence the total
time spent in the open arms in the elevated plus maze test, which was recapitulated in the CUS
experiments where neither CUS nor TAT-D1 treatment altered total time spent in the open
arms. This seems to be at odds with the robust effects of CUS and TAT-D1 peptide treatment
on anxiety-like reactivity in the novelty-suppressed feeding test. Such discrepancy in the
effects of CUS and the TAT-D1 peptide on anxiety-related behaviours as observed in the
elevated plus maze and novelty-suppressed feeding test may be explained by different forms of
anxiety triggered by CUS and antidepressants. Indeed, it was demonstrated that depressive-like
behaviour in the forced swim test and sucrose preference test correlated with anxiety-related
behaviour in the novelty-suppressed feeding test but not with the elevated plus maze (Bessa et
al., 2009).
The specificity of the TAT-D1 peptide to the D1-D2 heteromer has been thoroughly
characterized both in vitro and in vivo (Hasbi et al., 2014). The TAT-D1 peptide was
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comprised of the same 16 amino acid sequence as the D1R C-terminus, including the two
glutamic acid residues that the two arginine residues in the third intracellular loop of the D2R
interact with to form the D1-D2 heteromer (O’Dowd et al., 2012; Hasbi et al., 2014). The TAT-
D1 peptide was able to dose-dependently reduce the BRET and FRET signals elicited by the
D1-D2 heteromer when it was incubated with HEK293 cells stably co-expressing both
receptors and primary cultured striatal neurons, respectively (Hasbi et al., 2014). In addition,
the TAT-D1 peptide also reduced the co-immunoprecipitation of D1R and D2R in cells as well
as in native striatal tissues, and dose-dependently attenuated SKF 83959-induced calcium
mobilization in primary cultured striatal neurons (Hasbi et al., 2014). The specificity of the
TAT-D1 peptide towards the D1-D2 heteromer was further confirmed by findings showing that
the BRET signals elicited by D1R homoligomer, D2R homoligomer, or D2-D5 heteromer were
not affected by TAT-D1 administration (Hasbi et al., 2014). Furthermore, a TAT-scrambled
control peptide that contains the same amino acid residues as the TAT-D1 peptide but in
random order had no effect in the BRET, FRET, Co-IP and calcium mobilization experiments
(Hasbi et al., 2014). Collectively these findings indicate that the TAT-D1 peptide was able to
selectively disrupt the D1-D2 heteromer formation and antagonize its signalling. Therefore, the
use of the TAT-D1 peptide would allow us to exclusively determine D1-D2 heteromer-specific
physiological effects.
Physiologically, activation of the D1-D2 heteromer induces Gq-mediated and
phospholipase C-dependent intracellular calcium release that results in the phosphorylation of
calcium calmodulin kinase II and expression of BDNF in the NAc (Lee et al., 2004; Rashid et
al., 2007; Hasbi et al., 2009). In addition, expression of the major GABA-producing enzyme,
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GAD67, was also found to be significantly increased in the NAc following D1-D2 heteromer
stimulation (Perreault et al., 2012a), thereby potentially allowing D1-D2 heteromer to modulate
the inhibitory GABAergic tone in the NAc. Interestingly, although the proteins that are
modulated by the D1-D2 heteromer signalling are known for their involvement in the
regulation of psychostimulant reward (Anderson et al., 2008; Ghitza et al., 2010), evidence also
suggests their potential roles in the development of anxiety-and depression-like behavioural
phenotypes.
BDNF is well-known for its crucial involvement in the rewarding and addictive
properties of psychostimulants (Ghitza et al., 2010). Nevertheless, several studies have found
strong associations between BDNF expression and the development of anxiety- and
depression-like behaviours that are brain region-specific. For instance, reduced BDNF
expression has been documented in the hippocampus and the PFC of animals that underwent
chronic stress, as well as in post-mortem hippocampal tissue of depressed patients (reviewed in:
Duman, 2014). In contrast, depressed patients also displayed significantly increased levels of
BDNF in postmortem NAc that was not attributed to antidepressant administration (Krishnan et
al., 2007). In addition, powerful pro-depressive effects of BDNF signaling in VTA and NAc
have also been reported in rodents, highlighting the importance of BDNF in the mesolimbic
pathways of brain (reviewed in: Nestler and Carlezon, 2006; Russo and Nestler, 2013) in
mediating the pathophysiological effects of the disorder. Although BDNF in the NAc has been
traditionally thought to be derived from sources outside NAc such as VTA or cortex, and
transported to NAc via dopaminergic or glutamatergic projections respectively, we have now
shown that MSNs that express the D1-D2 heteromer provide a novel endogenous source for
BDNF in NAc (Hasbi et al., 2009). It has been postulated that the contribution of NAc to
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depression may reflect an enhancement of GABAergic signaling within the region manifesting
depression-like responses such as anhedonia (Nestler and Carlezon, 2006b; Tye et al., 2013),
an interesting hypothesis given the strong positive correlation between BDNF and GABAergic
signaling in a number of regions in the brain including prefrontal cortex, and more recently as
we showed in NAc upon activation of the D1-D2 heteromer (Perreault et al., 2012a)
Animals that displayed reduced immobility in the FST also exhibited reduced BDNF
expression in the NAc (Sequeira-Cordero et al., 2014) whereas animals displaying anhedonic
behavior in the sucrose preference test showed increased NAc levels of BDNF mRNA (Bessa
et al., 2013). Similarly, the depressive effects of social defeat stress in rodents are associated
with a rapid and long-lasting increase in BDNF signaling in NAc in susceptible mice that is
concomitant with elevated c-fos expression (Berton et al., 2006), an effect that is mimicked by
selective activation of the D1-D2 heteromer (Hasbi et al., 2009; Perreault et al., 2012a, 2015).
Coincident with an increase in BDNF protein in NAc, there was increased expression of the
BDNF receptor TrkB (Perreault et al., 2013), indicative of heightened BDNF signaling in this
region. Interestingly activation of the D1-D2 heteromer also resulted in weight loss
(unpublished data), a physiological response also seen in mice susceptible to social defeat
stress. In line with these findings, suppression of BDNF signaling in VTA attenuated the stress-
induced increase of BDNF in NAc and promoted resilience in social defeat tests (Berton et al.,
2006; Krishnan et al., 2007; Fanous et al., 2011; Wang et al., 2014). Similarly, blockade of
BDNF signaling in NAc, using a negative functional mutant of TrkB, induced an
antidepressant effect in the FST (Eisch et al., 2003) whereas infusion of BDNF into the NAc
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enhanced susceptibility to develop depression-like behaviours in animals that underwent social
defeat stress (Krishnan et al., 2007).
A potential involvement for the D1-D2 heteromer in prefrontal cortex (PFC) in
depression has been previously implicated (Pei et al., 2010). In the study by Pei et al, with the
use of a synthetic TAT-D2 peptide, disruption of the D1-D2 heteromer reduced both the total
immobility in the FST and the total number of escape failures in a learned helplessness
paradigm that involved chronic foot shock (Pei et al., 2010). Although the mechanism by
which D1-D2 heteromer disruption in PFC induced antidepressant effects in rodents was not
explored, evidence suggests it did not likely involve changes in BDNF or GAD67 as systemic
activation of the D1-D2 heteromer had no effect on the expression of either of these proteins in
the PFC (Perreault et al., 2012b). In addition, although enhanced glycogen synthase kinase-3
(GSK-3) activation in PFC has been implicated in the pathology of depression (Karege et al.,
2007), D1-D2 heteromer activation phosphorylated GSK-3 in PFC to suppress its activity
(Perreault et al., 2012b), thus negating a role for this kinase in mediating the antidepressant
effects of D1-D2 heteromer disruption in PFC. One possibility is that disruption of the D1-D2
heteromer in PFC regulates the activity of glutamatergic efferents, as the D1-D2 heteromer is
likely localized to cortical pyramidal neurons as a result of the high levels of D1R and D2R
coexpression in these neurons (Zhang et al., 2010). Nonetheless, although BDNF in NAc
appears to be a likely candidate in mediating the pro-depressive effects of the D1-D2 heteromer,
the molecular substrates that contribute to the antidepressant effects of D1-D2 heteromer
disruption in PFC remain to be elucidated.
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A potential caveat of the present study is the use of SKF 83959 to examine the effect of
D1-D2 heteromer stimulation on the expression of depression- and anxiety-like behavioural
phenotypes. Although SKF 83959 is a pharmacological agent that can stimulate the D1-D2
heteromer without concurrently activating the D1R or the D2R, this alleged dopamine agonist
has also been shown to have affinity for, or activates, other receptors including the dopamine
D5 receptor (Sahu et al., 2009; Perreault et al., 2012b), the α2C-adrenergic receptor, and the
serotonin 5HT-2C receptor (Chun et al., 2013). Therefore, the pro-depressive and anxiogenic
effect of SKF 83959 treatment as observed in the current study may possibly not be solely
mediated by the D1-D2 heteromer, but could also be partly due to the involvement of other
receptor systems as well (Holmes et al., 2001; Bagdy et al., 2002). However, the ability of the
highly selective TAT-D1 peptide to attenuate or even reverse the pro-depressive and
anxiogenic effects of SKF 83959 in all three behavioural paradigms validated the involvment
of the D1-D2 heteromer in the manifestation of depressive and anxiety-like behavioural
phenotypes, as we have shown that the TAT-D1 peptide did not affect the D5R or the 5HT-2C
receptor (Hasbi et al., 2014). In addition, SKF 83959 treatment has also been previously shown
to suppress amphetamine reward (Shen et al., 2015), thus implicating the D1-D2 heteromer in
the negative modulation of the brain reward system. This may explain why a dose of SKF
83959 that was insufficient to promote anxiety in the elevated plus maze was able to
completely prevent milk consumption in the novelty-induced hypophagia test, which may be a
result of simultaneous inhibition of milk reward and the emergence of anxiogenesis in response
to SKF 83959 treatment. However, the reduction in the latency to consume sweetened milk in
the novelty-induced hypophagia test following TAT-D1 peptide pre-treatment again verifies a
role for the D1-D2 heteromer in promoting anxiety-like behaviours.
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In summary, using agonist stimulation and selective antagonism by the use of a
heteromer disrupting TAT-D1 peptide, we have demonstrated that the dopamine D1-D2
receptor heteromer induced robust depression- and anxiety-like behaviour in rodents upon its
stimulation, a physiological function which has not been demonstrated for any other receptor
complex. More importantly, a potentially rapid onset of antidepressant-like activity produced
by selective inactivation of the D1-D2 heteromer may have great implication in the treatment
of depression and anxiety disorders as current interventions require chronic treatment to
achieve their therapeutic effects (Culpepper, 2010). Nevertheless, future studies will be
required to elucidate the exact mechanism by which the D1-D2 heteromer inactivation in the
NAc modulates the fast-onset antidepressant-like and anxiolytic effects as observed in the
current study.
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3. The dopamine D1-D2 receptor heteromer exerts tonic inhibitory effect on the
expression of amphetamine-induced locomotor sensitization
Abstract
A role for the dopamine D1-D2 receptor heteromer in the regulation of reward and
addiction-related processes has been previously implicated. In the present study, we examined
the effects of D1-D2 heteromer stimulation by the agonist SKF 83959 and its disruption by a
selective TAT-D1 peptide on amphetamine-induced locomotor sensitization, a behavioural
model widely used to study the neuroadaptations associated with psychostimulant addiction.
D1-D2 heteromer activation by SKF 83959 did not alter the acute locomotor effects of
amphetamine but significantly inhibited amphetamine-induced locomotor responding across
the 5 day treatment regimen. In addition, a single injection of SKF 83959 was sufficient to
abolish the expression of locomotor sensitization induced by a priming injection of
amphetamine after a 72-hour withdrawal. Conversely, inhibition of D1-D2 heteromer activity
by the TAT-D1 peptide enhanced subchronic amphetamine-induced locomotion and the
expression of amphetamine locomotor sensitization. Treatment solely with the TAT-D1
disrupting peptide during the initial 5 day treatment phase was sufficient to induce a sensitized
locomotor phenotype in response to the priming injection of amphetamine. Together these
findings demonstrate that the dopamine D1-D2 receptor heteromer exerts tonic inhibitory
control on neurobiological processes involved in sensitization to amphetamine, indicating that
the dopamine D1-D2 receptor heteromer may be a novel molecular substrate in addiction
processes involving psychostimulants.
Shen M.Y.F., Perreault M.L., Fan T., George S.R. The dopamine D1-D2 receptor heteromer
exerts a tonic inhibitory effect on the expression of amphetamine-induced locomotor
sensitization. Pharmacol Biochem Behav. 2015 Jan;128:33-40.
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1. Introduction
The dopamine D1-D2 receptor heteromer is a G protein-coupled receptor (GPCR)
complex that couples to the Gαq protein to elicit a phospholipase C (PLC)-dependent calcium
signal upon its activation (Rashid et al., 2007). It has been reported that a significant proportion
of D1 receptor (D1R)-expressing medium spiny neurons (MSNs) in the nucleus accumbens
(NAc) co-express the dopamine D1 and D2 receptors (17-25%) (Bertran-Gonzalez et al., 2008;
Matamales et al., 2009; Perreault et al., 2010; Gangarossa et al., 2013a) and approximately 90%
of these MSNs express the D1-D2 heteromer (Hasbi et al., 2009; Perreault et al., 2010). In
contrast, only 2-6% of D1R-expressing MSNs in the caudate putamen co-express the D1R and
D2R, of which only 25% of the neurons exhibit D1-D2 heteromer formation (Perreault et al.,
2010). The D1-D2 co-expressing neurons in the NAc extend efferent projections which directly
or indirectly influence the ventral tegmental area (VTA) (Perreault et al., 2012a), a region
widely known for its role in mediating addiction-like behaviours and reward through the
regulation of mesolimbic dopamine activity (Reviewed: Chen et al., 2010; Koob & Volkow,
2010).
