Dopamine D1 and D3 Receptor Polypharmacology in Cocaine Reward and Cocaine Seeking

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DOPAMINE D1 AND D3 RECEPTOR POLYPHARMACOLOGY IN COCAINE

REWARD AND COCAINE SEEKING

EWA GALAJ

A dissertation submitted to the Graduate Faculty in Psychology in partial fulfillment of

the requirements for the degree of Doctor of Philosophy,

The City University of New York, 2017 ii

Ewa Galaj

D1 AND D3 RECEPTOR POLYPHARMACOLOGY IN COCAINE REWARD AND

COCAINE SEEKING

EWA GALAJ

This manuscript has been read and accepted for the Graduate Faculty in Psychology in satisfaction of the dissertation requirements for the degree of Doctor of Philosophy.

______Date Dr. Robert Ranaldi______

Chair of Examining Committee

______Date Dr. Richard Bodnar______

Executive Officer

Supervisory Committee

Dr. Richard Bodnar______

Dr. Carolyn Pytte______

Dr. Kenneth Carr______

Dr. Grace Rossi______

THE CITY UNIVERSITY OF NEW YORK iv

ACKNOWLEDGEMENTS

I would like to express my deep appreciation and gratitude to my advisor, Dr. Robert

Ranaldi, for his patience and mentorship. I am truly fortunate to have had the opportunity to work with him and to learn what good science is. I would like to thank him for his patient teaching on how to think critically and to write in clear, focused manner. I am very grateful for his confidence and continuous support, especially in times of discouragement and doubt. Without his guidance and help this dissertation would not have been possible. I also want to thank the members of my dissertation committee: Drs.

Carolyn Pytte, Richard Bodnar, Kenneth Carr and Grace Rossi for their guidance on this project. I am very thankful to Dr. Rossi for the support and encouragement that she gave me on pursuing a doctoral degree. Very special thanks go to Monica Manuszak who provided exceptional dedication and assistance on many projects that we worked on together. I also would like to acknowledge other students in Dr. Ranaldi’s laboratory who provided technical support in various ways: Sandra Babic, Neal Suppersaad, Ali

Shukur, Jordan Schachar, David Arastehmanesh, Joseph Haynes and Ivonne Cruz.

Lastly, I want to thank my siblings, Beata Arnold and Marek Galaj, for their endless support in this challenging process.

This dissertation is dedicated to my mother, Rozalia Galaj.

PREFACE

Portions of this dissertation have been accepted for publication. Chapters 2 and 3 consist of text and data that have been accepted for publication by the peer-review journal

Psychopharmacology in August of 2016. The manuscript titled “Dopamine D1 and D3 receptor interaction in cocaine reward and seeking in rats” was co-authored with Drs.

Wayne Harding and Robert Ranaldi.

Dopamine D1 and D3 receptor polypharmacology in cocaine reward and cocaine seeking

ABSTRACT

Advisor: Professor Robert Ranaldi

Background: In the search for efficacious pharmacotherapies to treat cocaine addiction much attention has been given to agents targeting D1 or D3 receptors because of the involvement of these receptors in cocaine-related behaviors. D1 and D3 receptor partial agonists and antagonists have been shown to reduce cocaine reward, reinstatement of cocaine seeking and conditioned place preference (CPP) in rodents and non-human primates. However, translation of these encouraging results with selective D1 or D3 receptor agents has been limited due to a number of factors including toxicity, poor pharmacokinetic properties and extrapyramidal and sedative side effects.

Purpose: Given the role of D1 and D3 receptors in cocaine-related behaviors and the urgent need for effective anti-cocaine treatments, it is important that we continue to pursue new pharmacological approaches such as the combinations of compounds that have differential functions at multiple dopamine receptors. The aims of this dissertation were to evaluate the effects of the novel strategy of simultaneous treatments with a D3 receptor antagonist (NGB 2904) and D1 receptor partial agonist (SKF 77434) in animal models of drug addiction, including cue-induced reinstatement of cocaine seeking, cocaine conditioned place preference (CPP) and cocaine self-administration in rats. vii

Methods: In the reinstatement experiment, rats underwent cocaine self-administration training followed by extinction and a cue-induced reinstatement test. Prior to the reinstatement test rats were treated with one of several doses of NGB 2904, SKF 77434 or the combination of the two and their lever presses were measured. In the CPP experiment rats were conditioned to experience cocaine in one compartment of a CPP apparatus and saline in the other. After conditioning rats were treated with the same compounds alone or combined prior to the CPP test. The time spent in the cocaine-paired compartment prior to and after conditioning was measured as an indication of cocaine

CPP. In the self-administration experiment, rats were trained to self-administer cocaine under a progressive ratio (PR) schedule of reinforcement. After demonstrating stable baseline break points (BPs) rats were treated repeatedly with NGB 2904 or SKF 77434 alone or the combination of the two.

Results: The co-administration of NGB 2904 and SKF 77434, at doses which when administered individually produced no significant effects, prior to reinstatement or CPP tests significantly reduced lever pressing and time spent in the cocaine-paired environment, suggesting synergistic effects of the combined compounds on their abilities to reduce cocaine seeking. When administered to rats self-administering cocaine under a

PR schedule of reinforcement doses of NGB 2904 which were ineffective alone significantly potentiated the break point-reducing effects of SKF 77434.

Conclusions: The results of this study indicate that the combined treatment with a D1 receptor partial agonist and D3 receptor antagonist produces robust decreases in cocaine seeking and reward. These effects provide insight into a novel therapeutic approach to treat cocaine addiction. viii

TABLE OF CONTENTS

List of Figures.………………………………………………………..……….……..xi

1. Chapter 1- General Introduction …………………………………………………...... 1

1.1. Understanding drug abuse and addiction…………………………………………...... 1

1.2. Dopamine and drug reward…………………………………………………………...3

1.3. Dopamine receptors: subtypes, signaling and location…………………………..…...9

1.4. D1 receptors in reward ……………………………………………………………...13

1.5. The role of D1 receptors in conditioned place preference…………………………..18

1.6. D1 receptors and reinstatement of drug seeking…………………………………….21

1.7. D3 receptors in drug reward………………………………………….…...………...24

1.8. D3 receptors and CPP……………………………………………………………….30

1.9. D3 receptors and drug seeking………………………………………………………32

1.10. D1-D3 receptor interactions at the physical, biochemical and functional levels…..35

1.11. D1-D2 receptor heteromers……………………………………………………...…39

1.12. Current pharmacological strategies and possible directions in drug addiction

treatment………………………………………………………………………...…..43

1.13. Specific Aims………………...…………………………………………………….45

2. Chapter 2. Simultaneous D1 receptor partial agonism and D3 receptor antagonism

reduce cue-induced reinstatement of cocaine seeking and conditioned place

preference……………………………………………………………………………46

2.1. Introduction………………………………………………………………………….46

2.2. Methods………...……………………………………………………………………48

2.2.1. Subjects……………………………………………………………………………48 ix

2.2.2. Surgery…………………………………………………………………………….49

2.2.3. Apparatus………………………………………………………………………….50

2.2.4. Drugs………………………………………………………………………………51

2.2.5. Procedure………………………………………………………………………….51

Experiment 1: Cue-induced reinstatement of cocaine seeking……………………51

Experiment 2: Cocaine conditioned place preference…………………………….52

2.2.6. Data analysis………………………………………………………………………53

2.3. Results...………………………………………………………………...…………..54

Experiment 1: Cue-induced reinstatement of cocaine seeking……………….……54

Experiment 2: Cocaine conditioned place preference…………………………...... 56

2.4. Discussion…………………………...……………………………………………...61

3. Chapter 3. Interaction of D1 and D3 receptors in cocaine reward ……….....……...63

3.1. Introduction………………………………………...……………………………….63

3.2. Methods……………………………………………………...……………………..65

3.2.1. Subjects……………………………………………………………………………65

3.2.2. Surgery…………………………………………………………………………….65

3.2.3. Apparatus………………………………………………………………………….66

3.2.4. Drugs………………………………………………………………………………66

3.2.5. Procedure……………………………………………………………………….....67

Experiment 3: Cocaine self-administration under the PR schedule (cocaine

reward)………………………………………………………………………….…67

3.2.6. Data analysis………………………………………………………………………68 x

3.3. Results of Experiment 3: Cocaine self-administration under a PR schedule of

reinforcement (cocaine reward)……………………………………………….…..68

3.4. Discussion…………………………………………………………………...……....71

4. Chapter 4. General Discussion…………………………………………...……….…72

4.1. Conclusions……………………………………………………………………….…81

4.2. Implications and future directions…………………………………………...... ……82

5. References……………………………………………………………………………86

LIST OF FIGURES

Figure 1: Mean (± SEM) number of presses on the active and inactive levers averaged across the last three extinction sessions and during the cue-induced reinstatement test grouped by the dose and compound (of SKF 77434, NGB 2904 or a combination of the two) that rats were treated with ...... 58

Figure 2: Mean (± SEM) number of presses emitted by rats treated with vehicle/vehicle during the reinstatement test and rats responding for food and treated with 1/1 mg/kg of

NGB 2904/SKF 77434 …………………………………………………………….... 59

Figure 3: Mean (± SEM) time spent in the cocaine-paired compartment of the CPP apparatus during pre-exposure and preference tests for rats grouped by the dose and compound (of NGB 2904, SKF 77434 or NGB 2904/SKF 77434) that they were tested with……………………………………………………………………………………..60

Figure 4: Mean (± SEM) BPs reached during the baseline and test sessions for rats grouped by the dose and compound (of NGB 2904, SKF 77434 or NGB 2904/SKF

77434) that they were treated with………………………………………………………70 1

CHAPTER 1- GENERAL INTRODUCTION

2.1. Understanding drug abuse and addiction

Drug addiction has serious medical, legal, financial and social consequences.

Some of the most common medical complications related to drug abuse include cardiovascular and respiratory problems, kidney failure, hepatotoxicity, infectious diseases (HIV/AIDS, hepatitis B or C), seizures, stroke and coma (Riezzo et al. 2012;

Riordan and Williams 1998) and, sadly, the greatest cost of drug abuse is paid in human lives, lost either directly due to overdose or through drug using-related diseases (AIDS, hepatitis C, etc.). In recent years there has been a dramatic increase in heroin and prescription opiate use and overdose incidents, bringing the number of fatal overdoses to nearly 29,500 in 2014 (National Center for Health Statistics at the Centers for Disease

Control and Prevention, 2015) (Rudd et al. 2016). In addition to medical problems, drug users experience a series of legal problems ranging from fines, arrests, probation to extensive jail time. Drug-related crimes often involve theft, burglary, prostitution, drug dealing or domestic violence, all of which widen marginalization of addicts at the economic and social levels. Other consequences of drug addiction may include broken or strained relationships with friends and family, isolation, failure in education and financial problems.