We have previously shown that activation of the D1-D2 receptor heteromer modulated the
expression of proteins involved in drug addiction (Hasbi et al., 2009; Ng et al., 2010; Perreault
et al., 2010, 2012a), such as brain-derived neurotrophic factor (BDNF) and calcium-calmodulin
kinase II (CaMKII) in the NAc and VTA, and D1-D2 heteromer activation in NAc shell
enhanced production of the inhibitory neurotransmitter GABA in VTA (Perreault et al., 2012a).
These findings thus suggest a potential role for the D1-D2 heteromer in the regulation of
neuronal activity in the VTA and possibly as a regulator of brain reward processes.
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Since repeated amphetamine treatment was previously shown to enhance the functional
activity of the D1-D2 heteromer in rat striatum (Perreault et al., 2010), in this study we aimed to
further examine the potential involvement of the D1-D2 heteromer in processes linked with
addiction using the amphetamine-induced locomotor sensitization model in rats.
Psychostimulant-induced locomotor sensitization was proposed to be an animal model for drug
craving, and is characterized by a context-dependent, progressive augmentation of locomotor
responsiveness following repeated non-contingent administration of psychostimulants such as
cocaine and amphetamine (Robinson and Becker, 1986; Kalivas and Stewart, 1991; Robinson
and Berridge, 1993; Anagnostaras and Robinson, 1996). amphetamine-induced locomotor
sensitization is associated with neuroadaptations of the mesolimbic dopamine system that may
enhance the reinforcing properties of cocaine and amphetamine, as animals that were previously
sensitized with repeated amphetamine treatment showed increased acquisition of drug self-
administration (Mendrek et al., 1998; Suto et al., 2002; Vezina et al., 2002).
Once established, amphetamine locomotor sensitization has been reported to persist for
over a year (Paulson and Robinson, 1991), which may be a reflection of some of the long-term
neurobiological adaptations that accompany the persistent drug-seeking behaviours typically
seen in addicted patients (Robinson and Berridge, 2000). Similarly, animals that exhibited the
reinstatement of cocaine-seeking behaviour induced by a single exposure to amphetamine also
expressed locomotor sensitization (De Vries, 1998), suggesting that changes in the mesolimbic
dopamine system that accompany the expression of amphetamine locomotor sensitization may
also contribute to relapse of drug seeking.
The dopamine agonist SKF 83959 is a partial agonist for the D1-D2 heteromer with a
number of in vitro and in vivo studies demonstrating its ability to induce D1-D2 heteromer-
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mediated calcium signaling (Rashid et al., 2007; Verma et al., 2010), Gq and PLC activation
(Rashid et al., 2007), CaMKII phosphorylation (Ng et al., 2010) and BDNF production (Hasbi
et al., 2009). Validation of selectivity to the D1-D2 heteromer in these studies employed D1 and
D2 antagonists and dopamine receptor knockout mice. Recent studies, however, have indicated
that SKF 83959 has affinity for, or activates, a number of other receptors, such as the dopamine
D5 receptor, the α-adrenergic receptor 2C, and the serotonin 5HT-2C receptor (Sahu et al., 2009;
Perreault et al., 2012b; Chun et al., 2013). In addition, there are conflicting reports as to
whether SKF 83959 functions as an antagonist (Downes and Waddington, 1993; Cools et al.,
2002; Jin et al., 2003), a partial agonist (Lee et al., 2014), or has no effect (Lee et al., 2004;
Rashid et al., 2007) at the D1R. To assist in elucidating the physiological role of the D1-D2
heteromer, we developed a selective D1-D2 heteromer antagonist, the TAT-D1 peptide, which
occludes the interaction site between the two receptors (O'Dowd et al., 2012), thus inhibiting
D1-D2 heteromer expression and function and abolishing the physiological effects of D1-D2
heteromer activation by SKF 83959 without affecting other receptor oligomers such as D1-D1
homomers or D2-D5 heteromers (Hasbi et al., 2014). In the present study, we assessed the
effects of SKF 83959 on the expression of amphetamine locomotor responses and sensitization.
We only attributed an effect to be D1-D2 heteromer-specific when the TAT-D1 peptide
produced opposite behavioural output compared to SKF 83959. Our findings showed a novel
role for the D1-D2 heteromer in the suppression of amphetamine-induced locomotion and
locomotor sensitization.
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2. Materials and Methods
2.1 Animals
Ninety-six adult male Sprague-Dawley rats (Charles River, Canada), weighing 300-350 g
at the start of the experiment, were used. Rats were housed in polyethylene cages in a
temperature-controlled colony room, maintained on a 12-h light-dark cycle (lights on at 0700h),
with ad libitum access to food and water. Rats were handled daily for 5 days before the start of
the experiment. All treatments were performed during the light phase of the day-night cycle.
Animals were housed and tested in compliance with the guidelines described in the Guide to the
Care and the Use of Experimental Animals (Canadian Council on Animal Care, 1993), and
were approved by the Animal Care Ethics Committee of the University of Toronto.
2.2 Drugs
SKF 83959 hydrobromide (Tocris Bioscience) was dissolved in physiological saline
containing 5% DMSO, and was administered subcutaneously (s.c.). Amphetamine
hydrochloride (Sigma-Aldrich) was dissolved in physiological saline (0.9% NaCl), and was
administered intraperitoneally (i.p.). The TAT-D1 disrupting peptide was dissolved in saline
and administered into the intracerebroventricular (i.c.v.) space 15 minutes prior to vehicle, SKF
83959, or amphetamine injection. For non-drug injections, an equivalent volume of saline or
vehicle was administered. All systemic injections were given at a volume of 1.0 ml/kg just prior
to behavioural testing.
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2.3 Surgery
Rats were anesthetized with isoflurane (5%), administered analgesic ketoprofen (5 mg/kg,
s.c.) and secured in a stereotaxic frame. A cannula (22-gauge, Plastics One) was placed
unilaterally into the intracerebroventricular space close to the midline according to the
following stereotaxic coordinates: AP -0.8mm, ML + 1.3mm, DV – 3.7mm, and was secured by
dental cement anchored with four stainless steel screws (Plastics One) fixed on the dorsal
surface of the skull. AP and ML coordinates were taken from bregma, DV coordinate from the
dura (Paxinos and Watson, 1998). The animals were allowed to recover in their home cage for a
minimum of five days before the experiments were performed. Cannulae placement was
visually validated postmortem in brain slices.
2.4 Locomotor Activity Apparatus
The testing environment was a non-colony room containing 8 empty activity chambers that
are 20cm in height, 25cm in width, and 40cm in length. Two arrays of 16 infrared photocells
were attached along the longer sides of the polyethylene cages. The activity chambers were
interfaced to a computer that provided automated recording of horizontal locomotor activity
when both top and bottom infrared photocells were triggered. Ventilated polyethylene lids were
used to cover the activity chambers to prevent animals from escaping.
2.5 Locomotor Sensitization Protocol
The behavioural testing for locomotor sensitization to amphetamine was conducted using a
previously described protocol (Hall et al., 2008), which consisted of 3 phases:
Phase I: Habituation.
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Rats were first habituated to the activity chamber for 2 days for 30 minutes per day.
Phase II: The Sensitizing Regimen
In this phase, we examined the effects of D1-D2 heteromer stimulation and inactivation on
locomotion induced by acute and subchronic amphetamine treatment (1.5mg/kg, i.p.). Animals
were administered their designated drug treatment, VEH+SAL, SKF 83959+SAL, TAT-
D1+SAL, VEH+amphetamine, SKF 83959+amphetamine, TAT-D1+amphetamine, once daily
for 5 consecutive days. Immediately following each injection, horizontal locomotor activity was
monitored for 60 minutes. The dose of SKF 83959 (0.4 mg/kg, s.c.) given in this phase was
chosen based on our previous study showing repeated SKF 83959 treatment significantly
enhanced locomotor activity and grooming responses without desensitizing the D1-D2
heteromer (Perreault et al., 2010). The dose of the TAT-D1 peptide (300pmol, i.c.v.) was
previously shown to disrupt D1-D2 heteromer formation in vivo as indicated by the loss of co-
immunoprecipitation of D1R with D2R in rat NAc tissue (Hasbi et al., 2014).
Phase III: The Expression of Locomotor Sensitization
Here we examined the effect of D1-D2 heteromer stimulation/inactivation alone or with
amphetamine during the sensitizing regimen on the expression of locomotor sensitization to
amphetamine. All animals from Phase II underwent a 72 hour withdrawal following the 5th
injection, which was a time point previously shown to be sufficient to induce the expression of
locomotor sensitization in response to amphetamine challenge (Hall et al., 2008). Following
withdrawal, all animals were challenged with a priming injection of amphetamine (1.0 mg/kg,
i.p.), and their horizontal locomotor activity were measured for 60 minutes. In addition, a subset
of animals that were treated solely with amphetamine during Phase II received the co-
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administration of SKF 83959 (2.5 mg/kg, s.c.) or TAT-D1 peptide (300 pmol, i.c.v.) with the
priming amphetamine to examine the effect of acute D1-D2 heteromer stimulation or
inactivation on established locomotor sensitization to amphetamine. We increased the dose of
SKF 83959 for acute injection in the third phase, which was a dose that we have previously
shown to induce changes in CaMKII expression in rat striatum (Ng et al., 2010).
In addition, in order to elucidate the involvement of the D1-D2 heteromer on basal
locomotion, we performed a separate experiment in which animals were treated acutely with
VEH+SAL, SKF 83959 (0.4mg/kg, s.c.)+SAL, VEH+TAT-D1 (300pmol, i.c.v.), SKF
83959+TAT-D1 without habituation to the test chamber. The horizontal locomotor activity of
these animals was measured for 60 minutes immediately following SKF 83959 or VEH
treatment.
2.6. Data Analysis
All horizontal locomotor activity values are reported as mean ± s.e.m. All data was
analyzed for normality prior to ANOVAs using the Shapiro-Wilk test. The results shown in
Figure 1 were analyzed by Two-way ANOVA with D1-D2 heteromer Treatment (VEH, SKF
83959, TAT-D1 peptide) and amphetamine Treatment (SAL, amphetamine) as the between
subjects factors. The results shown in Figure 2 were analyzed by Repeated measures of
ANOVA with Injection as the within subject factor and Drug Treatments as the between subject
factor, followed by post-hoc test (Duncan multiple range test). The results shown in Figure 4
were analyzed by One-way ANOVA with Drug Treatments as the between subjects factor.
Planned comparisons between selected groups were performed using Student’s t-test. Planned
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comparisons were performed for Figure III-4A, Expression of amphetamine sensitization,
between the following groups: Saline vs. AMPH; AMPH vs. SKF+AMPH; AMPH vs. TAT-
D1+AMPH. Computations were performed using the SPSS statistical program. Statistical
criteria for significant differences were set at p<0.05.
3. Results
3.1 The effects of acute D1-D2 heteromer activation and disruption on basal and amphetamine-
induced locomotor activity
Using the horizontal locomotor activity data obtained from the 1st injection, we first
examined the effect of acute SKF 83959 administration or TAT-D1-induced D1-D2 heteromer
disruption on basal and amphetamine-induced locomotor activity (Figure III-1A-C). Two-way
ANOVA with D1-D2 heteromer Treatment and amphetamine Treatment as between subject
factors revealed a significant main effect of D1-D2 Treatment {F(2:82)=3.489, p<0.05} and a
significant main effect of amphetamine Treatment {F(1:82)=78.690, p<0.0001}, as well as a
significant D1-D2 Treatment X amphetamine Treatment interaction {F(2:82)=7.335, p<0.001}.
Planned comparisons between treatment groups were then made using Student’s t-test. As
shown in Figure 1A, over the 60 minute period, administration of the agonist SKF 83959 (0.4
mg/kg, s.c.) alone induced a significant increase in locomotion compared to saline-treated
controls (bar 1: 818.6 ± 70.8 vs. bar 2: 3314.4 ± 389.6, t(14)=6.30, p<0.001). Amphetamine
treatment alone induced a significant increase in locomotion (bar 1: 818.6 ± 70.8 vs. bar 4:
6081.2 ± 291.1, t(46)=8.00, p<0.001), which was not influenced by SKF 83959 co-treatment
(bar 5: 5124.9 ± 377.2). Similar to SKF 83959, acute pre-administration (15 min) of the TAT-
133
Figure III-1. The effects acute D1-D2 heteromer stimulation and inactivation on basal
and amphetamine-induced locomotor activity. A) The total horizontal activity over 60
minutes in units of beam breaks in response to acute administration of amphetamine (1.5 mg/kg,
i.p.), alone or in combination with SKF 83959 (0.4 mg/kg, s.c.) or TAT-D1 peptide (300 pmol,
i.c.v.). B) Time course of the acute locomotor response measured in 5-min intervals over 60
minutes. C) The total horizontal activity for the first 5 minutes. D) Total horizontal activity in
the absence of habituation in response to SKF 83959, TAT-D1 peptide, or SKF 83959 + TAT-
D1. Data represent means ± s.e.m. of n=7-11 rats/group with the exception of amphetamine,
n=40 rats/group, and controls n=16 rats/group. AMPH=amphetamine. (*p<0.05, ** p<0.01,
*** p<0.001 compared to Saline Control.)
0
200
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800SalineSKF 83959
AMPHSKF+AMPH
TAT-D1 TAT-D1+AMPH
5 10 15 20 25 30 35 40 45 50 55 60
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134
D1 peptide (300 pmol, i.c.v.) to rats, previously shown to disrupt the D1-D2 heteromer in rat
NAc (Hasbi et al., 2014), also elevated total locomotor activity compared to controls (bar 1:
818.6 ± 70.8 vs. bar 3: 2918.1 ± 317.6, t(14)=6.45, p<0.001) with no effect on acute
amphetamine-induced locomotion (bar 6: 6420.7 ± 440.2).
Examination of the time course of locomotion revealed that the acute locomotor activating
effects of SKF 83959 and TAT-D1 peptide steadily declined across the 60 minutes (Figure III-
1B, dotted lines). Conversely, the locomotor activity of animals treated with amphetamine
alone or in combination with SKF 83959 or TAT-D1 peptide remained elevated throughout the
testing period (Figure III-1B, solid lines). It is noteworthy that in the first 5 minutes of the
testing period (Figure III-1C), the animals treated with TAT-D1 peptide alone exhibited a very
robust increase in locomotor activity (t(22)=6.15, p<0.001) that was not apparent in the other
treatment groups. {D1-D2 Treatment: F(2,82)=17.6, p<0.0001; amphetamine: F(1,82)=0.2,
p<0.68; D1-D2 Treatment X amphetamine: F(2,82)=7.7, p<0.001}.