However, the impact of drug addiction is felt beyond the drug using population.

Illicit drug use costs our society annually $193 billion in healthcare, crime and lost work productivity (NIDA 2016). Each year 1.3 million visits to hospital emergency departments occur that are related to illicit drug misuse or abuse resulting in a total cost 2 of upwards of $5.6 billion. And this is only a portion of the total national expenditure of

$11.4 billion in healthcare. Furthermore, society endures a major drug-related burden of

$120.3 billion annually due to lost work productivity deriving from reduced motivation, poor labor participation, loss of employment, incarceration, hospitalization, special treatment programs and premature mortality (National Drug Intelligence Center, 2011).

The National Survey for Drug Use and Health (NSDUH) conducted by the Substance

Abuse and Mental Health Services Administration (SAMHSA) reports that in 2014, 27 million people in the United States were current users of illicit drugs including marijuana, cocaine, prescription-type pain relievers, heroin or methamphetamine and 7.1 million of the users met DSM-IV diagnostic criteria for substance use disorders (NSDUH 2015).

Despite the dangers and adverse consequences there has been a striking increase in the use of prescription-type pain relievers, heroin and methamphetamine in the last two decades. As drug abuse and addiction have reached epidemic proportions, urgent preventive measures and effective treatments are desperately needed.

In this dissertation introduction I will highlight the role of dopamine (DA) in drug seeking and reward with an emphasis on the importance of DA D1 and D3 receptors, independently and interactively, in drug related behaviors. In so doing I will focus on animal models of drug abuse and addiction. I will address challenges of traditional DA compounds, their limited clinical utility to treat addiction, and emphasize the urgent need for new strategies in pharmacotherapeutic development that would focus on compounds with more complex pharmacological profiles and differential actions at multiple DA receptor subtypes. This latter point constitutes the aims of this dissertation research.

1.2. Dopamine and drug reward

Drugs of abuse are addictive because they produce rewarding effects that reinforce (Pickens and Thompson 1968) and energize behavior. The notion that addictive properties of drugs of abuse are related to their rewarding effects has been elegantly demonstrated in self-administration studies. Laboratory animals readily self-administer cocaine (Pickens and Thompson 1968), amphetamine (Pickens and Harris 1968), heroin

(Blakesley et al. 1972) or nicotine (Hanson et al. 1979), at a steady rate that is directly related to the drug dose per infusion and continue to work for the drug until the end of the session. When given unlimited access, rats or monkeys respond for intravenous drug readily and to the point of convulsion or death (Bozarth and Wise 1985; Johanson et al.

1976). Under a progressive ratio (PR) schedule of reinforcement, in which the response requirement increases with each subsequent reward, rats will emit hundreds of responses for a drug infusion. And if the drug dose is reduced the animals will work less for the drug infusion; that is, the break point (BP; the response ratio at which animals stop responding for the drug) is lower. The BP is taken as in index of drug reward and a reduction in BP is interpreted as a reduction in drug reward (Richardson and Roberts

1996).

Additional early evidence that drugs of abuse are rewarding in their own right came from brain stimulation reward (BSR) studies. Early BSR studies showed that rats would work for electrical stimulation of certain, but not other, brain regions that are now widely considered “reward sites” (Corbett and Wise 1980; Gratton and Wise 1983). The strongest BSR comes from the medial forebrain bundle (at the level of the lateral and posterior hypothalamus) (Olds and Olds 1969) and ventral tegmental area (Olds et al. 4

1960) followed by nucleus accumbens (Phillips et al. 1975). Amphetamine (Phillips et al. 1975; Stein 1962), cocaine (Esposito et al. 1978), morphine (Olds and Travis 1960), heroin (Hubner and Kornetsky 1992), cannabis (Gardner et al. 1988) and other drugs have been shown to lower the threshold for BSR in these regions, indicating potentiation of reward. This suggests that drugs of abuse activate the same reward circuitry as intracranial self-stimulation and their abuse liability is directly related to the rewarding effects that these drugs can produce.

The rewarding effects of drugs derive from the activation of the DA mesocorticolimbic system with its origins in the ventral tegmental area (VTA) and projections to the forebrain regions including the nucleus accumbens (NAcc), amygdala, prefrontal cortex (PFC) and hippocampus (Fallon and Moore 1978). The biggest dopaminergic projections reach the NAcc where a vast number of DA receptors are expressed. Most drugs of abuse have the ability to enhance DA neurotransmission primarily in the NAcc and this mechanism of action is believed to be the underlying mechanism through which drugs produce rewarding effects and are liable for abuse (Wise

1996; 2004; Wise and Rompré 1989). This is true for cocaine (Church et al. 1987; Di

Chiara and Imperato 1988; Hurd and Ungerstedt 1989), amphetamine (Carboni et al.

1989; Di Chiara and Imperato 1988), nicotine (Di Chiara and Imperato 1988), opiates (Di

Chiara and Imperato 1988; Wise et al. 1995a), cannabis and ethanol (Di Chiara and

Imperato 1988; Khatib et al. 1988).

The studies of Wise et al. provided the first convincing evidence that DA is the primary neurotransmitter for reward. Rats previously trained to run and or press a lever for food (Wise et al. 1978), water (Gerber et al. 1981) or lateral hypothalamic BSR 5

(Fouriezos et al. 1978; Fouriezos and Wise 1976) did not continue to work long for these rewards under the treatment of pimozide, a DA receptor antagonist. When treated with pimozide during the acquisition period of a food-reinforced lever-press response rats did not acquire the lever press response (Wise and Schwartz 1981). On the other hand, pimozide increased lever pressing for amphetamine (Yokel and Wise 1975; 1976) and cocaine (de Wit and Wise 1977) in well-trained animals. The action of pimozide on heroin self-administration has been reported to be less effective (Gerber and Wise 1989), but other DA antagonists have been shown to disrupt heroin intake (Nakajima and Wise

1987). Generally, in animals self-administering heroin under fixed ratio (FR) schedules of reinforcement, low and medium doses of DA receptor antagonists cause an increase in stable drug intake while high doses inhibit responding. Such outcomes are typically interpreted as reductions in reward (de Wit and Wise 1977). In the conditioned place preference (CPP) paradigm, in which animals typically show preference for a compartment in which they previously experienced rewarding effects of drugs of abuse over another compartment in which they had not rats will spend less time in the heroin– associated compartment when treated with pimozide, indicating a reduction in heroin

CPP (Bozarth and Wise 1981a). Later development of other DA receptor antagonists with affinity for specific subtypes of DA receptors allowed deeper exploration of DA involvement in drug reward (see discussion below).

Several lines of research have identified sites implicated in the rewarding actions of drugs of abuse and they involve the VTA, NAcc, medial PFC (mPFC) and amygdala.

Early evidence came from lesion studies in which 6-hydroxydopamine (6-OHDA) injected into the VTA to damage cell bodies of DA neurons (Roberts and Koob 1982) 6 and into the NAcc to damage the terminal fields of DA neurons (Caine and Koob 1994b;

Lyness et al. 1979; Roberts et al. 1977; Roberts et al. 1980) disrupted intravenous self- administration of psychostimulants (e.g., cocaine and amphetamine). 6-OHDA lesions in the mPFC produced various outcomes: they either had no effect (Martin-Iverson et al.

1986), abolished self-administration (Goeders and Smith 1983; 1986) or increased supersensitivity to low doses of cocaine manifested as more reliable self-administration under a FR schedule of reinforcement (that was not observed in non-lesioned rats)

(Schenk et al. 1991) or as increased BPs under a PR schedule of reinforcement

(McGregor et al. 1996). Under the PR schedule of reinforcement, 6-OHDA lesions of mesocorticolimbic regions or intracranial treatment with DA receptor antagonists reduces

BPs for cocaine self-administration, suggesting a reduction in rewarding effects of the drug (Richardson and Roberts 1996). The effect of DA-selective lesions in the mesocorticolimbic system on opiate self-administration is ambivalent. 6-OHDA-induced lesions of the NAcc either failed to disrupt opiate self-administration (Pettit et al. 1984) or shifted the dose response curve to the right, indicating reduced drug reward (Smith et al. 1985). Interestingly, 6-OHDA-induced lesions of the NAcc and VTA blocked drug- primed reinstatement of morphine CPP (Wang et al. 2003) while destruction of DA cell bodies in the VTA blocked morphine-induced potentiation of BSR (Hand and Franklin

1985). The findings from the DA-selective lesion studies suggest that the integrity of the

VTA-NAcc pathway is required for psychostimulant, and to some extent, opiate reward.

Compelling evidence of DA involvement in drug reward comes from microdialysis and voltammetry studies in which each self-administered infusion of cocaine (Hurd et al. 1989; Pettit and Justice 1989), amphetamine (Ranaldi et al. 1999) or 7 heroin (Hemby et al. 1995; Wise et al. 1995a) is shown to increase the extracellular DA concentration in the NAcc. These in vivo neurochemical studies also link very convincingly the fluctuations in DA with the timing of drug self-administration behaviors. During a self-administration session the extracellular DA level is elevated by the initial responses for cocaine or amphetamine (loading phase) and is maintained at elevated levels by intermittent lever presses that occur at the point when the drug- elevated NAcc DA concentration begins to drop. Subsequent phasic (rapid) intra-session increases in DA concentrations occur in anticipation of (Kiyatkin and Stein 1994; 1995) and in response to drug infusion (Ranaldi et al. 1999; Wise et al. 1995b). Self- administered heroin provides a slightly different pattern of DA signaling. Microdialysis studies show that heroin produces slow and long lasting elevations in concentration of

DA in the NAcc that are maintained throughout the session and fall back to the baseline level only upon the termination of the session (Wise et al. 1995a). Voltammetry studies show that each heroin injection causes an initial decrease in DA signal followed by the rising DA signal while anticipation of the next heroin infusion causes short increases in

DA signaling (Kiyatkin et al. 1993). Moreover, electrophysiology studies show that the firing pattern of neurons in the NAcc corresponds with the pattern of a rat’s responding during cocaine self-administration. Neurons show a high rate of tonic firing during the loading phase and shift to a more evenly spaced pattern of firing during the remainder of the session as the rats’ responses for cocaine infusions shift from erratic to more evenly timed. Burst firing is evident prior to the initiation and 200 ms after each delivery of a cocaine infusion contingent upon the lever press (Carelli et al. 1993). 8

There have been reports that cocaine and heroin activate separate neuronal circuitries where heroin self-administration does not require the activation of the mesolimbic DA system (Ettenberg et al. 1982; Pettit et al. 1984; Vaccarino et al. 1985) but rather is mediated by direct stimulation of opiate receptors. This notion was supported by studies in which dopaminergic lesions (Pettit et al. 1984) and DA antagonists injected into the NAcc (Gerrits et al. 1994) were ineffective in disrupting heroin self-administration while opiate antagonists (i.e., naloxone) administered either systemically (Ettenberg et al. 1982) or directly into limbic regions increased heroin self- administration (Britt and Wise 1983; Vaccarino et al. 1985). It is now well documented that opiates activate the mesolimbic system (Gysling and Wang 1983) through its agonistic action on mu and delta opioid receptors located on the GABAergic axon terminals near dopaminergic cell bodies in the VTA (Devine et al. 1993; Johnson and

North 1992; Matthews and German 1984). The rewarding action of heroin and other opiates involve, at least in part, disinhibition of the mesolimbic DA system (Johnson and

North 1992) and subsequent enhanced DA release in the NAcc (Di Chiara and Imperato

1988).

Compelling evidence that drugs of abuse are rewarding because they activate the mesocorticolimbic system comes from studies in which rats learn to self-administer drugs directly into limbic (but not other) brain regions. Opiates are directly self-administered into the NAcc (Olds 1982) and VTA (Bozarth and Wise 1981b; Devine and Wise 1994;

Welzl et al. 1989). Cocaine has been reported to be self-administered into the mPFC

(Goeders and Smith 1983), olfactory tubercle (Ikemoto 2003), posterior but not anterior

VTA (Rodd et al. 2005), and the NAcc shell but not core (Carlezon et al. 1995; Goeders 9 and Smith 1983; Ikemoto 2003; McKinzie et al. 1999; Rodd-Henricks et al. 2002).

Phencyclidine (PCP) and amphetamine are readily self-administered into the orbitofrontal and mPFC and NAcc shell (Carlezon and Wise 1996; Hoebel et al. 1983;

Phillips et al. 1981; Phillips et al. 1994). Drug self-administration into mesolimbic regions can be attenuated by 6-OHDA lesions or DA receptor antagonism. Such findings provide further evidence for the role of DA, particularly within the mesolimbic and mesocortical systems, in drug reward. In addition, microinjections of opiates into the

VTA can establish CPP (Barr and Rossi 1992; Bozarth 1987; Phillips and LePiane 1980) and potentiate BSR (van Wolfswinkel and van Ree 1985). Intra-NAcc injections of amphetamine produce CPP (Carr and White 1983; Hemby et al. 1992) and enhance BSR

(Colle and Wise 1988). Microinjections of cocaine into the olfactory tubercle, the most ventral portion of the ventral striatum, and marginally into the NAcc shell have been shown to produce CPP and this effect is blocked by intracranial injections of DA receptor antagonists to these regions (Ikemoto 2003). Overall, specific sites within the mesocorticolimbic DA system have been demonstrated to play a crucial role in drug reward.

1.3. Dopamine receptors: subtypes, signaling and location

Five DA receptors have been identified and classified into two subfamilies (Cools and Van Rossum 1976; Kebabian 1978; Kebabian and Calne 1979) based on their homology, pharmacology and intracellular signaling properties (for a review see Missale

1998). DA receptors belong to a family of seven transmembrane domain G-protein 10 coupled receptors. The D1 receptor-like family includes D1 and D5 receptors that share

80% homology of their 7 transmembrane domains. The D2 receptor-like family comprises D2, D3 and D4 receptors in which D2 receptors share 75% homology of their protein structure with D3 receptors while only 53% homology with D4 receptors. The main structural differences among DA receptors are differences in size of the 3rd intracellular loop connecting transmembrane domains and of the carboxyl-terminal intracellular segment (i.e., the tail of the amino acid chain). The development of ligands selective for D1 or D2 receptor-like families has enabled the localization, function and pharmacological profile of these receptors to be investigated. Different DA receptor subtypes have been distinguished based on ligands showing selectivity for some but not other DA receptors. D1 and D2 receptor-like families have substantially distinct pharmacological profiles in terms of their binding properties while members of the same family share similarities, making it more difficult for DA agonists and antagonists to bind exclusively to a specific DA receptor subtype. For example, SCH 23390, a D1 receptor antagonist, has similar affinity for D1 and D5 receptors (but not for D2 or D3 receptors) making it possible to differentiate between D1 and D2 receptor-like families but not between receptor subtypes within the same family (for reviews of DA receptor structure and function see (Missale et al. 1998; Pivonello et al. 2007; Vallone et al. 2000). This is problematic for pharmacological research on drug addiction as it impedes our understanding of the function of specific DA receptors in drug-related behaviors and hinders our search for efficacious pharmacotherapeutics for drug addiction.

DA receptors have been categorized into D1 and D2 receptor-like families because of their stimulatory or inhibitory function. The D1 receptor-like family has the 11 ability to activate the second messenger enzyme, adenylyl cyclase (AC), by coupling to

G-protein Gsα or Golfα to increase the synthesis of the second messenger, cyclic adenosine monophosphate (cAMP). The D2 receptor-like family coupled to G-protein

Gi/oα has the ability to inhibit AC and the production of cAMP. The stimulation of cAMP results in the activation of protein kinase A (PKA) which phosphorylates a variety of target proteins involved in cellular metabolism (i.e., ion channels, synaptic vesicle protein, enzymes necessary for neurotransmitter synthesis) and gene expression; all of which are required for signal transduction. The second main function of DA receptors is to regulate Ca2+ signaling (CA2+ L-type current, concentration of intracellular Ca2+, Ca2+- dependent signaling pathways, etc.). In addition to PKA, DA receptors can activate several other protein kinases (e.g., PKC, ERK, CaMKII) that require cAMP to modulate intracellular Ca2+ concentration, alter ion channel properties or densities, regulate gene expression and protein synthesis causing structural changes at existing synapses as well as synaptogenesis. As a result, DA dependent-cAMP and other second–messenger (i.e.,

2+ Ca , cGMP, IP3) pathways facilitate neuronal communication and plasticity in response to experience. Drugs of abuse appear to activate and modify these pathways within the

DA mesolimbic system [for a review see (Lee and Messing 2008)]. For example, cAMP- dependent PKA activation in the NAcc is necessary for the acquisition but not expression of amphetamine CPP (Beninger et al. 2003; Gerdjikov et al. 2007). PKA activation

(through infusions of Sp-cAMPS into the NAcc) or inhibition (through infusions of Rp- cAMPS into the NAcc) increases or decreases, respectively, responding for cocaine under a PR schedule of reinforcement such that changes in BPs are relatively persistent, lasting several days (Lynch and Taylor 2005). Cocaine, morphine, nicotine and tetra- 12 hydrocannabinal (THC) have been shown to activate the ERK-pathway (which is critically involved in immediate early gene expression) in the NAcc, lateral bed nucleus of the stria terminalis, central amygdala and PFC. The ERK activation in these brain regions can be inhibited by the D1 receptor antagonist, SCH 23390 (Valjent et al. 2004), suggesting that ERK phosphorylation induced by drugs of abuse is mediated by D1 receptor stimulation.

The highest expression of D1 receptors has been found in the dorsal and ventral striatum followed by frontal cortex, amygdala, olfactory tubercle and subthalamic nucleus. D1 receptors have been identified pharmacologically in the substantia nigra pars reticulata and VTA, but D1 receptor mRNA in these regions has not been detected. D2 receptors have been found predominantly in the dorsal and ventral striatum, olfactory tubercle, and in the midbrain regions such as substantia nigra and VTA. D3 receptors are restricted to the mesolimbic areas such as the NAcc shell (the limbic portion) but not core

(the striatal portion of the striatum), Islands of Calleja and olfactory tubercle, all part of the ventral striatum that is deemed critical in reward and incentive motivation. The D4 receptors are highly expressed in the frontal cortex, amygdala, the olfactory bulb, hippocampus, hypothalamus and the midbrain. D5 receptors are restricted to the hippocampus, hypothalamus and thalamus with little or no amounts in the striatum.

Of particular interest are D1 and D3 receptors as both of them have been implicated in drug reward and drug seeking. To understand their specific roles in different aspects of addiction (reward, reinforcement, relapse, conditioning, etc.) and to assess the efficacy of various DA receptor ligands as potential pharmacotherapeutics, a number of animal models of drug abuse and addiction have been used. They include: 13

BSR, drug self-administration under different schedules of reinforcement, CPP and drug, stress or cue-induced reinstatement of drug seeking.

1.4. D1 receptors in reward

The role of D1 receptors in reward has been demonstrated in BSR and self- administration studies. The D1 receptor antagonist, SCH 23390, and partial agonist, SKF

38393, reduce (Nakajima and O'Regan 1991) while the D1 receptor agonists, SKF 82958 and A77636, facilitate BSR (Carr et al. 2001; Gilliss et al. 2002; Lazenka et al. 2016;

Ranaldi and Beninger 1994). In drug self-administration studies, D1 receptor antagonists, SCH 23390, A69024 or SCH 39166, increase cocaine intake but high doses of the antagonists decreases responding in rats and monkeys (Barrett et al. 2004; Britton et al. 1991; Caine and Koob 1994a; Caine et al. 1999; Corrigall and Coen 1991a; Hubner and Moreton 1991; Koob et al. 1987). A higher rate of cocaine intake under the treatment of SCH 23390 has been associated with a greater increase of DA oxidation current (i.e., a measure of extracellular DA concentration) in the NAcc. SCH 23390 alone does not affect self-administration of saline nor DA levels recorded during saline self-administration but instead, increases responding for cocaine and the magnitude of the related DA oxidation current (Egilmez et al. 1995). In addition, SCH 23390 also shifted the dose response curve for MDMA (ectasy; a drug that acts on monoamine systems) self-administration to the right, indicating a reduction in drug reward (Brennan et al.

2009; Daniela et al. 2004), and it decreased self-administration of methamphetamine

(Brennan et al. 2009) or nicotine (Corrigall and Coen 1991b) but only in the second half 14 of the test session, suggesting that it is not a motoric effect but likely a reduction in reward.

The effects of other D1 receptor agents on drug self-administration under FR schedules of reinforcement also have been evaluated. The D1 receptor full agonists, SKF

82958 or R-6-Br-APB, and the D1 partial agonist, SKF 77434, have been shown to increase latencies to the first response (without further effects on responding) or shift the dose response curve for cocaine-self-administration downward in rats (Caine et al. 1999) and monkeys (Caine et al. 2000). While the D1 receptor partial agonist, SKF 38393, failed to alter cocaine self-administration (Woolverton et al. 1984) another D1 receptor partial agonist, SKF 77434, produced differential effects (Mutschler and Bergman 2002).