Since D1-D2 heteromer stimulation by SKF 83959 and its inactivation by the TAT-D1
peptide both stimulated basal locomotion in habituated animals, we wanted to further resolve
the role of D1-D2 heteromer in modulating basal locomotion. Therefore, we next tested animals
in the absence of habituation, a process which may affect total horizontal locomotor activity
due to the animal’s natural tendency to explore a novel environment (Eilam and Golani, 1989).
One-way ANOVA revealed a significant main effect of Drug Treatments {F(4,56)=16.749,
p<0.0001}. As shown in Figure III-1D, Duncan’s post hoc analysis showed that vehicle-treated
rats indeed exhibited significantly higher locomotion in the absence of habituation (bar 1: 818.6
± 70.8 vs. bar 2: 2466.4 ± 193.4, p<0.01). The exposure to a novel environment also abolished
135
Figure III-2. The effects of subchronic D1-D2 heteromer stimulation and inactivation on
basal and amphetamine-induced locomotor activity. Repeated amphetamine treatment (1.5
mg/kg, i.p.) produced a slight increase in total locomotion over the 5 injections. Repeated SKF
83959 co-treatment (0.4 mg/kg, s.c.) attenuated, while repeated TAT-D1 peptide pre-treatment
(300pmol, i.c.v.) enhanced, amphetamine-induced locomotion over the 5 injections. Across
injections animals treated solely with SKF 83959 or TAT-D1 peptide did not significantly
differ from Saline controls. Data represent means ± s.e.m. of n=7-11 rats/group with the
exception of amphetamine, n=40 rats/group, and controls n=16 rats/group.
AMPH=amphetamine. (*p<0.05, compared to amphetamine group)
1 2 3 4 50
1000
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Injection
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136
the locomotor-activating effect of acute TAT-D1 peptide pre-treatment (bar 2: 2466.4 ± 193.4
vs. bar 3: 2970.8 ± 280.1), while the locomotion induced by SKF 83959 was maintained (bar 2:
2466.4 ± 193.4 vs. bar 4: 4112.3 ± 350.5, p<0.05). We further showed that pre-treatment with
TAT-D1 peptide did not attenuate the locomotor-activating effect of SKF 83959 in the absence
of habituation (bar 4: 4112.3 ± 350.5 vs. bar 5: 3806.8 ± 359.0). This demonstrates that
locomotor activation induced by SKF 83959 occurs via a mechanism not involving the D1-D2
heteromer.
3.2 The effects of D1-D2 heteromer activation and disruption on locomotor activity induced by
repeated amphetamine
To determine whether the D1-D2 heteromer could influence locomotor responding
induced by repeated amphetamine treatment, rats were administered 5 daily injections of
amphetamine alone or in combination with SKF 83959 or the TAT-D1 peptide, and locomotion
was monitored for 60 minutes following each drug injection (Figure III-2). Repeated measures
of ANOVA showed a significant within subject effect of Injection for the groups that received
amphetamine alone {F(4,40)=13.092, p<0.001} and amphetamine plus TAT-D1 {F(4,8)=5.106,
p<0.01}. Duncan’s post hoc analysis using Drug Treatment as the between subject factor
following repeated measures of ANOVA {F(5,83)=35.410, p<0.0001} further revealed that,
compared to the amphetamine alone group, co-administration of SKF 83959 significantly
diminished locomotor responsiveness to repeated amphetamine treatment (p<0.05) whereas
TAT-D1 co-treated rats exhibited significantly enhanced repeated amphetamine-induced
137
locomotion (p<0.05). Across injections, animals treated solely with SKF 83959 or TAT-D1
peptide did not significantly differ from saline controls.
We also examined the change in the temporal pattern of locomotor activity across the 5
injections for each of the treatments described in Figure 2. Repeated saline treatment had no
effect on the temporal pattern of locomotor activity, which showed a steady decline over the 60
minutes (Figure III-3, upper left panel). SKF 83959 treatment at the first injection produced an
initial excitation in locomotor activity that declined after the 15 minute mark (Figure III-3,
upper middle panel). With subsequent injections, this period of initial excitation gradually
diminished and ultimately disappeared. On the other hand, TAT-D1 peptide treatment
consistently produced a robust initial increase in locomotor activity within the first 5 minutes
across the 5 injections (Figure III-3, upper right panel). In contrast to SKF 838959 treatment,
repeated injections did not alter the temporal pattern of locomotor activity produced by the
TAT-D1 peptide.
The temporal pattern of locomotor activity for the first amphetamine injection steadily
increased and peaked at approximately 40 minutes (Figure III-3, lower left panel). With
successive amphetamine injections, a period of initial excitation emerged at the 10 minute mark,
followed by a period of secondary excitation that peaked at 40 minutes, indicating that repeated
injections altered the temporal pattern of locomotor activity associated with amphetamine
treatment. SKF 83959 co-treatment with amphetamine produced a similar temporal pattern of
locomotor activity over the 5 injections as the amphetamine alone group (Figure 3, lower
middle panel), although the overall locomotor activity was consistently lower in the SKF 83959
co-treated group. In contrast, TAT-D1 peptide pre-treatment with amphetamine progressively
138
Figure III-3. The time course of basal or amphetamine-induced locomotor responses
following D1-D2 heteromer stimulation or inactivation during the course of repeated
treatment. Horizontal activity is plotted at 5-min intervals. Lines depict overall group means.
AMPH=amphetamine. (n=8-11 rats/group with the exception of amphetamine, n=40 rats/group,
and controls n=16 rats/group).
0 10 20 30 40 50 600
200
400
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800
1000TAT-D1
Time (min)
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139
increased the maximum locomotor activity during the secondary excitation over the 5 injections
compared to the amphetamine alone group.
3.3 The effects of D1-D2 receptor heteromer activation and disruption on the expression of
amphetamine-induced locomotor sensitization
We next wanted to determine whether the D1-D2 heteromer could influence amphetamine-
induced expression of locomotor sensitization in response to a priming amphetamine injection
following a 72 hour withdrawal from the 5th
injection (Figure III-4). One-way ANOVA
revealed a significant main effect of Drug Treatment {F(7,84)=3.950, p<0.01}. As shown in
Figure III-4A, Planned comparisons further showed that the group treated with repeated prior
amphetamine injections (1.5 mg/kg, i.p.) exhibited significantly higher locomotor activity
compared to saline pretreated controls in response to the priming injection of amphetamine (1.0
mg/kg, i.p.) administered on the test day, indicative of locomotor sensitization to amphetamine
(bar 4: 6633.3 ± 354.3 vs. bar 1: 4723.0 ± 317.7, t(22)=3.46, p<0.01). Animals previously
administered SKF 83959 alone did not differ from saline pretreated controls in locomotor
activity following the priming amphetamine injection (bar 2 vs. bar 1). Interestingly, repeated
administration of TAT-D1 peptide alone elicited a sensitized locomotor response to the priming
amphetamine that was significantly higher than saline pretreated controls (bar 3: 7337.0 ± 920.4
vs. bar 1: 4723.0 ± 317.7, t(14)=2.69, p<0.05), and similar in magnitude to that observed in the
amphetamine-sensitized group.
Animals that received SKF 83959 plus amphetamine during the 5 day injection period did
not exhibit sensitized responding to the priming amphetamine on the test day (bar 5 vs. bar 1).
140
Figure III-4. The effects of D1-D2 receptor heteromer stimulation and inactivation on the
expression of amphetamine-induced locomotor sensitization. A) Total horizontal activity in
response to a priming injection of amphetamine (1.0 mg/kg, i.p.) for animals treated daily for 5
days with vehicle, SKF 83959 (0.4 mg/kg, s.c.), TAT-D1 peptide (300 pmol, i.c.v.),
amphetamine (1.5 mg/kg, s.c.), AMPH+SKF 83959 or AMPH+TAT-D1 peptide. Total
horizontal activity in response to priming amphetamine plus SKF 83959 (2.5mg/kg, s.c.) or
TAT-D1 (300pmol, i.c.v.) for amphetamine-sensitized animals are also shown. B) Time course
of the acute locomotor response to priming amphetamine in rats that received subchronic
pretreatment with Saline, SKF 83959 or TAT-D1 peptide. C) The time course showing the
effect of subchronic pretreatment with amphetamine, SKF 83959+amphetamine or TAT-
D1+amphetamine on priming amphetamine-induced locomotor sensitization. D) Time course
of the sensitized locomotor response to priming amphetamine alone, amphetamine+SKF 83959
or amphetamine+TAT-D1 for amphetamine pre-treated rats. Horizontal activity in 5-min
intervals is shown and is plotted in units of beam breaks. Data represent means ± s.e.m. of n=8-
16 rats/group. AMPH=amphetamine. (*p<0.05, ** p<0.01 compared to SAL group, #p<0.05
compared to amphetamine group.)
0
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Pretreatment:
Test: AMPH
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In contrast, animals that received prior exposure of amphetamine plus TAT-D1 for 5 days not
only produced a sensitized locomotor phenotype in response to the priming amphetamine
compared to the saline pretreated controls (bar 6: 8989.3 ± 994.0 vs. bar 1: 4723.0 ± 317.7,
t(14)=4.09, p<0.01), but the sensitized responding was also further significantly enhanced
compared to amphetamine-sensitized animals (bar 6: 8989.3 ± 994.0 vs. bar 4: 6633.3 ± 354.3.
t(22)=2.76, p<0.05). Acute co-administration of SKF 83959 with the priming amphetamine on
the test day (bar 7) abolished the expression of locomotor sensitization in animals pretreated
with amphetamine whereas acute TAT-D1 peptide pre-treatment with the priming amphetamine
did not affect the expression of locomotor sensitization to amphetamine (bar 8: 7948.5 ± 1179.5
vs. bar 1: 4723.0 ± 317.7, t(14)=2.64, p<0.05).
Lastly, we also examined the time course of the locomotion induced by the priming
amphetamine on the test day for each of the drug treatments described previously in Figure III-
4A. Prior repeated treatment solely with SKF 83959 or the TAT-D1 peptide did not alter the
dynamics of horizontal locomotor activity over the 60 minutes in response to the priming
amphetamine on the test day compared to the saline pretreated group (Figure III-4B). However,
post hoc analysis following repeated measures of ANOVA using Treatment as the between
subject factor {F(2,24)=3.31, p<0.056} showed a significant increase in locomotion over the 60
minutes following amphetamine priming for animals treated solely with repeated TAT-D1
peptide during the sensitizing regimen compared to those treated with repeated saline (p<0.05).
Prior SKF 83959 co-treatment or TAT-D1 pre-treatment with amphetamine during the
sensitizing regimen also did not affect the dynamics of horizontal locomotor activity over the
60 minutes in response to the priming amphetamine on the test day compared to the
amphetamine alone group (Figure III-4C). Nevertheless, SKF 83959 co-treatment and TAT-D1
142
pre-treatment with amphetamine consistently showed an opposing effect on priming
amphetamine-induced locomotion at each 5 minute time point throughout the 60 minute period
{F(2,34)=5.28, p<0.05}, with the TAT-D1 pre-treated group being significantly higher than the
amphetamine alone group (p<0.05) and SKF 83959 co-treatment showing a trend towards a
significant decrease (p<0.07). Similarly, acute co-treatment of SKF 83959 and pre-treatment of
TAT-D1 with the priming amphetamine also showed an opposing effect on priming
amphetamine-induced locomotion at each 5 minute time point throughout the 60 minute period
in animals previously sensitized with repeated amphetamine {F(2,36)=6.14, p<0.01} (Figure
III-4D), with SKF 83959 co-treatment being significantly lower than the amphetamine alone
group (p<0.05). Moreover, SKF 83959 co-treatment with the priming amphetamine also altered
the dynamics of horizontal locomotor activity starting from the 15 minute mark compared to the
amphetamine alone group, while TAT-D1 pre-treatment had no effect.
4. Discussion
In the present study we demonstrated that the dopamine D1-D2 receptor heteromer plays a
significant regulatory role in the locomotor activating effects of amphetamine and in locomotor
sensitization to amphetamine. Specifically, we showed that SKF 83959 co-treatment reduced
the magnitude of the locomotor response induced by repeated amphetamine administration and
abolished the expression of locomotor sensitization, with even a single treatment of the agonist
SKF 83959 being sufficient to abolish the sensitized phenotype of amphetamine-treated rats.
Conversely, inactivation of the D1-D2 receptor heteromer by a selective disrupting TAT-D1
peptide enhanced locomotion induced by repeated amphetamine treatment and augmented the
143
expression of sensitized locomotor responding to amphetamine priming, findings which
strongly suggest that the D1-D2 heteromer functions to tonically suppress the neurobiological
processes involved in these responses.
Amphetamine-induced locomotor sensitization has been associated with neuroadaptations
in the mesolimbic system that are critically involved in the regulation of reward and motivation
(Robinson and Becker, 1986; Henry and White, 1991; Kantor et al., 1999), and which have
been proposed to enhance the incentive salience, or “wanting”, of the drug itself, as well as
drug-related stimuli such as the environmental context associated with drug use, leading to
compulsive drug seeking behaviors that are typically seen in addicted individuals (Robinson
and Berridge, 2000; Schmidt and Beninger, 2006). Thus, these findings provide the first
evidence of a possible role for the receptor complex as a negative regulator of the physiological
processes associated with amphetamine addiction.
Numerous studies have demonstrated that amphetamine-induced locomotor sensitization is
a complex behaviour involving the close interplay between various neurotransmitter systems in
regions of the mesocorticolimbic circuitry. The initiation of amphetamine-induced locomotor
sensitization is thought to depend on enhanced VTA dopaminergic neuron excitability in the
form of long-term potentiation (LTP), which occurs as a consequence of reduced D2 auto-
receptor inhibition and reduced inhibitory GABAergic control in the VTA, as well as increased
glutamate release from the medial prefrontal cortex, following repeated non-contingent
amphetamine administration (White and Wang, 1984; Wolf and Xue, 1998; Saal et al., 2003).