Specifically, a low dose (1 mg/kg) of SKF 77434 shifted moderately the descending portion of the dose-effect function for cocaine self-administration to the right (thus it reduced cocaine reward) but did not alter the threshold reinforcing cocaine dose

(necessary for reliable self-administration) nor did it disrupt cocaine seeking or responding for food reinforcement. Higher doses of SKF 77434 shifted the dose-effect function for cocaine self-administration downward and disrupted food-maintained performance (Mutschler and Bergman 2002). Thus, a reduction in cocaine-intake with high doses of SKF 77434 most likely reflects non-specific motoric effects rather than a reduction in reward. When dose response curves were generated within each session of cocaine self-administration the D1 receptor agonists, SKF 82958 and R-6-Br-APB, shifted dose-effect functions downward, decreasing cocaine intake but the D1 receptor antagonist, SCH 39166, shifted the function rightward, increasing the total cocaine intake

(Barrett et al. 2004). Overall, these studies show that D1 receptor antagonists reduce 15 psychostimulant reward but D1 receptor agonists either disrupt cocaine self- administration, which may reflect non-specific motoric effects, or produce no effect.

Studies with intracranial injections have localized the sites of D1 receptor antagonist action within the mesocorticolimbic regions. Intracranial injections of SCH

23390 into the NAcc shell and core, central amygdala, dorsolateral bed nucleus of the stria terminalis (dlBNST), substantia nigra, mPFC but not posterior caudate nucleus, dorsal striatum, neostriatum or lateral septum increased self-administration of cocaine, which reflects a decrease in the magnitude of reward (Bari and Pierce 2005; Caine et al.

1995; Epping-Jordan et al. 1998; Maldonado et al. 1993; McGregor and Roberts 1993;

1995; Quinlan et al. 2004).

The role of D1 receptors in reward has been demonstrated in studies using multiple schedules of reinforcement where two or more reinforcement schedules are typically presented one at a time in an alternating sequence each day, and each schedule component is associated with different discriminative stimuli and rewards. Under the multiple FR 15 schedules of reinforcement rats responded for sucrose or alternatively for cocaine. When treated with SCH 23390 rats showed an increase in self-administration of cocaine but not sucrose (Stairs et al. 2010). Higher doses of SCH 23390 reduced responding for cocaine without altering responding for food (Caine and Koob 1994a).

Similarly, under the multiple FR5 schedules of reinforcement for nicotine and sucrose

(on alternating days), SCH 23390 decreased nicotine intake in the second half of the session (Stairs et al. 2010). This suggests that D1 receptor antagonists may reduce rewarding effects of psychostimulants without altering responding for food.

With the PR schedule of reinforcement, systemic (Depoortere et al. 1993; Hubner 16 and Moreton 1991) or intra-VTA, NAcc, mPFC, but not intra-amygdala or striatum,

(McGregor and Roberts 1993; 1995; Ranaldi and Wise 2001) injections of SCH 23390 produced dose-dependent reductions in BP in rats self-administering cocaine.

Interestingly, the attenuating effects of intra-PFC SCH 23390 on PR responding were observed even 24 hours later for low, but not moderate, doses of cocaine (Olsen and

Duvauchelle 2006). Under the same schedule of reinforcement responding for nicotine

(Sorge and Clarke 2009) or the combination of heroin and cocaine (speedball) (Cornish et al. 2005) was reduced by systemic or intra-accumbens injections of SCH 23390, respectively. D1 receptor agonists produced the opposite effects on PR responding in rhesus monkeys such that systemic administration of SKF 81297 shifted heroin or cocaine dose-response functions to the left, suggesting enhancement in drug reward

(Rowlett et al. 2007).

In the two lever choice paradigm, where animals are trained to discriminate drug from saline, the D1 receptor antagonists, SCH 23390, SCH 39166, or D1 receptor partial agonists, SKF 77434 or SKF 83959, can reduce the discriminative stimulus effects of cocaine (Anderson and van Haaren 2000; Barrett and Appel 1989; Katz et al. 1999;

Kleven et al. 1990; Platt et al. 2000; Sinnott and Nader 2001), amphetamine (Callahan et al. 1991; Kamien and Woolverton 1989) or methamphetamine (Munzar and Goldberg

2000; Tidey and Bergman 1998) and moderately nicotine (Corrigall and Coen 1994) but not heroin (Corrigall and Coen 1989). Discriminative stimulus effects of drugs of abuse often are related to subjective effects experienced by human addicts. Thus, understanding the underlying mechanisms of discriminative stimulus effects of drugs of abuse in animal models allows us to understand a mechanism of drug reward and 17 stimulus effects that perpetuate drug taking. The D1 receptor full agonists, SKF 81297,

SKF 82958 or R6-Br-APB, and at least one D1 receptor partial agonist, SKF 38393, either failed to engender (Chausmer and Katz 2002; Kleven et al. 1990; Platt et al. 2000;

Sinnott and Nader 2001) or increased cocaine-appropriate responding (Chausmer and

Katz 2002; Spealman et al. 1991). Although D1 receptor full agonists appear to reduce the discriminative stimulus effects of psychostimulants they themselves mimic the discriminative stimulus effects of cocaine when substituted for the drug and also are self- administered (Grech et al. 1996; Spealman et al. 1991; Witkin et al. 1991), therefore suggesting they might produce reinforcing effects of their own. This limits severely their therapeutic utility to treat drug addiction.

Of clinical interest are D1 receptor partial agonists, as they do not produce cocaine-like behavior (i.e., when substituted for cocaine they fail to induce cocaine-like responses or only partially substitute for cocaine) (Barrett and Appel 1989; Callahan et al.

1991; Kleven et al. 1990). In addition to their lower abuse potential [(Grech et al. 1996;

Weed and Woolverton 1995) but see (Self and Stein 1992)], D1 receptor partial agonists produce mild extrapyramidal, undesirable effects (Platt et al. 2000).

In a second order schedule of reinforcement one schedule of reinforcement is superimposed on another such that an animal is trained to respond according to the lower order schedule (e.g., FR 10) for secondary rewards (cues associated with primary (drug) reward) and the higher order schedule (e.g., FI 5) for primary rewards (cocaine paired with cues). In the first order schedule (in this example FR 10) cues motivate animals to continue to work toward completion of the second order schedule (i.e., FI 5), which ultimately provides the reward (in this example the first response that occurs at the end of 18

5 minutes is reinforced by a cocaine infusion). Under this type of schedule, as the dose of cocaine is increased, rates of responding first increase and then either decrease or level off. Systemic injections of D1 receptor partial agonists or antagonists shift dose response curves for cocaine self-administration in monkeys to the right, indicating a reduction in drug reward (Bergman et al. 1990; Grech et al. 1996). SCH 23390 injected directly into the caudal portion of the basolateral amygdala decreased, while SKF 81297, a D1 receptor agonist, increased cocaine seeking under a second order schedule of reinforcement when cocaine and cues were available. SCH 23390 infused into the rostral part of the basolateral amygdala decreased, while SKF 81297 increased cocaine seeking when only cues were available (Mashhoon et al. 2009). This suggests that D1 receptors in the rostral part of the basolateral amygdala are involved in cocaine seeking while D1 receptors in its caudal part are involved in reward.

1.5. The role of D1 receptors in conditioned place preference

Drug cues can be powerful stimuli that maintain and or trigger drug seeking, approach or preference for an environment in which the animal experienced drug effects.

Pharmacological studies have demonstrated that D1 receptors are involved in mediating cue-driven behaviors. SCH 23390 administered during conditioning blocks the acquisition of cocaine (Akins et al. 2004; Cervo and Samanin 1995; Nazarian et al. 2004;

Pruitt et al. 1995), nicotine (Acquas et al. 1989), amphetamine (Acquas and Di Chiara

1994; Hiroi and White 1991; Hoffman and Beninger 1989), ethanol (Pina and

Cunningham 2014) and morphine CPP (Acquas et al. 1989; Acquas and Di Chiara 1994; 19

Shippenberg and Herz 1987). Interestingly, rodents with an earlier history of methamphetamine or phencyclidine exposure can develop phencyclidine CPP but naive rodents will develop conditioned place aversion (CPA) with phencyclidine (Noda and

Nabeshima 1998). SCH 23390 can block the establishment of CPP and CPA induced by phencyclidine (Acquas et al. 1989; Noda et al. 1998), indicating a role of D1 receptors in drug-related learning. A single trial with intravenous amphetamine paired with one compartment of a CPP apparatus has been shown to be enough to establish CPP but not in rats pretreated with SCH 23390 (Bardo et al. 1999). The establishment of nicotine

(Spina et al. 2006) or morphine CPP (Fenu et al. 2006) with a single conditioning session can be blocked by microinjections of SCH 39166, a D1 receptor antagonist, into the

NAcc shell but not into the core while SCH 23390 infused into the dorsal hippocampus can block the acquisition of single-trial cocaine CPP (Kramar et al. 2014). Intra- accumbens injections of SCH 23390 during conditioning block the acquisition of cocaine

(Baker et al. 1998) and ethanol CPP (Young et al. 2014). Co-administration of SCH

23390 and amphetamine into the NAcc blocked the acquisition of amphetamine CPP

(Liao 2008). Microinjections of SKF 38393, a D1 receptor partial agonist, into the central amygdala (Zarrindast et al. 2003) or dorsal hippocampus (Rezayof et al. 2003) during conditioning with ineffective or low doses of morphine either produced or enhanced CPP, respectively. Overall, the findings suggest that stimulation of D1 receptors is necessary for the establishment of CPP and that D1 receptor antagonists or partial agonists administered systemically or into specific mesolimbic regions can block the establishment of drug CPP. 20

However, D1 receptor agents alone can produce CPA or CPP. Low, but not high, doses of SCH 23390 produced CPA (Acquas and Di Chiara 1994; Shippenberg and Herz

1987). Systemic injections of SKF 38393 alone produced CPA that could be counteracted by SCH 23390 (Hoffman and Beninger 1989) while intra-accumbens microinjections of SKF 38393 produced CPP (White et al. 1991). The D1 receptor full agonists, SKF 82958, ABT-431, SKF 81297, but not the partial agonist, SKF 77434, all when administered alone, produced CPP (Abrahams et al. 1998; Graham et al. 2007).

Again, this poses severe limitations in translation of such agents into clinical settings.

There is some evidence that D1 receptors might be involved in the expression of

CPP. Systemic or intra-accumbens microinjections of SCH 23390 attenuate dose- dependently the expression of amphetamine CPP (Hiroi and White 1991; Liao et al.

1998), but not of cocaine (Adams et al. 2001; Cervo and Samanin 1995; Liao et al. 1998) nor of ethanol (Young et al. 2014). SCH 23390 also failed to block the expression of

MDMA-CPP (Vidal-Infer et al. 2012). In fact, SCH 23390 attenuated cocaine induced locomotor activity but not cocaine conditioned activity (Adams 2001). Microinjections of

SCH 23390 into the central amygdala (Zarrindast et al. 2003) or dorsal hippocampus

(CA1) (Rezayof et al. 2003) decreased the expression of morphine CPP [but see

(Haghparast et al. 2013) for no effect of intra CA1-SCH 23390 on intra-VTA morphine induced CPP ]. The expression of morphine single trial CPP was not affected by intra-

NAcc injections of SCH 23390 (Fenu et al. 2006). Intra-VTA (Galaj et al. 2014b), but not intra-hippocampus (Kramar et al. 2014), infusions of SCH 23390 blocked the expression of cocaine CPP. Systemic injections of the D1 receptor agonists, SKF 81297,

SKF 82958 and ABT-431 can block the expression and reinstatement of cocaine CPP 21

(Graham et al. 2007). Stress-induced reinstatement of cocaine CPP can be blocked by the microinjection of SCH 23390 or SKF 81297 into the mPFC while cocaine-induced reinstatement of cocaine CPP can be prevented only by SCH 23390 and not SKF 81297

(Sanchez et al. 2003). SCH 23390 also blocks drug-induced reinstatement of MDMA

CPP (Vidal-Infer et al. 2012). The D1 receptor partial agonist, SKF 38393, has been shown to facilitate the extinction of cocaine CPP. Mice that demonstrated cocaine CPP and later were restricted to the cocaine side (while in a cocaine-free state) under the treatment of SKF 38393 for 3 days no longer showed CPP during a subsequent preference test (Sabioni et al. 2012). These findings suggest that D1 receptors are necessary for the acquisition and reinstatement of CPP but the role of D1 receptors in the expression of CPP is less well understood.

1.6. D1 receptors and reinstatement of drug seeking

A host of pharmacological studies have demonstrated the involvement of D1 receptors in drug seeking induced by cues, stress or drug priming. The D1 receptor antagonist, SCH 23390, can reduce cue or context induced reinstatement of cocaine

(Alleweireldt et al. 2002a; Crombag et al. 2002), nicotine (Liu et al. 2010) or heroin seeking (Bossert et al. 2007; Shaham and Stewart 1996). The D1 receptor full agonist

SKF 81297, but not the D1 receptor partial agonist, SKF 38393, attenuated cue-induced reinstatement of cocaine seeking (Alleweireldt et al. 2002a). These similar effects of D1 receptor antagonists and full agonists on cue-induced reinstatement and CPP are quite surprising given the facts that these drugs produce opposing effects in studies with a two 22 lever choice procedure to discriminate cocaine from saline or self-administration studies involving second order or PR schedules of reinforcement. It is possible that these discrepancies derive from the differences in test conditions (i.e., animals being tested under the influence of drug or drug free) or that the paradigms reflect different aspects of addiction (i.e., incentive motivation versus drug reinforcement).

Specific locations of D1 receptors involved in drug seeking have been identified.

SCH 23390 injected into the prelimbic cortex, dorsolateral caudate-putamen (dlCP),

NAcc shell (but not core) or dorsomedial caudate-putamen (dmCP) attenuated cue- (Gao et al. 2013; See 2009) or context-induced reinstatement of opiate seeking (Bossert et al.

2007; Bossert et al. 2009). On the other hand, SCH 23390 injected directly into the

NAcc core or shell attenuated context-induced reinstatement of ethanol seeking

(Chaudhri et al. 2009) but when infused into the central amygdala SCH 23390 had no effect on cue-induced reinstatement of cocaine seeking (Thiel et al. 2010). Cocaine seeking induced by non-contingent priming injections of cocaine or with cue presentations in rats and monkeys has been attenuated by the D1 receptor antagonist,

SCH 39166, the D1 receptor full agonists, SKF 81297 and SKF 82958, and also the D1 receptor partial agonists, SKF 883959 and SKF 38393 (Khroyan et al. 2000; Khroyan et al. 2003; Self et al. 1996a).

D1 receptors have also been implicated in drug-primed drug seeking. SCH

23390 attenuated drug induced reinstatement of heroin (Shaham and Stewart 1996),

MDMA (ectasy) (Schenk et al. 2011) and cocaine seeking [(Green and Schenk 2002;

Norman et al. 1999) but see (Schenk and Gittings 2003) for no effect]. Drug-primed reinstatement of heroin seeking was blocked by SCH 23390 injected directly into the 23 prelimbic cortex (See 2009). Drug (cocaine, SKF 81297 or 7-OH-DPAT) primed reinstatement of cocaine seeking was blocked by microinjections of SCH 23390 into the

NAcc shell but not core or lateral septum (Anderson et al. 2003; Bachtell et al. 2005;

Schmidt and Pierce 2006). Systemic injections of SKF 81297 or SKF 82958 alone (Dias et al. 2004) or intra-central amygdala microinjections of SKF-38393 (Thiel et al. 2010) failed to induce reinstatement of cocaine seeking. When SKF 81297 was injected into the mPFC it caused a small but significant increase in cocaine primed cocaine seeking

(Schmidt et al. 2009). Systemic injections of SKF 82958 blocked drug-primed reinstatement of cocaine seeking (Self et al. 1996a). SCH 23390 injected into the dorsal

PFC blocked cocaine-induced reinstatement of cocaine seeking but it had no effect on cocaine self-administration under an FR5 schedule of reinforcement (Sun and Rebec

2005). The D1 receptor agonist, ABT-431, failed to block drug-primed cocaine seeking but it disrupted cocaine self-administration by suppressing the initiation of responding and producing an extinction-like pattern of responding (Self et al. 2000).

In addition to their involvement in cue and drug-primed drug seeking, D1 receptors play a role in stress-induced drug seeking. SCH 23390 attenuated reinstatement of heroin seeking induced by food deprivation (Tobin et al. 2009) but not by foot shock

(Shaham and Stewart 1996). Microinjections of SCH 23390 into the NAc shell, dmPFC, or basolateral amygdala, but not into the NAc core or the ventromedial PFC, attenuated reinstatement of heroin seeking induced by food deprivation (Tobin et al. 2013).

Injections of SCH 23390 into the prelimbic or orbitofrontal cortical regions blocked drug- or stress-induced cocaine seeking while blockade of D1 receptors in the prelimbic cortex attenuated stress-induced cocaine seeking (Capriles et al. 2003). Basolateral amygdala 24 injection of SCH 23390 blocked cocaine seeking (See et al. 2001).

Thus, these studies provide evidence that D1 receptors play a role in numerous aspects of addiction: reward (as demonstrated by BSR and self-administration studies), drug cue conditioning (as revealed by the acquisition of CPP), to some degree in drug- cue driven conditioned response (as evaluated in the expression and reinstatement of CPP and second order schedule responding), drug seeking (as evident in the reinstatement and second order schedule responding paradigms) and discriminative stimulus effects (as seen in the drug/saline choice paradigm).

1.7. D3 receptors in drug reward

D3 receptors are highly involved in drug-related behavior: particularly in drug seeking and conditioned responding. But there is some evidence that these receptors may also play a role in drug reward. The early indication that D3 receptors play a role in reward came from Caine and Koob’s work (Caine and Koob 1993; 1995; Caine et al.

1997). Rats self-administering cocaine under an FR schedule of reinforcement when treated with 7-OH-DPAT or quinelorane, pramipexole or PD 128,907, all D3 receptor agonists, maintained a relatively stable pattern of cocaine self-administration but at a lower rate (Caine and Koob 1993; 1995; Caine et al. 1997). A decrease in the stable rate of drug intake – or seen another way, an increase in the intervals between self- administered injections – without interruptions in self-administration patterns is interpreted as enhancement in drug reward. Under a PR schedule of reinforcement 7-

OH-DPAT shifted the cocaine-dose effect function to the left also suggesting enhancing 25 effects in cocaine reward (Caine and Koob 1995).

The role of D3 receptors in reward has been demonstrated in BSR studies. NGB

2904 and SB-277011A, both selective D3 receptor antagonists, can increase thresholds for BSR enhanced by cocaine (Vorel et al. 2002; Xi et al. 2006), methamphetamine

(Spiller et al. 2008) or nicotine (Pak et al. 2006), indicating a reduction in reward and implicating D3 receptors in cocaine reward. Other D3 receptor antagonists, YQA14 and low doses of S33138 (Peng et al. 2009; Song et al. 2014) or low doses of the D3 receptor partial agonist, BP-897, (Spiller et al. 2008) have been effective at reducing cocaine- enhanced BSR. The D3 receptor antagonist, PG01037, has been reported to block enhancement of BSR (Higley et al. 2011b). Interestingly, when these D3 receptor agents were administered alone they failed to affect BSR thresholds (Song et al. 2014; Spiller et al. 2008; Xi et al. 2006), suggesting that D3 receptor stimulation itself is not involved in

BSR per se but is involved in drug reward itself. Thus, this demonstrates a lack of potential of D3 receptor antagonists and partial agonists for abuse and renders them possible candidates as pharmacotherapeutics for drug addiction.

Although the findings of BSR studies suggest that D3 receptors may be directly involved in drug reward, other studies indicate that the role of D3 receptors in drug reward is ambivalent. As mentioned already, one of the more powerful demonstrations that drugs of abuse produce rewarding effects is the fact that they are readily self- administered by laboratory animals. Under low response requirements, as in low FR schedules of reinforcement, D3 receptor antagonists either reduced (e.g., SB-277011A,

YQA14, U-99194A) (Gal and Gyertyan 2003; Song et al. 2011) or did not affect (e.g.,

SB-277011A, PG01037, NGB 2904) cocaine self-administration (Caine et al. 2012; Gal 26 and Gyertyan 2003; Xi et al. 2006). Similarly, animals self-administering cocaine under treatment with D3 receptor partial agonists either increased their drug intake (e.g., BP-

897, cariprazine) (Gal and Gyertyan 2003; Roman et al. 2013) or showed no change in their self-administration pattern (e.g., CJB090) (Achat-Mendes et al. 2009). Also, under an FR1 schedule of reinforcement D3 receptor knockout mice showed either a similar pattern of cocaine self-administration to that of wild-type counterparts (Caine et al. 2012) or enhancement in rate of self-administration (Song et al. 2012). SB-277011A and a novel D3 receptor antagonist, Compound 16, have been shown to reduce heroin self- administration in wild-type mice but not in D3 receptor knock out mice (Boateng et al.

2015). The D3 receptor partial agonist, CBJ090, but not the D3 receptor antagonist,

PG01037, reduced FR1 responding for methamphetamine in rats with short and long access to drug self-administration (Orio et al. 2010). BP-897 and SB-277011A failed to alter self-administration of nicotine under low schedules of reinforcement (Andreoli et al.

2003; Khaled et al. 2010). Thus, the findings from self-administration studies using low

FR schedules of reinforcement are inconclusive and raise the possibility that D3 receptor stimulation may not be directly involved in drug reward but instead may be critically important for incentive motivation and conditioned behavior related to drug reward. And there is some evidence for this claim.