This subsequently results in increased dopamine release in the NAc in response to amphetamine
challenge that act on supersensitized D1 receptors (Robinson et al., 1988; Wolf et al., 1994),
leading to the expression of amphetamine-induced locomotor sensitization. It should be noted
144
that the increased dopamine release into the NAc was also shown to be dependent on the
enhanced activity of CaMKII in the NAc following amphetamine challenge in cocaine-
sensitized rats (Pierce and Kalivas, 1997). In addition to increased dopamine release,
amphetamine challenge also results in increased glutamate release into the NAc from the
medial PFC in amphetamine-sensitized animals, the attenuation of which has been shown to
block the expression of amphetamine-induced locomotor sensitization (Kim et al., 2005).
There are several potential mechanisms by which the D1-D2 heteromer stimulation may
counteract the neuroadaptations associated with amphetamine-induced locomotor sensitization.
We have previously demonstrated that D1-D2 heteromer stimulation by a single systemic SKF
83959 injection significantly increased the expression of a major GABA producing enzyme,
glutamic acid decarboxylase (GAD67), in the VTA (Perreault et al., 2012a), potentially
enhancing GABAergic control in the VTA and preventing LTP of VTA dopaminergic neurons
known to be associated with the initiation of amphetamine-induced locomotor sensitization. In
addition, we have also shown that repeated stimulation of the D1-D2 heteromer significantly
reduced the expression of CaMKII in the NAc (Perreault et al., 2010), which may prevent the
enhanced dopamine release in the NAc upon amphetamine challenge and thereby abolish the
expression of amphetamine-induced sensitization. Another possibility is that the D1-D2
heteromer may reduce AMPA receptor activity in the NAc, as enhanced glutamatergic activity
was shown to be required for the expression of this behaviour (Kim et al., 2005). This idea is
supported by our previous work which showed repeated administration of SKF 83959
significantly reduced phosphorylation of AMPA receptor subunit GluA1 at the Ser831 site in
the NAc (Perreault et al., 2010), a site shown to be critical for inducing a long-lasting
potentiation of acute amphetamine-induced locomotion (Loweth et al., 2010).
145
Perhaps the most intriguing finding in the present study is the fact that subchronic D1-D2
heteromer inactivation by the TAT-D1 peptide alone was able to produce a sensitized
locomotor phenotype following amphetamine priming to a magnitude comparable to that which
was observed in amphetamine-sensitized animals, suggesting that the D1-D2 heteromer exerts
tonic suppression on the neurological processes associated with the expression of amphetamine-
induced locomotor sensitization. Although the mechanism through which the D1-D2 heteromer
exerts such effect remains unclear, a study suggests the potential involvement of cyclin-
dependent kinase 5 (cdk5). In the study by Taylor et al., animals that received subchronic intra-
NAc administration of cdk5 inhibitor roscovitine also exhibited a sensitized locomotor
phenotype upon cocaine priming to a degree similar to that produced by cocaine-sensitized
animals (Taylor et al., 2007). Furthermore, co-administration of roscovitine with cocaine during
the induction phase significantly enhanced the expression of cocaine locomotor sensitization
induced by cocaine priming (Taylor et al., 2007), which remarkably mirrors the effect of TAT-
D1 peptide co-administration on the expression of amphetamine locomotor sensitization in the
current study. Taken together, it is possible that cdk5 may be a downstream effector of the D1-
D2 heteromer signaling, and thus D1-D2 heteromer inactivation by the TAT-D1 peptide had
similar effect on the expression of psychostimulant-induced locomotor sensitization as the cdk5
inhibitor roscovitine. This is an interesting hypothesis that will be addressed in future studies.
We have previously demonstrated that the D1-D2 heteromer has representations in the
mesolimbic, striatonigral and striatopallidal pathways (Perreault et al., 2010, 2011) and that
medium spiny neurons that express the D1-D2 heteromer also express both GABA and
glutamate (Perreault et al., 2012a). In addition, the acute effects of D1-D2 heteromer activation
in the NAc on the expression of proteins involved in GABA and glutamate transmission were
146
shown to be pathway specific, with enhanced GABA-related protein expression in NAc/VTA
and increased glutamate-related protein expression in the dorsal striatum (Perreault et al.,
2012a). It is therefore possible that the D1-D2 heteromer may exert dual modulation on
amphetamine-induced locomotion by differentially regulating glutamate and GABA
transmission in several nuclei within the mesocorticolimbic and the basal ganglia circuitry
(Perreault et al., 2011, 2012a). This ability of the D1-D2 heteromer to potentially regulate both
GABA and glutamate neurotransmission would allow for the “fine tuning” of neuronal activity
within the mesolimbic direct and indirect dopamine pathways (Lobb et al., 2010). This line of
reasoning is supported by our previous study which showed increased functional activity of the
D1-D2 heteromer in the striatum of amphetamine-sensitized rats (Perreault et al., 2010), which
may be a potential negative-feedback compensatory process in an attempt to normalize
disturbances in dopaminergic transmission induced by subchronic amphetamine treatment.
A potential limitation of the present study is the use of SKF 83959 to examine the
behavioural effects of D1-D2 heteromer activation. No selective agonists for the D1-D2
heteromer are available, and while SKF 83959 has been shown to definitively induce the Gq-
PLC-linked calcium signaling mediated by the D1-D2 heteromer, its pharmacology has been
shown to extend to include a few other receptors including the dopamine D5 receptor (Sahu et
al., 2009; Perreault et al., 2012b; Chun et al., 2013; Guo et al., 2013). While the use of the
highly selective disrupting TAT-D1 peptide that exclusively targets the specific interaction sites
between the D1R and the D2R allows us to conclusively isolate D1-D2 heteromer-specific
effects on specific amphetamine-induced locomotor responses, off-target effects of SKF 83959
are also clearly evident. Indeed, we showed herein that acute locomotor activation induced by
147
SKF 83959 was not regulated by the D1-D2 heteromer as the TAT-D1 peptide did not abolish
these locomotor effects in the absence of habituation to the testing chamber.
It is also important to note that SKF 83959 attenuated, but did not abolish, amphetamine-
induced locomotion during the five day sensitizing phase, indicating that other receptors also
likely contribute to locomotor activation by this psychostimulant. In addition, we did not
directly demonstrate that TAT-D1 could abolish the effects of SKF 83959 on subchronic
amphetamine-induced locomotion as we were concerned that administering all three agents
simultaneously may overwhelm the in vivo system, as both SKF 83959 and amphetamine affect
various neurotransmitter systems via multiple mechanisms of action (Fleckenstein et al., 2007;
Perreault et al., 2012b; Chun et al., 2013; Guo et al., 2013). Nonetheless, the ability of SKF
83959 and the highly specific TAT-D1 peptide to opposingly regulate amphetamine-induced
locomotor activation and sensitization argues for a pivotal role for the D1-D2 heteromer in
mediating these behaviours.
In the present study, we demonstrated that the dopamine D1-D2 receptor heteromer exerts
tonic suppression on the locomotor sensitization behaviour. Despite its lack of clear face
validity, psychostimulant-induced locomotor sensitization results in neurobiological alterations
in the mesolimbic dopamine system that have been suggested to be the underlying molecular
basis for the subsequent manifestation of drug-seeking behaviours, which makes
psychostimulant-induced locomotor sensitization an important model for the study of drug
addiction (Robinson and Berridge, 2000; Steketee and Kalivas, 2011). Indeed, not only have
behaviorally sensitized animals been shown to concomitantly exhibit conditioned place
preference and drug self-administration (Lett, 1989; Pierre and Vezina, 1997, 1998; Vezina,
2004), but the neurocircuitry of psychostimulant-induced locomotor sensitization was also
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shown to largely overlap with that of the reinstatement model of drug self-administration
(Steketee and Kalivas, 2011), suggesting that the persistent neuroadaptations associated with
psychostimulant sensitization may also contribute to eventual drug relapse. Therefore, the
identification of the dopamine D1-D2 receptor heteromer as a negative modulator of locomotor
sensitization to amphetamine may have implications in the understanding and treatment of drug
addiction.
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5. Regulation of c-fos expression by the dopamine D1-D2 receptor heteromer
Abstract
The dopamine D1 and D2 receptors form the D1-D2 receptor heteromer in a subset of
neurons and couple to the Gq protein to regulated intracellular calcium signaling. In the present
study the effect of D1-D2 heteromer activation and disruption on neuronal activation in rat
brain was mapped. This was accomplished using the dopamine agonist SKF 83959 to activate
the D1-D2 heteromer in combination with a TAT-D1 disrupting peptide we developed, and
which has been shown to disrupt the D1/D2 receptor interaction and antagonize D1-D2
heteromer-induced cell signaling and behaviour. Acute SKF 83959 administration to rats
induced significant c-fos expression in nucleus accumbens that was significantly inhibited by
TAT-D1 pretreatment. No effects of SKF 83959 were seen in caudate putamen. D1-D2
heteromer disruption by TAT-D1 did not have any effects in any striatal subregions, but
induced significant c-fos immunoreactivity in a number of cortical regions including the
orbitofrontal cortex, prelimbic and infralimbic cortices and the piriform cortex. The induction
of c-fos by TAT-D1 was also evident in the anterior olfactory nucleus, as well as the lateral
habenula and thalamic nuclei. These findings show for the first time that the D1-D2 heteromer
can differentially regulate c-fos expression in a region-dependent manner either through its
activation or through tonic inhibition of neuronal activity.
Perreault M.L.*, Shen M.Y.F.*, Fan T., George S.R. Regulation of c-fos expression by the
dopamine D1-D2 receptor heteromer. Neuroscience. 2015 Jan 29;285:194-203. *These
authors contributed equally to this work
The work on the immunohistostaining in this study was done by Dr. Melissa Perreault.
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1. Introduction
The dopamine D1 and D2 receptors (D1R and D2R) can form a heteromeric receptor
complex, the D1-D2 receptor heteromer, that exhibits pharmacological and functional
properties distinct from its constituent receptors (Lee et al., 2004, Rashid et al., 2007, Hasbi et
al., 2009). The distribution of the dopamine D1-D2 receptor heteromer has only been partially
characterized thus far, with the major focus being directed to the striatal subregions. In rodents
and non-human primates an abundance of neuroanatomical evidence now suggests that the
D1R and D2R are coexpressed in a subset of striatal medium spiny neurons (Meador-Woodruff
et al., 1991, Surmeier et al., 1992, Lester et al., 1993, Surmeier et al., 1996, Aubert et al., 2000,
Lee et al., 2004, Deng et al., 2006, Bertran-Gonzalez et al., 2008, Hasbi et al., 2009, Matamales
et al., 2009, Perreault et al., 2010, Gangarossa et al., 2013), with low receptor coexpression
(~4-6% of neurons) in caudate putamen (CP) and higher coexpression levels in the nucleus
accumbens (NAc) (~17-30% of neurons) (Bertran-Gonzalez et al., 2008, Perreault et al., 2010,
Gangarossa et al., 2013). Approximately 90% of coexpressing neurons in NAc expressed the
D1-D2 heteromer with only about 25% in CP (Perreault et al., 2010). While D1-D2 heteromer
expression in other brain regions has for the most part not been determined, it is expressed in
globus pallidus (Perreault et al., 2011), in the medial prefrontal cortex (mPFC) (Pei et al.,
2010). The dopamine D1-D2 receptor heteromer has been linked to Gq-mediated
phospholipase C activation and intracellular calcium signaling (Lee et al., 2004, Rashid et al.,
2007, Hasbi et al., 2009), the activation of calcium calmodulin kinase II (Rashid et al., 2007,
Ng et al., 2010), the expression and release of brain-derived neurotrophic factor (BDNF) in
NAc (Hasbi et al., 2009, Perreault et al., 2012) and reduced activation of glycogen synthase
kinase-3β (GSK-3β) in the PFC (Perreault et al., 2013). Furthermore, activation of the D1-D2
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heteromer in NAc shell was shown to regulate the expression of protein markers of GABA and
glutamate in ventral tegmental area and substantia nigra (Perreault et al., 2012) suggesting that
activation of the D1-D2 heteromer may exert local effects as well as have farther reaching
effects through efferent projections.
A role for the dopamine D1-D2 receptor heteromer in the regulation of neuronal
activity has not been explored but can be examined through the expression of immediate early
genes, such as c-fos, which have often been used as a measure of neuronal activation within
circuits (Perez-Cadahia et al., 2011). The dopamine agonist SKF 83959, which is a partial
agonist for the D1-D2 heteromer (Rashid et al., 2007), has been shown to induce dorsal striatal
Fos expression at high doses (Wirtshafter and Osborn, 2005). SKF 83959 has often been used
to activate the D1-D2 heteromer with dopamine receptor knockout mice (D1R-/- and D5R-/-)
used to validate selectivity as SKF 83959 also activates the PLC-coupled D5R (Sahu et al.,
2009, Perreault et al., 2013). However recent reports indicate that this compound also exhibits
affinity at other receptors such as the serotonin 5HT-2c receptor (Chun et al., 2013), may act as
an allosteric modulator at the sigma-1 receptor (Guo et al., 2013), and there are conflicting
reports as to whether SKF 83959 functions as an antagonist (Downes and Waddington, 1993,
Cools et al., 2002, Jin et al., 2003), a partial agonist (Lee et al., 2014), or has no effect (Lee et
al., 2004, Rashid et al., 2007) at the D1R To assist in elucidating the physiological role of the
D1-D2 heteromer, we developed a selective D1-D2 heteromer antagonist, the TAT-D1 peptide,
which occludes the interaction site between the two receptors (O'Dowd et al., 2012), thus
inhibiting D1-D2 heteromer expression and function and abolishing the physiological effects of
D1-D2 heteromer activation by SKF 83959 (Hasbi et al., 2014). Therefore in the present study,
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using SKF 83959 together with TAT-D1, we sought to address the involvement of the D1-D2
heteromer in regulating neuronal activation as indexed by the induction of c-fos expression.