When work demands for drug self-administration are increased, as they are in higher FR (e.g., FR 10) and PR schedules of reinforcement, SB-277011A, NGB-2904,

YQA14, SR 21502 significantly reduced responding for cocaine (Galaj et al. 2014a; Song et al. 2011; Xi and Gardner 2007; Xi et al. 2005). In recent years our lab has tested a novel highly selective D3 receptor antagonist, SR 21502, (Ananthan et al. 2014) in 27 various animal paradigms of drug abuse. We found that rats self-administering cocaine under a PR schedule of reinforcement showed a dose-dependent reduction in BP when treated with SR 21502. The D3 receptor antagonist did not produce non-specific motoric effects as it did not affect spontaneous locomotor activity, PR responding for food nor did it incapacitate rats in making more responses for higher cocaine doses (i.e., greater drug reward) (Galaj et al. 2014a). Rats treated with any one dose of SR 21502 were able to reach higher BPs under the same dose of SR 21502 if the cocaine dose was raised (from

0.75 to 1.0 or 1.5 mg/kg). Thus, we concluded that the observed effects of SR 21502 are unlikely to be non-specific motoric effects but rather, like with other D3 receptor antagonists, reflect a reduction in cocaine reward.

Although systemic injections of D3 receptor antagonists reduce cocaine self- administration under PR schedules of reinforcement, U99194A, a D3 receptor antagonist, injected directly into the core or shell of the NAcc did not reduce BPs in rats self- administering cocaine under a PR schedule of reinforcement (Bari and Pierce 2005). A high dose (56 mg/kg) of SB-277011A attenuated nicotine self-administration under a PR schedule of reinforcement, however the same dose also reduced locomotor activity and reduced PR responding reinforced by food in separate groups of rats (Ross et al. 2007).

Therefore, the attenuating effect of SB-277011A on nicotine seeking cannot be easily interpreted as a reduction in motivation in drug taking. Treatment with SB-277011A,

YQA14 or PG01037 lowered BPs in PR responding for methamphetamine but it failed to alter methamphetamine self-administration under FR 1 or 2 schedules of reinforcement

(Chen et al. 2014; Higley et al. 2011a; Higley et al. 2011b). CBJ090 and PG01037 reduced PR responding in rats with extended access to methamphetamine while only 28

CBJ090 produced reliable attenuating effects in rats with short access to drug self- administration (Orio et al. 2010). As mentioned earlier, a reduction in responding under these schedules of reinforcement is interpreted as a reduction in reward (Richardson and

Roberts 1996) (Arnold and Roberts 1997) and D3 receptor antagonists are effective in producing such effects. To the best of my knowledge, no study has tested D3 receptor agents against heroin self-administration under a PR schedule of reinforcement.

The role of D3 receptors in reward has been demonstrated in studies using multiple schedules of reinforcement. Under a multiple VI-60 schedule of reinforcement, rats that responded for sucrose and alternatively for intravenous cocaine when treated with the D3 receptor partial agonist, OS-3-106, showed decreased responding for cocaine and increased latencies to emit the first response for cocaine (Cheung et al. 2013). In the same study, the novel D3 receptor partial agonists, OS-3-106 and WW-III-55, reduced

PR responding for cocaine, suggesting that the incentive motivation to self-administer cocaine and drug reward require stimulation of D3 receptors.

D3 receptor agents have also been tested in the drug/saline and drug/food choice paradigms. Rhesus or squirrel monkeys that were trained to discriminate cocaine from saline injections showed poor discrimination under treatment with CJB090, a D3 receptor partial agonist, (Achat-Mendes et al. 2009; Martelle et al. 2007b) while NGB 2904 produced no effect (Martelle et al. 2007b). Similarly, food deprived mice that learned to discriminate cocaine from saline showed an increase in cocaine-appropriate responding when treated with the D3 receptor agonists, pramipexole and quinpirole (Collins et al.

2014). PG01037, a D3 receptor antagonist, shifted responding for cocaine to food and decreased total cocaine intake (John et al. 2015b), but other D3 receptor agents (e.g., 29

PG619, NGB 2904 or PG01037) did not disrupt cocaine/food choice or methamphetamine/food choice (John et al. 2015a; John et al. 2015b; Martelle et al.

2007b).

Studies with a second order schedule of reinforcement provide supporting evidence for the notion that cue-controlled drug seeking is mediated by D3 receptor stimulation. Rats self-administering cocaine under a second order schedule of reinforcement when treated with SB-277011A show a dose-dependent reduction in responding during the pre-drug interval and drug intervals as well as an increased latency to achieve the first cocaine conditioned cue presentation and cocaine infusion (Di Ciano et al. 2003). However, intracranial injections of SB-277011A directly into the basolateral amygdala but not into the shell of the NAcc and the dorsal striatum have been reported to decrease responses for cocaine during the pre-drug interval of a second order schedule of reinforcement (Di Ciano 2008). In this study intra-amygdala SB-277011A also affected inactive lever pressing and did not alter the latency to reach the first cue presentation and cocaine infusion, making the results more difficult to interpret. Under this schedule, systemic treatment with the D3 receptor antagonists or partial agonists PNU 9919A, BP-

897 or CJB 090, but not NGB 2904, decreased cocaine seeking before the first infusion of cocaine in rats and monkeys (Claytor et al. 2006; Martelle et al. 2007a; Pilla et al.

1999). CJB 090 produced non-selective effects as it also reduced food-maintained responding (Martelle et al. 2007b). These studies suggest that D3 receptors are involved in cue-controlled cocaine seeking.

1.8. D3 receptors and CPP

A host of pharmacological studies have demonstrated that D3 receptor antagonists can reduce the expression of cocaine (e.g., SB-277011A, SR 21502, YQA14, Compound

35) (Cervo et al. 2005; Hachimine et al. 2014; Micheli et al. 2007; Song et al. 2013;

Vorel et al. 2002), heroin (e.g., SR 21502, SB-277011A) (Ashby et al. 2003; Galaj et al.

2015), morphine (e.g., YQA14) (Hu et al. 2013), nicotine (e.g., SB-277011A, Compound

35) (Micheli et al. 2007; Pak et al. 2006) and methamphetamine CPP (Sun et al. 2016).

The D3 receptor partial agonist, BP-897, has been shown to produce similar generally attenuating effects on the expression of cocaine [(Duarte et al. 2003) but see (Cervo et al.

2005)], morphine [(Frances et al. 2004a) but see (Duarte et al. 2003)], amphetamine

(Aujla and Beninger 2005) and nicotine CPP (Le Foll et al. 2005). However, conflicting results have been reported on the effects of these agents on the acquisition of CPP. When administered during conditioning SB-277011A blocked the acquisition of heroin (Ashby et al. 2003) or cocaine (Vorel et al. 2002) CPP, although an absence of effect on cocaine conditioning (Gyertyan and Gal 2003) has also been reported. BP-897 either had no effect (Gyertyan and Gal 2003) or blocked the acquisition of cocaine CPP (Duarte et al.

2003); and it either enhanced (Frances et al. 2004b) or had no effect of the acquisition of morphine CPP (Duarte et al. 2003). YQA14 blocked the acquisition of cocaine CPP

(Song et al. 2013) but had no effect on methamphetamine (Sun et al. 2016) nor morphine

CPP (Hu et al. 2013). The acquisition of morphine CPP was enhanced by 7-OH-DPAT, a

D3 receptor agonist, but attenuated with high doses of PNU-99194, a D3 receptor antagonist that also induced CPP in itself (Frances et al. 2004b). It appears then that D3 31 receptors are critical for the expression of an already established CPP – conditioned behavior that requires incentive motivation and which is triggered by conditioned stimuli

(drug cues) – but are less important for the acquisition of CPP, a process that might be more dependent on the effects of unconditioned stimuli (drug reward) per se.

To investigate the role of D3 receptors in conditioned behavior and incentive motivation we recently investigated whether D3 receptor antagonism can affect the extinction of cocaine CPP. Rats with established cocaine CPP were repeatedly treated with SR 21502 in the presence of conditioned cues and later retested drug-free for CPP

(Galaj et al. 2016). In animals with established cocaine CPP treatment with SR 21502 in the cocaine-paired environment (i.e., in the presence of cocaine cues), but not in the home cage (i.e., in the absence of cocaine-paired cues), reduced the previously established cocaine CPP. SR 21502 itself did not produce preference or aversion for one or the other compartment of the CPP apparatus in cocaine naive rats. Similar findings have been reported with SB-277011A on extinction of cocaine CPP (Ashby et al. 2015a) and with

YQA14 on the extinction of methamphetamine CPP (Sun et al. 2016). Thus, it appears that D3 receptor antagonism can facilitate extinction of psychostimulant CPP without producing aversive effects of its own. It also suggests that behaviors driven by drug conditioned cues and drug-related incentive motivation require stimulation of D3 receptors.

D3 receptor antagonism can reduce incentive motivation for drug and drug-related stimuli. It has been demonstrated that D3 receptor antagonism reduces reinstatement of cocaine CPP. After cocaine CPP was established, rats were repeatedly exposed to cocaine-conditioned cues drug free to extinguish their cocaine CPP (Ashby et al. 2015b; 32

Rice et al. 2013). A single injection of morphine (Rice et al. 2013) or cocaine or temporal food deprivation (Ashby et al. 2015b) reinstated the extinguished cocaine CPP and this effect was reduced when SB-277011A was administered prior to the reinstatement test session. YQA14 has been reported to block drug-induced reinstatement of methamphetamine (Sun et al. 2016) or heroin CPP (Hu et al. 2013).

These studies suggest that D3 receptors are necessary to elicit drug- or stress-induced reinstatement of CPP.

Conditioned cues can drive cocaine-related behavior and such behavior appears to be mediated, at least in part, by D3 receptor stimulation. Rodents that were treated chronically with cocaine display hyperlocomotion in the presence of cocaine cues. The

D3 receptor antagonists, SB-277011A or ABT-127, can block the expression of the conditioned response (cue-induced hyperlocomotion) (Banasikowski et al. 2010; Le Foll et al. 2002) but failed to block the acquisition of this conditioned behavior (Banasikowski et al. 2010). Nicotine cue-induced hyperlocomotion can be attenuated by SB-277011A and BP-987 (Le Foll et al. 2003b; Pak et al. 2006).

1.9. D3 receptors and drug seeking

Strong evidence for the notion that D3 receptors are involved in incentive motivation underlying drug seeking comes from studies using the reinstatement paradigm. D3 receptor agents of antagonistic action can reduce reinstatement of drug seeking primed by a single injection of the drug. Highly selective D3 receptor antagonists, SB-277011A, NGB 2904, low doses of partially selective D3 receptor 33 antagonists, S33138 and the D3 receptor partial agonist, BP-897, can cause a reduction in cocaine-primed reinstatement of lever pressing in rats with a history of cocaine self- administration (Gilbert et al. 2005; Peng et al. 2009; Vorel et al. 2002; Xi et al. 2006). In monkeys, the D3 receptor antagonist, PG01037, attenuated cocaine-primed reinstatement of cocaine seeking and the D3 receptor agonist, PD 128907, produced at least a partial reinstatement of cocaine seeking (Achat-Mendes et al. 2010). Overall, there is strong agreement that D3 receptor antagonists and partial agonists can effectively reduce reinstatement of cocaine seeking. However, Anderson et al. (2006) reported that intracranial injections of U99194A, a D3 receptor antagonist, into the shell or core of the

NAcc failed to alter cocaine-primed drug seeking (Anderson et al. 2006). SB-277011A has been shown to reduce reinstatement of methamphetamine (Higley et al. 2011a) and alcohol seeking (Heidbreder et al. 2007), triggered by drug or cue/alcohol, respectively.