2. Materials and Methods
2.1 Animals
Sixty-eight adult male Sprague-Dawley rats (Charles River, Canada), weighing 300-350
g at the start of the experiment, were used. Rats were housed in polyethylene cages in a
temperature-controlled colony room, maintained on a 12-h light-dark cycle (lights on at 0700),
with ad libitum access to food and water. Rats were handled daily for 5 days before the start of
the experiment. All treatments were performed during the light phase of the day-night cycle.
Animals were housed and tested in accordance with the guidelines described in the Guide to the
Care and the Use of Experimental Animals (Canadian Council on Animal Care, 1993), and
were approved by the Animal Care Ethics Committee of the University of Toronto.
2.2 Drugs and Peptides
SKF 83959 hydrobromide (Tocris Bioscience) was dissolved in physiological saline
containing 5% DMSO, and was administered subcutaneously (0.4, 2.5 mg/kg, s.c.). Haloperidol
(0.5 mg/kg, Sigma Aldrich) was used as a positive control for c-fos immunochemistry in
striatum, dissolved in a 0.3% tartaric acid in water, and administered intraperitoneally (i.p.). For
non-drug injections, an equivalent volume of vehicle was administered and all injections were
given at a volume of 1.0 ml/kg. The TAT-D1 disrupting peptide, or TAT-scrambled peptide
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control (TAT-Sc) (Hasbi et al., 2014), was dissolved in saline and administered into the
intracerebroventricular space (300 pmol/4µL, i.c.v.) 15 minutes prior to vehicle or SKF 83959.
2.3 Surgery
Rats were anesthetized with isoflurane, administered analgesic ketoprofen (5 mg/kg)
and secured in a stereotaxic frame. A cannula was placed unilaterally into the
intracerebroventricular space close to the midline according to the following stereotaxic
anterior-posterior (AP), mediolateral (ML), and dorsoventral (DV) coordinates: AP -0.8mm,
ML + 1.3mm, DV – 3.7mm. AP and ML coordinates were taken from bregma, DV coordinate
from skull surface (Paxinos and Watson, 1998). The animals were allowed to recover in their
home cage for a minimum of five days before the experiments were performed.
2.4 Grooming
Grooming activity was monitored for 30 minutes immediately following SKF 83959
(0.4 mg/kg) injection. Animals were placed in clear cages containing no bedding
(20x20x45cm3). The measurement of grooming behavior followed a previously described
protocol (Culver et al., 2000). The animal’s grooming was scored for 30 second intervals, for a
total of 4 minutes (2 minutes sampled from the first 15 minutes of testing and 2 minutes
sampled from the last 15 minutes of testing). Ventilated polyethylene lids were used to cover
the cages to prevent animals from escaping.
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2.5 Immunochemistry
Ninety minutes following SKF 83959 (2.5 mg/kg) or vehicle injection, brains were
rapidly removed and frozen in isopentane (−60°C) and stored at −80°C until cryostat sectioning.
Serial sections through the prefrontal cortex (PFC, Bregma 3.2 mm), CP/NAc (Bregma
1.6 mm), ventral pallidum (Bregma 0.2), globus pallidus (Bregma -0.8 mm), lateral
hypothalamus (Bregma -1.8), habenula/hippocampus/thalamus/amygdala (Bregma -3.6),
substantia nigra/ventral tegmental area (Bregma −5.6 mm) and rostromedial tegmental nucleus
(Bregma -6.8 mm) were cut coronally at 16-μm thickness and mounted onto gelatin-coated
glass slides. Sections were air dried and stored at −35°C until use. Immunochemistry for c-fos
was performed as previously described (Sundquist and Nisenbaum, 2005) with the following
changes. Slides were brought to room temperature, immersed in 4% paraformaldehyde and
washed several times in TBS-Tween (0.05%). Slides were then placed in methanol containing
0.3% hydrogen peroxide, washed, and blocked by a 10 minute incubation (humid chamber) in a
solution of 10% goat serum in TBS-T. Tissue was incubated with anti-rabbit c-fos primary
antibody (1:250, Cell Signaling) in antibody diluent (1% BSA in TBS-T) for 2 hours in humid
chamber, washed, and then incubated with a biotinylated goat anti-rabbit secondary antibody
(Vector Laboratories) in antibody diluent for 45 mintues followed by several washes. Sections
were reacted with avidin-biotin peroxidase complex (Vectastain Elite Kit; Vector Laboratories)
for 30 minutes and washed. c-fos positive nuclei were visualized with VIP (Vector
Laboratories), washed and dehydrated, cleared in xylene and coverslipped with Vectamount
(Vector Laboratories). Images were obtained at 10X magnification using an Axioplan2
microscope (Carl Zeiss).
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2.6 Statistical Analysis
Values are reported as mean ± s.e.m. The grooming data was measured in seconds and
the immunochemistry data was collected by cell counting of c-fos positive nuclei (4
fields/animal). Comparisons of means for time spent grooming and cell counts in NAc and CP
were performed by ANOVA, with Treatment as the between subjects factor, followed by
Bonferroni post-hoc tests. Comparisons of means in all other brain regions were performed by
Student’s t test (two-tailed, unpaired). Statistical significance set at P<0.05 and computations
were performed using the SPSS/PC+ statistical package.
3. Results
3.1 Effects of dopamine D1-D2 receptor heteromer activation on striatal c-fos expression
A role for the dopamine D1-D2 heteromer in the induction of c-fos expression in NAc
core and shell (Figure IV-1A) was first evaluated. Administration of the D1-D2 receptor
heteromer agonist SKF 83959 or the D2 receptor antagonist haloperidol resulted in a
homogenous distribution of highly labelled c-fos positive nuclei in NAc core (Figure IV-1B)
and in NAc shell. In contrast, disruption of the dopamine D1-D2 heteromer by administration
of TAT-D1 resulted in little to no c-fos expression in either NAc subregion. Quantification of
the number of immunoreactive cells (Figure IV-1C&D) showed a significant increase in highly
labeled c-fos positive cells compared to TAT-Sc-treated controls in both the NAc core (22.1 ±
2.0 vs. 0.4 ± 0.2 cells/field, p<0.0001) and NAc shell (22.6 ± 2.2 vs. 0.2 ± 0.2 cells/field,
P<0.0001) in response to SKF 83959, and these effects of SKF 83959 on c-fos expression in
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Figure IV-1: Regulation of c-fos expression in rat nucleus accumbens (NAc) by the
dopamine D1-D2 receptor heteromer. A) Schematic showing regions of sampling for c-fos
positive nuclei. B) Representative images showing the effect of D1-D2 heteromer activation
and disruption on c-fos immunoreactivity in NAc core. Magnification of select immunopositive
nuclei (solid arrow) and nuclei considered negative (dashed arrow) are shown inset. C, D) SKF
83959 (2.5 mg/kg) administration resulted in a significant increase in the number of c-fos
positive cells in both NAc core and shell and to a magnitude similar to that observed with
haloperidol (HALO) which was used as positive control. The effects of SKF 83959 were
significantly inhibited by pretreatment with a TAT-D1 disrupting peptide. TAT-D1 alone had
no effect on c-fos levels. (Bars shown represent means ± s.e.m. ***p<0.001 compared to TAT-
Sc controls, ###
p<0.001 compared to SKF 83959-treated rats. Scale bar 100 µm).
A) B)
C) D)
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NAc were of similar magnitude to that seen with haloperidol treatment. Pre-administration of
TAT-D1 significantly reduced SKF 83959-induced c-fos expression (NAc Core: 22.1 ± 2.0 vs.
10.1 ± 0.9; NAc Shell: 22.6 ± 2.0 vs. 13.3 ± 0.9 cells/field) indicating that the effects of SKF
83959 were mediated by the D1-D2 heteromer. Overall TAT-Sc treatment had very little effect
on c-fos expression in either NAc subregion. ANOVA revealed a significant effect of
Treatment {F(4, 83)=82.9, p<0.0001}.
SKF 83959 has been demonstrated to induce oral movements and grooming behaviour
when administered systemically (Downes and Waddington, 1993, Deveney and Waddington,
1995, Perreault et al., 2010, Perreault et al., 2012) or directly into NAc shell (Perreault et al.,
2012). To determine whether these effects were mediated by the D1-D2 heteromer, we
assessed a role for the D1-D2 heteromer on SKF 83959-induced grooming behaviour (Figure
IV-2). We showed that the increased grooming response induced by SKF 83959 was abolished
by pretreatment with TAT-D1 (87.4 ± 8.4 vs. 48.3 ± 6.6 seconds, p=0.003) whereas TAT-D1
alone did not influence the amount of time animals spent grooming compared to controls (51.3
± 6.7 vs. 46.8 ± 7.0 seconds, p=1.00) {Treatment: F(3, 44)=7.2, p<0.0001}.
We next evaluated the effects of SKF 83959 and TAT-D1 on c-fos immunoreactivity in CP
(Figure IV-3A), a region with very little dopamine D1-D2 heteromer expression. Similar to that
observed in NAc, haloperidol-treated rats exhibited homogeneous expression of c-fos-positive
nuclei in CP, whereas SKF 83959 and TAT-D1, and TAT-Sc had little to no effect on c-fos in
this region (Figure IV-3B-D). Furthermore, in contrast to haloperidol which showed robust c-
fos labelling in many of the nuclei, c-fos positive cells in CP of SKF 83959 or TAT-D1/SKF
83959-treated rats were only lightly labelled (Figure IV-3C). TAT-D1 pretreatment had no
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Figure IV-2. Grooming induced by SKF 83959 in rats is mediated by the dopamine D1-
D2 heteromer. A single systemic injection of SKF 83959 (0.4 mg/kg) induced a significant
increase in the amount of time spent grooming compared to TAT-Sc-treated rats. TAT-D1
pretreatment abolished SKF 83959-induced grooming but did not influence grooming
behaviour when administered alone (n=12 rats/group). (Bars shown represent means ± s.e.m.
and are expressed in seconds (s). ***p<0.001 compared to TAT-Sc controls).
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Figure IV-3: Regulation of c-fos expression in rat caudate putamen (CP) by the dopamine
D1-D2 receptor heteromer. A) Schematic showing area of sampling for c-fos positive nuclei.
B) Representative images showing the effect of D1-D2 heteromer activation and disruption on
c-fos immunoreactivity in CP. Select immunopositive nuclei chosen for magnification are in
the boxed area. C) Magnification of select c-fos positive cells showing the intensity of antibody
labeling between Treatment groups. D) SKF 83959 (2.5 mg/kg) induced a very modest
increase in the number of c-fos positive cells CP that was not abolished by pretreatment with
TAT-D1. In contrast, haloperidol (HALO) induced a robust increase in the number of c-fos
positive cells. (Bars shown represent means ± s.e.m. ***p<0.001 compared to TAT-Sc
controls. Scale bar 100 µm).
A) B)
C) D)
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effect on the small amount of c-fos labelling induced by SKF 83959 {Treatment: F(4, 83)=82.9,
p<0.0001}.
3.2 Effects of dopamine D1-D2 heteromer disruption on c-fos expression in anterior olfactory
nucleus and cortical subregions
SKF 83959 induced c-fos expression in a number of regions of the brain that was not
attributed to the D1-D2 heteromer. This significantly confounded the findings in animals that
received both TAT-D1 and SKF 83959. We therefore chose to focus selectively on a role for
D1-D2 heteromer disruption on c-fos expression for the remainder of the experiments. The
effects of acute TAT-D1 administration on c-fos expression in were evaluated in anterior
olfactory nucleus (AOP) and a number of cortical subregions (Figure IV-4). Dopamine D1-D2
heteromer disruption resulted in increased c-fos immunoreactivity in AOP and most of the
cortical regions including orbitofrontal cortex (OFC), the infralimbic and prelimbic cortices (IL,
PL) and piriform cortex (PiC), indicating the release of a tonic inhibitory effect. In the AOP,
TAT-D1-induced c-fos expression was relatively dense, with medium to high labelling (Figure
IV-4A). Quantitative data showed an approximate 5-fold increase in the number of positive
nuclei compared to TAT-Sc-treated controls (20.6 ± 2.0 vs. 3.5 ± 1.2 cells/field; t(32)=7.9,
p<0.0001). The distribution of c-fos immunoreactivity in OFC by TAT-D1 (Figure IV-4B) was
similar to that of AOP with relatively strong and dense labelling compared to TAT-Sc controls
(20.7 ± 2.2 vs. 2.1 ± 0.6 cells/field; t(32)=9.4, p<0.0001).
Administration of TAT-D1 resulted in c-fos expression in both the IL and PL that was
significantly higher than controls (IL: 9.1 ± 1.0 vs. 2.1 ± 0.6 cells/field; t(32)=6.3, PL: 10.9 ±
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Figure IV-4. Effects of dopamine D1-D2 heteromer disruption on c-fos immunoreactivity
in rat cortex. Representative images and quantitative data showing significantly increased c-
fos staining in A) the anterior olfactory nucleus (AOP) and B) the orbitofrontal cortex (OFC)
following administration of the TAT-D1 disrupting peptide. C) TAT-D1 treatment resulted in
intense labeling of c-fos cells in the infralimbic cortex (IL) but fewer cells were labelled than
that observed in AOP and OFC. D) c-fos immunoreactivity was increased in the prelimbic
cortical region following TAT-D1 administration. E) No effect of TAT-D1 on c-fos expression
in cingulated cortex (Cg). F) TAT-D1 increased c-fos levels in piriform cortex (PiC). G)
Schematic showing regions of sampling for c-fos positive nuclei. Select magnified neurons
displaying c-fos positive (solid arrow) and negative (dashed arrow) are shown inset.
(***p<0.001 compared to TAT-Sc controls. Scale bar 100 µm).
A)
B)
C)
D) E) F) G)
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1.7 vs. 1.7 ± 0.5 cells/field; t(32)=6.0, p<0.0001; Figure IV-4C&D) but with a more sparse
distribution than that observed in AOP and OFC. Where c-fos immunoreactivity was present,
nuclei were highly labelled. Only modest effects of TAT-D1 peptide on c-fos expression were
seen in the cingulate cortex (Figure IV-4E). In the PiC, TAT-D1 significantly increased c-fos
expression with mild to medium intensity labelling (14.9 ± 2.1 vs. 2.2 ± 0.7 cells/field;
t(32)=6.5, p<0.0001; Figure IV-4F).
3.3 Effects of dopamine D1-D2 heteromer disruption on c-fos immunoreactivity in lateral
habenula and other regions
Disruption of the dopamine D1-D2 heteromer by TAT-D1 induced c-fos expression in
the lateral habenula (LHb), but not medial habenula (MHb), as well as the thalamic nuclei
(Figure IV-5). Medium intensity labelling occurred predominantly in the ventral LHb with
sparse labelling in the dorsal area (Figure IV-5B). Quantification of the number of
immunoreactive nuclei revealed a significantly higher number of c-fos positive cells in LHb
following TAT-D1 treatment (13.4 ± 1.8 vs. 1.7 ± 0.5 cells/field; t(26)=7.1, p<0.0001). TAT-
D1 induced a homogenous distribution of c-fos expression, of medium to high intensity, in the
mediodorsal thalamic nucleus with modest staining observed in the paraventricular thalamic
nucleus (PVP) (Figure IV-5C). The number of c-fos immunoreactive cells were significantly
higher in the thalamic regions following treatment with TAT-D1 as compared to TAT-Sc
treatment (17.8 ± 1.7 vs. 3.0 ± 0.7 cells/field; t(26)=8.8, p<0.0001).
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Figure IV-5: Effect of dopamine D1-D2 heteromer disruption on c-fos expression in rat
lateral habenula and thalamus. A) Schematic showing area of sampling for c-fos positive
nuclei. B) Representative images and quantitative data showing significantly increased c-fos
staining in the lateral habenula (LHb) but not the medial habenula (MHb) following D1-D2
heteromer disruption by TAT-D1. C) TAT-D1 significantly increased c-fos expression in the
thalamic nuclei including the mediodorsal thalamic nucleus and paraventricular nucleus. Select
magnified neurons displaying c-fos positive (solid arrow) and negative (dashed arrow) are
shown inset. ***P<0.001 compared to TAT-Sc controls. Scale bar 100 µm.
A)
B)
C)
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No significant effects of TAT-D1 on c-fos expression were observed in any other brain
region including the ventral pallidum, globus pallidus, lateral hypothalamus, hippocampus,
amygdala, substantia nigra, ventral tegmental area and rostromedial tegmental nucleus.
4. Discussion
In the present study we showed that the activation state of the dopamine D1-D2
receptor heteromer contributed to neuronal activation in a region-specific manner as evidenced
by increased c-fos expression. Specifically, whereas activation of the D1-D2 heteromer
induced c-fos in the NAc core and shell, its disruption resulted in significantly elevated c-fos
immunoreactivity in a number of cortical regions, as well as the LHb and thalamus. These
findings suggest that the D1-D2 heteromer plays a dual role in the regulation of neuronal
activity, increasing activity in some regions and exerting tonic suppression on activity in others.
Recent studies have indicated that SKF 83959 has affinity for, or activates, a number of
different receptors (Sahu et al., 2009, Chun et al., 2013, Perreault et al., 2013) highlighting the
critical importance of using TAT-D1 in this study. It was especially obvious in the cortical
regions whereby SKF 83959 significantly induced c-fos expression, an effect not attenuated by
pretreatment with TAT-D1 (data not shown), and likely mediated by dopamine D5R (Perreault
et al., 2013), or other receptors. This was additionally confounded by our findings which
showed that D1-D2 heteromer disruption by TAT-D1 induced c-fos expression in several
subregions of the cortex. We therefore chose to examine the effects of SKF 83959 solely in
striatal regions where administration of TAT-D1 individually had no effect. In CP very
minimal effects of the 2.5 mg/kg dose of SKF 83959 were observed, a finding consistent with
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previous reports showing a lack of effect on c-fos expression of another dopamine agonist
linked to PLC activation, SKF 38393 (potentially induced through activity at the D1-D2
heteromer), in this region in normal rats (Robertson et al., 1991, Paul et al., 1992, LaHoste et
al., 1993). In NAc core and shell a marked increase in c-fos levels were observed following
SKF 83959 administration and this effect was significantly inhibited, but not abolished by
pretreatment with TAT-D1. These findings in NAc indicate that the observed effects on c-fos
expression were mediated in large part by the D1-D2 heteromer. The effects of D1-D2
heteromer activation in CP and NAc on c-fos expression are also consistent with the known
distribution of the receptor complex in striatum. In CP, coexpression of the D1R and D2R is
very low (~4-6% of cell bodies) (Bertran-Gonzalez et al., 2008, Perreault et al., 2010) with
only about 25% of these coexpressing neurons showing D1-D2 heteromer formation (Perreault
et al., 2010), and thus only ~1-2% of CP neurons in total expressing the D1-D2 heteromer. In
contrast, in NAc expression of the D1-D2 heteromer is much higher with ~17-30% of neurons
coexpressing the D1R and D2R (Bertran-Gonzalez et al., 2008, Perreault et al., 2010,
Gangarossa et al., 2013) and the majority of these (>90%) also expressing the D1-D2
heteromer (Perreault et al., 2010). These findings are also consistent with our previous data
showing that SKF 83959 could induce grooming behaviour upon injection into NAc shell
(Perreault et al., 2012). In the present study we were able to conclusively identify the D1-D2
heteromer as mediating SKF 83959-induced grooming but could not establish a role for the
receptor complex in basal grooming behaviour as TAT-D1 alone had no effect.
In NAc, the TAT-D1 peptide greatly reduced, but could not completely abolish SKF
83959-induced c-fos expression. There are two possible explanations. Firstly, it may be that the
dose of SKF 83959 was too high to be completely inhibited by TAT-D1. While this is certainly
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a possibility, our previous behavioural study showed a complete loss of SKF 83959-induced
effects in the forced swim test with TAT-D1 pretreatment when using the same doses (Hasbi et
al., 2014). Another possibility is that some of the effects of SKF 83959 on c-fos expression in
NAc were mediated by a receptor other than the D1-D2 heteromer. SKF 83959 has been
shown to have affinity for the serotonin 5HT-2c receptor, as well as the α-adrenergic-2b
receptor (Chun et al., 2013). Although activation of signaling was not shown at these receptors
by SKF 83959, it cannot be ruled out as a possible contributor to the observed effects. The
dopamine D5R, which has lower expression in striatum being confined to cholinergic
interneurons, is also a potential candidate as SKF 83959 has been reported to activate D5R-
mediated PLC signaling (Sahu et al., 2009) and to induce D5R-mediated BDNF expression in
mPFC (Perreault et al., 2013). Nonetheless, our evidence clearly demonstrates a significant role
for the D1-D2 heteromer in mediating the effects of SKF 83959 on c-fos expression in NAc.
Disruption of D1-D2 heteromer activity by TAT-D1 resulted in the induction of c-fos in
numerous cortical regions including the PL and IL regions of the mPFC, OFC and PiC.
Significant coexpression of the D1R and D2R in mPFC has been reported (Zhang et al., 2010)
whereby double transgenic Drd1a-tdTomato/Drd2-EGFP mice were used to show that almost all
of the pyramidal neurons that expressed the D1R also expressed the D2R. That same year using
coimmunoprecipitation, the existence of the D1-D2 heteromer in the mPFC was confirmed (Pei
et al., 2010). The dopamine D1-D2 heteromer in PFC has now been shown to play a
significant role in depressive-like behaviour (Pei et al., 2010) and to suppress GSK-3β activity
by a mechanism likely involving activation of Akt (protein kinase B) (Perreault et al., 2013).
Increased activation of cortical GSK-3β has been linked to cognitive decline in schizophrenia
(Kozlovsky et al., 2005) and to contribute to the neurodegenerative process in Alzheimer’s
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disease (Llorens-Maritin et al., 2014). Although expression of the D1-D2 heteromer has not yet
been established in OFC and PiC, the present findings indicate that the involvement of this
complex in these regions may worthy of further investigation, especially given their importance
in reward and decision making processes (Noonan et al., 2012) as well as social interactions
(Zenko et al., 2011). Indeed, one study reported social interaction impairment induced solely
by coactivation, not individual receptor activation, of D1R and D2R in the PiC (Zenko et al.,
2011).
Significant increases in c-fos were observed in the LHb, as well as the PVP and
mediodorsal thalamic nuclei, following acute TAT-D1 treatment. The LHb and thalamus has
received much attention in recent years due to their emerging role in drug addiction (Matzeu et
al., 2014, Velasquez et al., 2014). The acute administration of cocaine was shown to result in
increased c-fos expression in LHb neurons (Zahm et al., 2010) and notably, evidence now
suggests a relationship between c-fos levels in LHb and cocaine reinstatement. For example, in
one study a significant correlation between activation of LHb neurons and cocaine-primed
reinstatement in a conditioned place preference paradigm in mice has been identified (Brown et
al., 2010). Similarly, the propensity for cue-induced relapse in rats trained to self-administer
cocaine was shown to be related to increased c-fos expression in LHb neurons, as well as
specific nuclei of the thalamus including the PVP and mediodorsal nuclei (James et al., 2011).
Conversely, inactivation of the PVP abolished the expression of cocaine CPP in rats (Browning
et al., 2014). Together these findings suggest a positive contribution of LHb and thalamic
nuclei to cocaine-induced behaviours and thus highlight a potential contribution of the D1-D2
heteromer in the regulation of processes involved in addiction.
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In summary, the current study demonstrates that the dopamine D1-D2 receptor
heteromer regulates neuronal activity in different regions of the brain, and does so in a highly
selective manner. Whereas activation of the D1-D2 heteromer increases c-fos
immunoreactivity in some regions such as NAc, its disruption promotes c-fos expression in
others such as in PFC, LHb and thalamus. These results indicate an important functional
duality of the D1-D2 heteromer in the regulation of neuronal output through direct activation or
tonic inhibition. Although the mechanisms by which this is achieved have not been elucidated,
these findings are indicative of the inherent inhibitory and excitatory characteristics of medium
spiny neurons that express the D1-D2 heteromer in striatum (Perreault et al., 2012). Future
studies examining more closely the neuroanatomical distribution of D1-D2 heteromer-
expressing neurons and their projections could provide important insights into how neuronal
activity is being regulated by this receptor complex.
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6. General Discussion
The overall purpose of the present thesis was to characterize a functional role for the
D1-D2 heteromer in the brain reward system and was accomplished using a series of model
systems in rodents for addiction, depression, and anxiety. The present findings showed that D1-
D2 heteromer stimulation was able to suppress reward-seeking behaviour under conditions of
elevated dopamine release (Chapter I&III), whereas it promoted anhedonic, depression- and
anxiety-like behaviour under basal dopamine tone (Chapter II). In addition, it was
demonstrated that the D1-D2 heteromer modulated the neuronal activity of several regions
within the mesocorticolimbic system (Chapter IV), a circuitry significantly involved in the
reward pathways, and thus supporting a role for the D1-D2 heteromer in the regulation of
reward. Collectively, these findings demonstrate a novel role for the D1-D2 heteromer in the
negative regulation of brain reward function, and suggest that the dysregulation of D1-D2
heteromer activity may contribute to the pathophysiology of neuropsychiatric disorders such as
drug addiction and depression.
Given the highly comorbid nature of drug addiction and depression, it is not surprising
that studies have identified that the two neuropsychiatric disorders share a similar
neurobiological pathology, namely, a dysfunctional mesolimbic reward system (Nestler and
Carlezon, 2006b; Russo and Nestler, 2013). Drug addiction and depression may be viewed as
the two extreme ends of the hedonic spectrum, with drug addiction being the pathological
consequence of inducing reward system hyperactivity, and depression being characterized by
hypoactive brain reward function. This idea is supported by studies that examined the
functional activity of the brain reward system using intracranial self-stimulation (ICSS), which
demonstrated that drugs of abuse sensitized, whereas exposure to depression-inducing chronic
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stressors dampened, brain reward function (West and Michael, 1986; Moody and Frank, 1990;
Vogel et al., 1990). In addition, while the mesolimbic dopaminergic activity is consistently
enhanced by drugs of abuse (Bradberry and Roth, 1989; Pellegrino and Druse, 1992; Nisell et
al., 1994; Stewart and Rajabi, 1996), recent evidence also showed that its inhibition promoted
depression-like symptoms in rats that underwent chronic stress (Tye et al., 2013), reinforcing
the notion that bidirectional modulation of the brain reward system may contribute to the
pathogenesis of drug addiction and depression. In this sense, as D1-D2 heteromer stimulation
and inactivation respectively induced a depression-like behavioural phenotype and promoted
reward-seeking behaviours, the D1-D2 heteromer may be a single molecular entity that
functions to regulate brain reward function, and thus may be a suitable therapeutic target for
drug addiction and depression though its activation or inactivation depending on the
neuropsychiatric disorder being evaluated.
6.1 The D1-D2 heteromer is a negative modulator of psychostimulant and natural reward
In Chapter I, it was shown that D1-D2 heteromer stimulation by the agonist SKF 83959
abolished cocaine reward in the CPP model, prevented cocaine-induced locomotor sensitization,
and attenuated cocaine intake in the SA model. More importantly, in all three behavioural
models examined, selective inactivation of D1-D2 heteromer consistently produced an opposite
behavioural effect compared to its stimulation, thereby providing strong support for a role for
the D1-D2 heteromer in the negative modulation of cocaine reward. Moreover, the inhibitory
effect of the D1-D2 heteromer was not limited to cocaine-mediated behaviours as D1-D2
heteromer stimulation similarly prevented the locomotor sensitization to amphetamine (Chapter
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III, Shen et al. 2014b) and attenuated the consumption of sucrose solution in the sucrose
preference test (Hasbi et al. unpublished data), whilst selective D1-D2 heteromer inactivation
enhanced amphetamine sensitization and sucrose intake. These results indicate that the D1-D2
heteromer negatively modulated psychostimulant and natural reward, and prevented the
behavioural sensitization that are associated with repeated exposure to psychostimulants.