SB-277011A and compound 35 can attenuate drug-primed nicotine seeking (Andreoli et al. 2003; Micheli et al. 2007). No pharmacological study has tested D3 receptor agents against drug-primed heroin seeking.

D3 receptor antagonists and partial agonists also have the ability to reduce reinstatement of drug seeking triggered by drug cues. Compounds such as SB-277011A,

NGB 2904, YQA14, RGH-188 (cariprazine), and BP-897 reduced cue-induced reinstatement of cocaine seeking (Cervo et al. 2007; Gilbert et al. 2005; Roman et al.

2013; Song et al. 2014). Recently our lab reported that SR 21502 reduced dose dependently active lever pressing during a cue-induced reinstatement test in rats with a history of cocaine (Galaj et al. 2014a) or heroin self-administration (Galaj et al. 2015).

These reductions in lever pressing were unlikely due to motoric incapacity as SR 21502 34 did not affect pressing on the inactive lever nor did it cause a significant reduction in spontaneous locomotor activity. In fact, animals that worked for food under a PR schedule of reinforcement but treated with a high dose (15 mg/kg) of SR 21502 were able to make at least twice as many responses as the vehicle-treated (and certainly more responses than the 15 mg/kg of SR 21502-treated group) in the cue-induced reinstatement of the heroin seeking group (Galaj et al. 2015). SB-277011A, but not BP-897, has been shown to block context- and cue-induced nicotine seeking in rodents (Khaled et al. 2010;

Sabioni et al. 2016). And reduced cue-induced methamphetamine seeking has been reported in animals treated with YQA14 or PG01037 (Chen et al. 2014; Higley et al.

2011b).

Several studies have demonstrated the importance of D3 receptors in stress- induced reinstatement of drug seeking. When treated with SB-277011A, or YQA14 rats show a reduction in cocaine seeking (Song et al. 2014; Xi et al. 2004). However, NGB

2904 has been reported to have no effect on reinstatement of extinguished lever pressing triggered by food deprivation (Tobin et al. 2009).

Thus, pharmacological studies have demonstrated that D3 receptor stimulation is necessary primarily for drug-related conditioned behaviors driven by cues (as demonstrated in CPP studies, studies with second order schedules of reinforcement, conditioned locomotor activity studies and cue-induced reinstatement studies) and drug seeking that can be initiated by stress, cues and drug (as demonstrated in the response reinstatement paradigm). The role of D3 receptors in reward is more ambivalent - studies with BSR or PR schedules of reinforcement show their importance in reward but such 35 findings are not corroborated by studies with low FR schedules of reinforcement or of acquisition of drug CPP.

1.10. D1-D3 receptor interactions at the physical, biochemical and functional levels

Within the last decade increasing attention has been given to D1 and D3 receptor interactions within the mesolimbic system. Medium spiny neurons (MSNs) represent a major constituent of the neuronal population in the dorsal and ventral striatum (90-95%)

(Freund et al. 1984; O'Donnell and Grace 1993) and at least half of them express DA receptors (Weiner et al. 1991). Although classical thinking has depicted the D1 receptor- like family of DA receptors as being expressed on the GABAergic Substance P dynorphinergic MSNs (Le Moine et al. 1991) and the D2 receptor-like family (including

D3 receptors) as primarily expressed on GABAergic enkephalinergic neurons (Le Moine and Bloch 1996), new evidence indicates that D1 and D3 receptors often co-localize on the same GABAergic SP dynorphinergic MSNs in the ventral striatum, particularly in the

Islands of Calleja and NAcc shell (Le Moine and Bloch 1996; Ridray et al. 1998;

Schwartz et al. 1998), brain structures deemed critical for reward and motivation.

D1 and D3 receptors of close proximity can interact physically by forming heteromers that are composed of at least two receptor subunits (Fiorentini et al. 2008;

Marcellino et al. 2008). DA receptors, as part of the G-protein couple receptor (GPCR) family can form heteromers between adjacent GPCRs of the same subfamily (e.g.,

D1/D2; D1/D3) or receptors of an entirely distinct subfamily (e.g., D1/NMDA).

Increasing attention has been given to D1 and D3 receptor heteromers as potential pharmacotheraputic targets to treat neuropsychiatric disorders such as Parkinson disease 36 and schizophrenia. D1-D3 receptor heteromerization is believed to account for changes in the receptors’ ability to bind ligands and modulate intracellular signaling cascades.

For example, SCH 23390 shows higher binding to the D1 receptors in human embryonic kidney cells (HEK 293 T) that express D1-D3 receptor heteromers than to D1 receptors in HEK 293 T cells expressing only D1 receptors (Fiorentini et al. 2008). Similarly, in the HEK 293 T cells and striatal MSNs expressing both D1 and D3 receptors, stimulation of D3 receptors with 7-OH-DPAT potentiated the affinity of the DA agonist SKF 38393 for D1 receptors. However, stimulation of D1 receptors with SKF 81297 had no effect on the 7-OH-DPAT affinity for D3 receptors (Marcellino et al. 2008), suggesting that the

D1-D3 receptor interaction is not reciprocal. Moreover, D1-D3 receptor heteromerization modulates the AC signaling pathway. DA can stimulate cAMP formation to a greater degree in HEK 293 T cells expressing D1 and D3 receptors than in cells expressing only D1 receptors and this effect is counteracted by SCH 23390 or sulpiride (Fiorentini et al. 2008). Guitart and colleagues did not confirm that D1-D3 receptor heteromers potentiate D1-receptor mediated AC signaling but they have demonstrated other positive cross talk between D1 and D3 receptors at the level of mitogen-activated protein kinase (MAPK) signaling (i.e., ERK 1/2 signaling pathway)

(Guitart et al. 2014). Specifically, synergistic phosphorylation of ERK1/2 was observed with simultaneous administration of D1 and D3 receptor agonists, and later was blocked by either D1 or D3 receptor antagonists suggesting that stimulation of D3 receptors within the D1-D3 receptor heteromer is crucial for potentiation of D1 receptor-mediated signaling to occur.

D1-D3 receptor heteromers also modulate the expression of Substance P mRNA 37 in neurons of the Islands of Calleja and NAcc shell. Co-administration of SKF 38393 and quinpirole, a D1 receptor partial agonist and D2/3 receptor agonist, respectively, in reserpine treated rats potentiated the small enhancing effect of SKF 38393 on the expression of Substance P mRNA in neurons expressing D1 and D3 receptors and this synergistic effect was abolished by nafadotride, a preferential D3 receptor antagonist

(Ridray et al. 1998). This suggests that simultaneous stimulation of D1 and D3 receptors can potentiate D1 receptor-mediated intracellular signaling. Similarly, D3 receptor knockout mice exhibit a decrease in the expression of the immediate early gene, c-fos, in response to a D1 receptor agonist (Jung and Schmauss 1999). This provides additional evidence of the functional interaction between D1 and D3 receptors in the ventral striatum.

Chronic treatment with L-dopa for parkinsonism has been shown to reduce the expression of D3 receptors in the NAcc shell and to promote the expression of D1

(Berthet et al. 2009) and D3 receptors in the dorsal striatum, where normally D3 receptors are absent (Bordet et al. 1997; Solis et al. 2015). One aversive consequence of chronic L-dopa treatment is dyskinesia which has been linked to the overexpression of

D3 receptors on D1-containing neurons of the dorsal striatum (Solis et al. 2015) and to an increase in D1-D3 receptor heteromers (Farre et al. 2015). With chronic L-dopa treatment D1 receptors become more sensitive to DA and heteromerization with D3 receptors appears to be a mechanism that controls D1 receptor sensitivity (Farre et al.

2015). Compared to their wild type counterparts, mice lacking D3 receptors showed milder symptoms of dyskinesia after L-dopa treatment and transgenic mice with overexpression of D1 receptors are more responsive to treatments involving D3 receptor 38 antagonism aimed at reducing dyskinesia symptoms (Solis et al. 2015). Similarly, treatment with D3 receptor partial agonists or antagonists attenuates L-dopa–induced dyskinesia in MPTP–treated monkeys (Bezard et al. 2003; Visanji et al. 2009) or in rats; all of which points to the direct interaction between D1 and D3 receptors.

Thus, the D1-D3 receptor interactions at the physical and biochemical levels might account for synergistic effects of D1 and D3 receptor ligands at the behavioral level. The D3 receptor agonist, PD 128907, can potentiate SKF 38393, a D1 receptor partial agonist, induced locomotor activity in reserpinized mice (but not in mice lacking

D3 receptors) and this effect can be counteracted by D3, but not D2, receptor antagonism

(Marcellino et al. 2008) (note: reserpinized mice are used to isolate postsynaptic effects).

Compared to their wild type counterparts, mice lacking D3 receptors showed higher locomotor activity induced by cocaine (Karasinska et al. 2005; Xu et al. 1997) and co- administration of D1 and D2 receptor agonists, respectively, can enhance this response even more (Xu et al. 1997). Deletion of D1 and D3 receptors blocks CPP established with low doses of cocaine (Karasinska et al. 2005). Thus, it appears that D3 receptors might be important in modulating (increasing or decreasing) neuronal signaling at the D1 receptor level and controlling striatal function (i.e., reward and movement).

It is known that repeated exposure to cocaine decreases sensitivity of autoreceptors in the VTA and increases sensitivity of D1 receptors to DA and DA receptor agonists in the NAcc (Henry et al. 1989). Chronic cocaine is also associated with upregulation of D3 receptor mRNA in the NAcc as seen in cocaine-overdosed individuals (Segal et al. 1997) and an increase in 7-OH-DPAT binding to D3 receptors in the ventromedial striatum in cocaine addicts (Staley and Mash 1996). Similar results 39 have been reported in animal studies in which acute or chronic exposure to cocaine (Le

Foll et al. 2002; Neisewander et al. 2004), nicotine (Le Foll et al. 2003a), morphine

(Spangler et al. 2003) or drug cues also enhances D3 receptor expression in the rodent striatum and the VTA and substantia nigra. It is possible then that up-regulation of D3 receptors increases the sensitivity of D1 receptors, and thus potentiates D1 receptor- mediated signaling involved in drug taking and seeking. This intriguing hypothesis warrants further investigation.