Despite the traditional view that the D1R- and D2R-expressing MSNs are largely
segregated in the NAc (Gerfen et al., 1990; Harrison et al., 1990; Le Moine and Bloch, 1995;
Surmeier et al., 2007), evidence has also emerged that supported the existence of D1R/D2R co-
expressing MSNs (Bertran-Gonzalez et al., 2008, 2010; Matamales et al., 2009; Perreault et al.,
2010; Gangarossa et al., 2013a), within which the D1-D2 heteromer is expressed (Perreault et
al., 2010, 2011; Hasbi et al., 2014). This specific population of D1R/D2R co-expressing MSNs
in the NAc was shown to extend efferent projections to nuclei within both the direct D1R-
expressing and the indirect D2R-expressing pathways (Deng et al., 2006; Wang et al., 2006,
2007; Perreault et al., 2011), and has thus been suggested to function as a third neuronal
pathway within the basal ganglia circuitry that maintains homeostatic balance between the
neuronal output from the direct and indirect pathways (Perreault et al., 2011). This notion is
supported by the finding showing that the D1R/D2R co-expressing MSNs also possess a
unique GABA/glutamate co-expressing phenotype (Perreault et al., 2012a), which may allow
the D1-D2 heteromer to differentially regulate the inhibitory GABAergic and excitatory
glutamatergic activities in the direct and indirect pathways.
Given the established critical involvement of D1R- and D2R-expressing MSNs in the
modulation of psychostimulant reward (Nestler and Carlezon, 2006b; Thomas et al., 2008;
Steketee and Kalivas, 2011; Baik, 2013), it would be expected that stimulation of the D1R/D2R
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co-expressing MSNs could similarly have a modulatory influence on the rewarding effects of
psychostimulants. Coincidentally, stimulation of the D1-D2 heteromer, which results in a Gαq-
mediated and PLCβ-dependent calcium signalling (Lee et al., 2004; Rashid et al., 2007; Hasbi
et al., 2009), may be a means to activate the D1R/D2R co-expressing MSNs in the NAc since it
has been shown that PLCβ-mediated PIP2 hydrolysis in the striatum resulted in the inhibition of
KCNQ potassium channels, leading to enhanced MSN activity in the striatum (Zhang et al.,
2003; Shen et al., 2005). Supporting this notion, the DREADD technology uses engineered
muscarinic M3 receptors (hM3Dq) that signals through Gαq-mediated, PLCβ-dependent
calcium signalling to selectively stimulate neurons on which it is virally expressed (Alexander
et al., 2009). Therefore, the ability for D1-D2 heteromer stimulation to inhibit psychostimulant-
induced reward-seeking behaviours indirectly implicated a novel physiological role for the
D1R/D2R co-expressing MSNs in the negative modulation of psychostimulant reward.
Nevertheless, although D1-D2 heteromer stimulation was shown to increase c-fos induction in
the NAc, indicating neuronal activation in this region (Chapter IV, Perreault et al. 2015),
electrophysiology studies would be beneficial in determining the relationship between D1-D2
heteromer stimulation and the neuronal activity of the specific D1R/D2R co-expressing MSNs
in the NAc.
In addition, it is possible that the negative modulatory effect of the D1-D2 heteromer on
psychostimulant reward may also extend to influence natural reward, since D1-D2 heteromer
stimulation and inactivation respectively attenuated and enhanced the consumption of sucrose
solution, and repeated D1-D2 heteromer stimulation prevented animals from gaining weight
(Hasbi et al. unpublished finding). Indeed, it has been proposed that the neurobiology
underlying psychostimulant and natural reward may overlap to some degree (Cooper et al.,
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1992; Hodge et al., 1994; Hajnal and Norgren, 2001; Gambarana et al., 2003; Halpern et al.,
2013; Cameron et al., 2014), and thus repeated exposure to natural reward stimuli may
similarly result in neuroadaptations in the mesolimbic dopamine system as those induced by
repeated psychostimulant exposure to contribute to the pathophysiology of eating disorders and
obesity (Volkow et al., 2008; Grosshans et al., 2011). Therefore, the D1-D2 heteromer may
function to inhibit the fundamental brain reward mechanism, allowing its stimulation to
simultaneously dampen both psychostimulant and natural reward. Interestingly, this notion
also raises the possibility that D1-D2 heteromer stimulation under basal dopamine level, that is,
without an external reward stimulus, may result in excessive suppression of brain reward
function, potentially leading to an aversive and depressive state. Indeed, the present findings
also showed the development of depression- and anxiety-like behaviour following D1-D2
heteromer activation under normal physiological conditions.
6.2 D1-D2 heteromer stimulation induced a depressive-like state
In Chapter II, it was shown that D1-D2 heteromer stimulation by SKF 83959 promoted
depression- and anxiety-like behavioural phenotypes in the forced swim test, novelty-induced
hypophagia test, and in the elevated plus maze, effects suppressed or abolished by the TAT-D1
peptide. In addition, the TAT-D1 peptide by itself in many instances produced anxiolytic and
antidepressant-like activity in these behavioural paradigms. More importantly, acute D1-D2
inactivation by the TAT-D1 peptide also elicited rapid antidepressant-like reactivity in animals
that underwent chronic unpredictable stress, which models chronic depressive states (Willner et
al., 1987b), as measured in the sucrose preference test, fruit loop test, and the novelty-
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suppressed feeding test. These findings together strongly suggest that activation of the D1-D2
heteromer, under normal physiological conditions, can significantly suppress brain reward
function, leading to a depressive and anxiety-like state.
Although the majority of studies in the depression field have largely focused on
noradrenergic and serotonergic signalling in the hippocampus and the frontal cortex
(Dranovsky and Hen, 2006; Duman and Monteggia, 2006; Müller and Holsboer, 2006; Albert
et al., 2014; Mahar et al., 2014), emerging evidence has also implicated a role for the
mesolimbic dopamine system in the pathophysiology of depression (Nestler and Carlezon,
2006b; Krishnan and Nestler, 2008; Russo and Nestler, 2013). Indeed, although the
hippocampus and the frontal cortex are undoubtedly involved in the cognitive and memory
aspects of depression, it does not encompass the core symptom of depression: the reward
deficits, which likely involves the mesolimbic dopamine system due to its well-established role
in regulating brain reward function (Nestler and Carlezon, 2006b). In addition, the fact that
drug addiction and depression are highly comorbid further suggests that the disturbances in the
mesolimbic dopamine system may be a common mechanism underlying the pathophysiology
of neuropsychiatric disorders characterized by reward dysfunction (Conway et al., 2006).
Specfically, findings showing that withdrawal from psychostimulants induced a
depressive-like behavioural phenotype in both humans and rodents (Barr and Markou, 2005b;
D’Souza and Markou, 2010) effectively placed the mesolimbic dopamine system between the
two reward-related neuropsychiatric disorders. Numerous studies have suggested that
withdrawal from psychostimulants results in neuroadaptations in the brain reward circuit that
would subsequently contribute to the manifestation of depressive behaviours (Markou and
Koob, 1992; Wise and Munn, 1995; Coffey et al., 2000; Barr and Phillips, 2002; McGregor et
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al., 2005). Indeed, common alterations in the mesolimbic dopamine system were observed in
animals with a depressive-like behavioural phenotype and those that underwent
psychostimulant withdrawal, such as reduced basal extracellular dopamine concentration in the
NAc (Paulson et al., 1991; Rossetti et al., 1992, 1993; Gambarana et al., 1999), and decreased
firing of VTA dopaminergic neurons (Henry et al., 1989; Ackerman and White, 1992; Tye et
al., 2013).
The theoretical perspective of the emergence of depressive behaviours following
psychostimulant withdrawal can be explained by the “opponent process theory” of drug
addiction. Originally proposed by Solomon and Corbit, the opponent process theory postulates
that, whenever there is a disturbance in the neutrality of the brain reward system, an “opponent
process” ensues in an attempt to maintain homeostatic balance in the brain reward system
(Solomon and Corbit, 1974). According to the theory, the “opponent process” is slow to onset,
gradually reaches an asymptote, and slow to decay. In the case of psychostimulant exposure,
the excessive stimulation of the brain reward system by psychostimulants is counteracted by
neuroadaptations that inhibit the brain reward system in order to restore homeostatic balance.
Since the opponent process is slow to decay, the compensatory inhibition of the brain reward
system persists during psychostimulant withdrawal, thus dampening basal reward function to
result in an anhedonic and depressive state, which is subsequently proposed as a negative
reinforcement that drives addicts to seek and obtain psychostimulants in order to avoid the
aversive effect (Koob and Le Moal, 2001).
In this sense, the D1-D2 heteromer functions very much like an opponent process in
that its stimulation attenuated reward-seeking behaviours induced by external stimuli such as
psychostimulants and sucrose, and induced an anhedonic and depressive state in the absence of
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an external rewarding stimulus due to excessive inhibition of brain reward function. In support
of this idea, enhanced functional activity of the D1-D2 heteromer was found in the NAc of
amphetamine-sensitized rats (Perreault et al., 2010), which may potentially be a compensatory
opponent process activated in an attempt to normalize disturbances in dopaminergic
transmission induced by subchronic amphetamine treatment. Thus, the heightened D1-D2
heteromer function following repeated amphetamine administration may be one of the
underlying mechanisms for the depressive-like behavioural phenotypes that have been
observed during amphetamine withdrawal (Markou et al., 2005; D’Souza and Markou, 2010).
It is an intriguing idea that the expression and functional activation of the D1-D2 heteromer
may be transient, which functions to suppress or enhance the reward pathways as required to
maintain balance and avoid the pathogenic changes that contribute to addiction and depression,
an idea that fits with the previously suggested theory of a homeostatic role for D1R/D2R
coexpressing MSNs in the regulation striatonigral and striatopallidal output (Perreault et al.,
2011).
6.3 The D1-D2 heteromer modulation of reward: A potential role for BDNF and GAD67
Amongst the various proteins that are regulated by the D1-D2 heteromer signalling
pathway, BDNF and GAD67 were shown to be the common molecular substrates underlying
both drug addiction and depression (Nestler and Carlezon, 2006b; Russo and Nestler, 2013;
Sanchez-Catalan et al., 2014). Whereas systemic D1-D2 heteromer stimulation increased the
expression of BDNF in the NAc (Hasbi et al., 2009; Perreault et al., 2013), D1-D2 heteromer
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stimulation selectively in the NAc shell enhanced the expression of the major GABA-
producing enzyme, GAD67, in the VTA (Perreault et al., 2012a).
BDNF is well known for its role in mediating the rewarding effect of cocaine (Ghitza et
al., 2010; McGinty et al., 2010). In particular, infusion of BDNF into the NAc was shown to
enhance cocaine-seeking whereas the adenovirus-mediated knockdown of endogenous BDNF
in the NAc had the opposite effect (Graham et al., 2007b). Although these findings would
appear to be in contradiction with the previous findings which showed that D1-D2 heteromer
stimulation increased BDNF levels in the NAc (Hasbi et al., 2009; Perreault et al., 2012a), the
effects of BDNF on cocaine reward was shown to be dependent upon the subset of NAc MSNs
in which it signals (Lobo et al., 2010a). We therefore posit that D1-D2 heteromer stimulation
increased BDNF levels selectively in the D1R/D2R co-expressing MSNs in the NAc that
contributed to reduced cocaine reward. As BDNF signaling through TrkB in D1R-expressing
MSNs suppresses reward (Lobo et al., 2010a), its release from D1R/D2R-expressing MSNs
would act predominantly on the D1R MSN subtype to achieve this. We further suggest that
increased BDNF signaling in D1R-expressing MSNs may subsequently lead to increased Cdk5
activity (Bogush et al., 2007), a kinase that was shown to reduce the motivation to obtain
cocaine when expressed in the NAc (Taylor et al., 2007).
In addition to its effect on cocaine-seeking, BDNF has been shown to have region-
specific effects on depression-like behaviours. Whereas BDNF was shown to exert
antidepressant-like activity when expressed in the hippocampus (Schmidt and Duman, 2010;
Kavalali and Monteggia, 2012), BDNF expression in the NAc was found to be strongly pro-
depressive in various animal models for depression (Eisch et al., 2003; Krishnan et al., 2007;
Fanous et al., 2011; Wang et al., 2014). Moreover, depressed patients also displayed
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significantly increased levels of BDNF in postmortem NAc that was not due to antidepressant
administration (Krishnan et al., 2007). Therefore, the pro-depressive behavioural effects of D1-
D2 heteromer stimulation as observed in the current study could be attributed to the ability of
the D1-D2 heteromer to provide an endogenous source of BDNF in the NAc (Hasbi et al.,
2009). Although a role for Cdk5 activity in NAc in depression has not yet been elucidated, it is
possible that the suppression of cocaine reward, and the induction of a depression-like
phenotype, may occur through a similar mechanism.
Another potential mechanism by which the D1-D2 heteromer regulates reward is
through the regulation of GAD67 expression in VTA which could result in the suppression of
dopaminergic activity within the mesolimbic reward system by increasing the inhibitory
GABAergic modulation of VTA dopamine neurons. Several studies have shown that the
inhibition of VTA dopaminergic activity attenuated reward-seeking behaviours (Friedman et al.,
2011; Van Zessen et al., 2012; Ilango et al., 2014). For instance, increasing the excitability of
GABAergic synapses on to VTA dopaminergic neurons was shown to suppress cocaine-
seeking behaviours (Friedman et al., 2010). Similarly, optogenetic stimulation of VTA
GABAergic neurons was shown to reduce dopamine release in the NAc and subsequently
disrupted the consumption of sucrose solution (Van Zessen et al., 2012).
More recently, studies have also implicated a role for VTA GABAergic neurons in the
induction of depression-like behaviours (Lecca et al., 2014; Proulx et al., 2014). The activity of
GABAergic neurons in the VTA that synapse on NAc-projecting dopaminergic neurons was
shown to be increased in animals that exhibited behavioural models for depression (Li et al.,
2011, 2013), as well as in animals that were congenitally depressed (Yang et al., 2008),
suggesting that reducing dopaminergic activity in the mesolimbic reward system may
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contribute to the pathogenesis of depression. Supporting this, a recent study by Tye et al (2013)
showed that the chronic unpredictable stress model of depression resulted in a reduction of
VTA-NAc dopaminergic neuron firing, and optogenetic phasic stimulation of these neurons
induced an antidepressant-like effect in the tail suspension test and sucrose preference test.
Consequently, the induction of GAD67 expression in the VTA following D1-D2 heteromer
simulation may result in an increase in GABAergic activity in the same region as GAD67 is the
predominant GABA-producing enzyme, thereby contributing to the induction of depression-
like behaviours as observed in the current study. Similarly, the rapid antidepressant-like
activity of acute D1-D2 heteromer inactivation in the chronic unpredictable stress model of
depression shown herein is in line with the findings from the Tye et al. (2013) study in that
increasing VTA dopaminergic activity, potentially as a result of reduced D1-D2 heteromer-
mediated GABAergic influence in the same region, exerted antidepressant-like behavioural
effect. Nevertheless, the exact effect of D1-D2 heteromer-mediated GAD67 induction on the
activity of specific dopaminergic neurons in the VTA (NAc- or PFC-projecting) remains to be
investigated.
Collectively, the ability of the D1-D2 heteromer to modulate the expression of proteins
that are critically involved in both reward-seeking and depression-like behaviours reinforces
the notion that this receptor complex may be a link between drug addiction and major
depression, two highly comorbid neuropsychiatric disorders (Davis et al., 2008).
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6.4 The D1-D2 heteromer is a novel molecular substrate for aversions
Perhaps the most intriguing finding in the current study was that agonist stimulation of
the D1-D2 heteromer was aversive, whereas its selective inactivation was rewarding (Chapter
I). This suggests that the D1-D2 heteromer is constantly stimulated by the basal dopamine
outflow in the NAc to exert tonic inhibition on brain reward function, possibly to maintain a
neutral hedonic perception in the absence of an external rewarding stimulus. The identification
of a dopamine receptor complex in the NAc that induces aversion is unprecedented.
Nevertheless, the concept of the mesolimbic dopamine system to modulate both reward and
aversion was recently introduced in light of the advancements in molecular technology that
allowed for cell type-specific analysis of the brain reward circuitry.
The VTA dopaminergic neurons that project to the NAc exhibit two distinct patterns of
firings: phasic/burst firing that is responsible for stimulating the low affinity D1R, and tonic
firing that is crucial for modulating the high affinity D2R (Mirenowicz and Schultz, 1994, 1996;
Grace et al., 2007). Phasic/burst firing of the VTA-NAc dopaminergic neurons via optogenetics
produced CPP, whereas tonic firing of these neurons had no effect (Tsai et al., 2009),
indicating that D1R stimulation in the NAc is critical in generating reward. On the other hand,
aversive stimuli were shown to reduce the tonic firing of VTA-NAc dopaminergic neurons
(Mirenowicz and Schultz, 1996; Ungless et al., 2004; Brischoux et al., 2009), resulting in
blunted tonic dopamine release in the NAc that eventually led to CPA (Liu et al., 2008). Since
D2R in the NAc is more sensitive to changes in the tonic firing of VTA-NAc dopaminergic
neurons (Grace et al., 2007), such a finding indirectly implicated a role for the D2R in the NAc
in mediating aversions. In addition, although individual optogenetic stimulation of the D1R-
and D2R-expressing MSNs in the NAc was shown to be ineffective in eliciting CPP or CPA
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(Lobo et al., 2010b), it was found that selective blockade of the GABAergic neurotransmission
mediated by the D1R- and D2R-expressing MSNs respectively prevented reward and aversive
conditioning (Hikida et al., 2010). Together these findings indicate opposing roles for the D1R
and D2R neuronal systems in the NAc in modulating hedonic perception, with the D1R
facilitating reward and the D2R promoting aversion. Herein we additionally report a novel
mechanism by which dopaminergic transmission in the mesolimbic system could mediate
aversion: via the stimulation of the D1-D2 heteromer.
As aversive stimuli were found to reduce the tonic firing of VTA-NAc dopaminergic
neurons (Mirenowicz and Schultz, 1996; Ungless et al., 2004; Brischoux et al., 2009), it raised
the possibility that the attenuation of the neuronal activity of VTA-NAc dopaminergic neurons
may be an underlying mechanism of inducing aversions. Indeed, it was found that the
rostromedial tegmental nucleus (RMTg), which sends GABAergic projections to VTA
dopaminergic neurons, showed increased phasic activation and c-fos expression following the
presentation of aversive stimuli (Jhou et al., 2009a). Furthermore, optogenetic stimulation of
GABAergic neurons in the VTA resulted in CPA (Tan et al., 2012), indicating the GABA-
mediated inhibition of VTA dopaminergic neurons is responsible for aversion. Therefore, the
CPA induced by D1-D2 heteromer stimulation may be a consequence of increased GABAergic
activity in the VTA (Perreault et al., 2012a), which would subsequently reduce the firing of
VTA-NAc dopaminergic neurons, thereby resulting in an aversion.
The major GABAergic innervations onto VTA-NAc dopaminergic neurons originate
from the RMTg and local interneurons (Nair-Roberts et al., 2008; Jhou et al., 2009a). Notably,
the activity of these VTA-projecting GABAergic neurons is in turn modulated by excitatory
glutamatergic inputs from the lateral habenula (Lhb) (Jhou et al., 2009b; Omelchenko et al.,
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2009; Lammel et al., 2012; Stamatakis and Stuber, 2012). As a result, stimulation of the Lhb
was shown to inhibit up to 90% of VTA dopaminergic neurons (Matsumoto and Hikosaka,
2007), whereas its lesioning enhanced dopamine release in the NAc (Lecourtier et al., 2008).
The potent inhibitory influence of the Lhb on VTA dopaminergic neurons thus makes it a
critical nucleus that could mediate aversion. Indeed, Lhb neurons were strongly excited by
aversive stimuli (Matsumoto and Hikosaka, 2009), and CPA was elicited by deep brain
stimulation (DBS) or optogenetic activation of Lhb neurons (Friedman et al., 2011; Lammel et
al., 2012). Interestingly, a recent study demonstrated that the activity of Lhb is modulated by a
unique population of neurons from the VTA that co-express both GABA and glutamate (Shabel
et al., 2014). While the co-expression of GABA and glutamate in the same subset of neurons is
not yet a universally accepted phenomenon, we have previously demonstrated that D1-D2
heteromer expressing neurons in the NAc also co-express both GABA and glutamate (Perreault
et al., 2012a), thereby making the D1-D2 heteromer also a highly possible modulator of Lhb
activity. Therefore, in addition to increasing GAD67 expression in the VTA, the D1-D2
heteromer may have also induced aversive perception via the modulation of Lhb activity. This
notion is supported by the finding that selective D1-D2 heteromer inactivation regulated c-fos
induction in the Lhb (Chapter IV, Perreault et al. 2015), however additional studies will need to
provide definitive evidence for direct or indirect projections from NAc to Lhb.
6.5 Significance and Conclusion
In summary, we have identified the novel dopamine receptor complex, the D1-D2
heteromer, as a negative modulator of brain reward function. We postulate that, on a hedonic
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spectrum (Figure 8), the D1-D2 heteromer is tonically active at basal state in the absence of an
external rewarding stimulus to exert constant suppression of brain reward function to maintain
a neutral hedonic perception. Stimulation of the D1-D2 heteromer in the presence of external
rewarding stimuli such as psychostimulants and natural rewards will attenuate, whereas its
inactivation will enhance, the rewarding effect (positive hedonic value) induced by these
stimuli. In contrast, stimulation of the D1-D2 heteromer in the absence of an external
rewarding stimulus will result in an aversive state (negative hedonic value) and a depression-
like behavioural phenotype as a consequence of excessive suppression of brain reward function.
As the D1-D2 heteromer also modulated the neuronal activity of various nuclei that are
involved in brain reward function, including the NAc, prelimbic and infralimbic regions of the
mPFC, the orbitofrontal cortex, and the Lhb, these findings together suggest the D1-D2
heteromer may be a novel molecular substrate through which the mesolimbic dopamine system
could self-regulate the reward processes via homeostatic inhibition.
The identification of the D1-D2 heteromer as a negative modulator of brain reward
processes has profound implications for the understanding and treatment of neuropsychiatric
disorders characterized by a dysfunctional reward system, such as drug addiction and
depression. Using pharmacological agents that stimulate or inactivate the D1-D2 heteromer,
brain reward function could be respectively suppressed or enhanced to treat specific
pathological conditions accordingly. For instance, D1-D2 heteromer stimulation was shown to
abolish cocaine- and cue-induced reinstatement of drug self-administration, and thus could be a
potential method to prevent relapse to drug-seeking in human addicts. On the other hand, D1-
D2 heteromer inactivation was found to elicit rapid antidepressant-like activity in animals with
chronic stress-induced depressive behavioural phenotype, thus signifying its utility in treating
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Figure 7: The proposed role of the D1-D2 heteromer in the modulation of brain reward
function. The exposure to psychostimulants or natural reward results in a positive hedonic
value to induce a euphoric feeling, whereas depressed patients have negative hedonic value and
thus are less sensitive to reward perception. The stimulation of the D1-D2 heteromer reduces
the hedonic value to promote depression- and anxiety-like behaviour, whereas its stimulation
increases the hedonic value to promote psychostimulant-induced behaviours. The ability of the
D1-D2 heteromer to exert bidirectional modulation of the brain reward function makes it a
novel therapeutic target for the treatment of drug addiction and depression.
Positive
Negative
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ic S
pec
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m Natural Reward
Psychostimulant Reward
and Behaviour
Aversion
Depression
Anxiety
Homeostatic Point
D1-D2 Heteromer
Stimulation
D1-D2 Heteromer
Inactivation
D1-D2 Heteromer
Stimulation
D1-D2 Heteromer
Inactivation
185
patients with major depression. Since currently no effective intervention is available for drug
addiction or major depression, the D1-D2 heteromer may be a potential novel therapeutic target
for the treatment of these pathological conditions.
7. Future Directions
Our study thus far has implicated the D1-D2 heteromer in the negative modulation of
brain reward function, allowing its stimulation to suppress drug-seeking behaviours and its
inactivation to exert antidepressant-like activity. Nevertheless, additional studies are required
to further characterize the physiological role of the D1-D2 heteromer in reward- and
depression-related behaviours.
Although behavioural studies have clearly implicated the D1-D2 heteromer in reward-
seeking and depression-like behaviours, the precise molecular substrates through which the
D1-D2 heteromer signalling exerted its behavioural effects remain to be elucidated. It would be
imperative to determine the exact involvement of proteins downstream from the D1-D2
heteromer signalling, such as BDNF, DARPP-32, Cdk5, and GAD67, on the behavioural
effects of the D1-D2 heteromer as described in the current study. This can be achieved using
adenovirus-mediated knock-down of these proteins in specific brain regions, namely the NAc
and the VTA, and then examine reward- and depression-related behavioural effects of D1-D2
heteromer stimulation.
To further support the notion that the D1-D2 heteromer is a novel receptor complex that
functions to modulate brain reward function, studies should be conducted that assess the effects
186
of D1-D2 heteromer stimulation and inactivation on intracranial self-stimulation behaviour, a
well-validated behavioural model that directly reflects the activity of brain reward circuitry.
In addition, given that D1R- and D2R-expressing MSNs in the NAc have differential
effects on reward-seeking behaviours, the effect of direct stimulation of D1R/D2R co-
expressing MSNs should also be examined to establish the physiological function of this
unique subset of neurons in the NAc. This question can be easily addressed with the aid of
recent advancement in molecular techniques, such as optogenetics and designer receptors
exclusively activated by designer drugs (DREADDs). On the other hand, the effect of silencing
D1R/D2R co-expressing MSNs on brain reward function can be examined by injecting a
doubled-floxed Cre-dependent adeno-associated virus type 2 (AAV2) vector that expresses
D1R siRNA under the control of D1 receptor promoter into D2R-Cre mice, and behavioural
analyses will be performed on these mice.
Lastly, the projection targets of the D1R/D2R co-expressing MSNs remain largely
unknown, and it would be important to establish the network of nuclei that are interconnected
by this unique subset of NAc MSNs in order to further deduce how the D1-D2 heteromer
exerts its behavioural effects. In this respect, anterograde or retrograde tracing experiments
could be performed to determine the potential projection targets of the D1R/D2R co-expressing
MSNs.
The completion of these studies will allow us to firmly establish the role of the D1-D2
heteromer in the modulation of brain reward function, which in turn would make this novel
receptor complex an attractive therapeutic target for neuropsychiatric disorders that are
characterized by a dysfunctional reward system, such as drug addiction and major depression.
187
Nevertheless, as currently no pharmacological agent yet exists that could selectively stimulate
the D1-D2 heteromer, it would be imperative to conduct a comprehensive screening of existing
dopamine receptor agonists or use bioinformatics and in silico modeling strategies to search for
a selective agonist for this receptor complex before its modulatory effect on brain reward
function can be utilized in clinical settings.
Furthermore, in addition to its potential therapeutic benefits for the treatment of drug
addiction and major depression, studies have additionally suggested possible physiological role
for the D1-D2 heteromer in other neuropsychiatric disorders. For instance, the ability of D1-D2
heteromer stimulation to increase the production of BDNF (Hasbi et al., 2009; Perreault et al.,
2012a), which is crucial for neuronal growth and maturation, may have great implications in
the management of neurodegenerative disorders such as Alzheimer’s disease. In addition, the
D1-D2 heteromer has also been shown to signal through GSK-3 (Perreault et al., 2014), a
important mediator of the therapeutic effect of lithium for the the treatment of schizophrenia
(Beaulieu et al., 2004). Comprehensive behavioural and biochemical studies would be required
to explore the potential therapeutic benefits of the D1-D2 heteromer beyond drug addiction and
depression as suggested by these early studies.
188
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