Given the involvement of D1 and D3 receptors in drug-related behaviors, their co- localization and the existence of D1-D3 receptor heteromers, revolutionary therapeutic approaches are emboldened to treat addiction and other neuropsychological disorders.

D1-D3 receptor heteromers, with their distinct pharmacological and cell-signaling properties, make possible the expansion of the search for effective anti-addiction therapeutics into areas involving polypharmacological approaches leading to the discovery of new agents that would simultaneously, but differentially, target D1 and D3 receptors. Such compounds possibly could produce more robust effects against drug taking and relapse with minimal unwanted side effects, as often seen with traditional D1 or D2 receptor agents.

1.11. D1-D2 receptor heteromers

It is noteworthy to point out that D1 receptor-mediated signaling can be also potentiated through D2 receptors, as D1 and D2 receptors also co-localize and form heteromers. Co-activation of both D1 and D2 receptors leads to increases in intracellular 40

Ca2+ levels, something that is not observed with selective simulation of D1 or D2 receptors (Lee et al. 2004; Rashid et al. 2007). Stimulation of D1-D2 receptor heteromers coupled to Gq protein in the ventral striatum (Perreault et al. 2010; Rashid et al. 2007) can activate phospholipase C and IP3 that are necessary for intracellular calcium release (Lee et al. 2004) and activation of CaMKII (Hasbi et al. 2010; Ng et al. 2010).

CaMKII activation is important for brain-derived neurotrophic factor (BDNF) expression

(Hasbi et al. 2009; Perreault et al. 2012; Perreault et al. 2013) and phosphorylation of

AMPA receptors (Derkach et al. 1999) – both of which are involved in drug-mediated synaptic plasticity (Wolf and Ferrario 2010). D1-D2 receptor heteromers have been shown to have greater affinity for DA in response to amphetamine exposure (Perreault et al. 2010).

The synergistic effects of D1 and D2 receptor agonists within the NAcc shell have been reported on locomotor activity (Dreher and Jackson 1989; Essman et al. 1993), jaw movements (Cools et al. 1995) and neuronal firing (White 1987). Similar effects were observed on reward such that SKF 38393 or quinpirole alone were not self-administered into the NAcc shell by rats but a combination of the D1 receptor partial agonist and the

D2 receptor agonist produced a synergistic effect on intra-accumbens self-administration that was later abolished by either SCH 23390 or sulpride, D1 or D2 receptor antagonists, respectively (Ikemoto et al. 1997). Although it is not known whether D1-D2 receptor heteromers are involved in the synergistic effects observed in these studies, the findings suggest that simultaneous stimulation of D1 and D2 receptors produces robust effects.

L-stepholidine, an active ingredient in the Chinese herb Stephania that functions as a D1 receptor partial agonist and D2 receptor antagonist (Jin et al. 2002) or as 41

D1/D2/D3 receptor antagonists (Meade et al. 2015), has been shown to reduce drug- primed (Ma et al. 2014) and cue-induced reinstatement of heroin seeking (Yue et al.

2014) as well as the acquisition, but not the expression, of morphine CPP (Wang et al.

2007). L-stepholidine, when co-administered with L-dopa, blocked the development of

L-dopa induced dyskinesia and reduced already expressed symptoms in 6-OHDA lesioned rats (Mo et al. 2010). In addition, L-stepholidine reduces amphetamine- and

PCP-induced locomotor activity (Natesan et al. 2008). Its analogue, L- tetrahydropalmatine (l-THP), an alkaloid substance extracted from the herbs Stephania and Corydalis, functions as a D1 and D2/3 receptor antagonist and has the ability to reduce cue-, cocaine- and stress-induced cocaine seeking in rats (Figueroa-Guzman et al.

2011; Mantsch et al. 2007) as well as heroin seeking (Yue et al. 2012). In self- administration studies, low to moderate doses (1, 3 and 10 mg/kg) of l-THP increased, but a high dose (20 mg/kg), decreased FR2 responding for cocaine (Xi et al. 2007). L-

THP also reduced BPs in responding for cocaine under a PR schedule of reinforcement

(Mantsch et al. 2010; Xi et al. 2007) and, in a multiple schedule of reinforcement with alternating 30-min FR4 cocaine and 15-min FR4 food reinforcement, produced a rightward and downward shift in the dose-response curve for cocaine self-administration at doses that failed to alter responding for food (Mantsch et al. 2007). In addition l-THP reduces oxycodone (Liu et al. 2009), cocaine and methamphetamine CPP (Su et al.

2013). L-THP has been reported to block the substitution of 7-OH-DPAT for cocaine

(Mantsch et al. 2007) and it lowered cocaine-enhanced BSR at doses that alone failed to increase self-stimulation thresholds (Xi et al. 2007). Together, these studies suggest l-

THP and L-stepholidine can reduce drug seeking and reward through simultaneous 42 antagonism of D1, D2 and D3 receptors.

The synergistic or potentiating effects from simultaneous stimulation of D1-D2 or

D1-D3 receptors seen in in vitro studies do not always translate to in vivo studies. For example, simultaneous microinjections of SKF 38393 or CY208 243 (another D1 receptor agonist) and quinpirole into the NAcc produced at least additive effects on locomotor activity when compared to those induced by the individual agonists (Dreher and Jackson 1989). Co-administration of D1 and D2/3 agonists, SKF 82958 and quinpirole, respectively, did not potentiate the enhancing effect of SKF 82958 on BSR but rather produced effects that resembled those of the individual agonists: enhancement of BSR at low stimulation frequencies (similar to the effect of SKF 82958 alone) and a decrease in BSR at high stimulation frequencies (similar to the effect of quinpirole alone)

(Lazenka et al. 2016). Therefore, more research is needed to fully understand whether the physical and biochemical interactions between DA receptors translate to DA- mediated drug-related behavior.

The existence of D2-D3 receptor heteromers in the striatum has also been reported and is of clinical relevance to treat Parkinson disease, schizophrenia and possibly drug addiction. D2-D3 receptor heteromers have unique functional properties that permit development of compounds with a higher benefit to undesirable properties ratio. In vitro studies with cultured cells provide promising results. D2 receptor partial agonists, aripiprazole, S33592 and N-desmethylclozapine (NDMC), lose their agonistic properties and can act as antagonists to D2 receptors (thus fully inhibit cAMP production) in cells co-expressing excess D3 receptors but not in cells expressing only

D2 receptors or a 1:1 ratio of D2:D3 receptors (Novi et al. 2007). Similarly, 43 antiparkinsonian agents, preferential D3 versus D2 receptor agonists S32504, ropinirole or pramipexole, show amplified potencies to suppress forskolin-stimulated cAMP production in transfected cells COS-7 expressing D2 and D3 receptors compared to cells expressing only D2 or D3 receptors (Maggio et al. 2003). The expression of D2 receptors with a mutant D3 receptor (that cannot signal) restores the ability of D3 receptor agonists to inhibit adenylyl cyclase activity (Scarselli et al. 2001). Thus, DA receptors display different functions in the presence of D2-D3 receptor heteromers such that partial agonists can be transformed into antagonists and preferential D3 over D2 receptor agonists can become more selective for D3 receptors. These interactions may provide possible solutions to develop better compounds that would effectively alleviate

Parkinsonian or psychosis symptoms while minimizing extrapyramidal side effects as often seen with traditional D2 receptor antagonists or L-dopa treatment. Perhaps such compounds could become effective therapeutic agents for drug addiction.

1.12. Current pharmacological strategies and possible directions in drug addiction treatment

Given that the involvement of DA in drug reward and drug seeking has been well documented, interference with the activation of DA receptors has been a main focus of pharmacotherapeutic approaches to drug addiction for the last 3 decades. Compounds that function as selective D1 or D2 receptor antagonists have been effective at reducing drug reward but their potential clinical utility is fairly gloomy due to the adverse side effects that they produce ranging across sedation, anhedonia, motoric incapacity, 44 increases in prolactin production, and the ability of animals and drug users to compensate for reduced rewarding effects of the drug by self-administering the drug more frequently.

Clinical trials with selective D1 or D2 receptor antagonists for the treatment of cocaine addiction have failed for this reason (Haney et al. 2001). Although D1 receptor agonists have been shown to reduce cocaine seeking and cocaine self-administration, the compounds themselves have high abuse potential which limits severely their clinical utility. Compounds with high affinity and selectivity for D3 receptors have provided favorable outcomes in rodents and non-human primate models of drug addiction but their poor bioavailability, short half-life (e.g., SB- 277011A) (Reavill et al. 2000) and propensity to increase blood pressure (e.g., GSK 598809), especially when combined with psychostimulants (Appel et al. 2015), have limited their translation to human studies. Clinical trials with the D3 receptor antagonist, GSK 598809, produced mixed results such that it transiently alleviated craving for cigarettes but increased cigarette consumption and puffs/cigarette when subjects were allowed to smoke freely (Mugnaini et al. 2013) and in healthy individuals GSK 598809 increased prolactin levels.

In recent years the search for improved pharmacotherapeutic agents to treat cocaine addiction has focused some attention on agents that work as partial agonists of the D1 or D3 receptors. Unfortunately, no suitable D1 receptor partial agonists are available for clinical testing at this time due to toxicity and blood brain barrier penetration issues. Given the urgent need for effective anti-cocaine treatments, the discovery of D1-D3 receptor heteromers and the importance of these receptors in drug addiction, the development of agents with more complex polypharmacological profiles and with differential actions at multiple DA receptors seems like a promising avenue to 45 explore.

1.13. Specific Aims

This dissertation is an attempt at exploring D1-D3 receptor interactions in cocaine reward and seeking. In this particular study we investigated whether simultaneous D1 receptor partial agonism and D3 receptor antagonism can reduce cocaine reward and cocaine seeking. We also assessed whether any interaction effects would be additive, potentiating or synergistic. Specific aim 1 of this dissertation was to test the combination of a selective D1 receptor partial agonist, SKF 77434 and a selective D3 receptor antagonist, NGB 2904 on cocaine seeking. Specific aim 2 was to test the effects of the combination of these compounds on cocaine reward.

CHAPTER 2. SIMULTANEOUS D1 RECEPTOR PARTIAL AGONISM AND D3

RECEPTOR ANTAGONISM REDUCE CUE-INDUCED REINTATEMENT OF

COCAINE SEEKING AND CONDITIONED PLACE PREFERENCE

2.1. Introduction

Cocaine, like other drugs of abuse, can activate the brain’s reward circuit to produce rewarding effects (Wise and Rompré 1989) that reinforce behavior (Pickens and

Thompson 1968) and energize an organism to continue to take and seek the drug. As such the rewarding effects of cocaine are thought to play a strong role in its addiction liability (Wise 1996; 2004; Wise and Bozarth 1987; Wise and Rompre 1989). One of the major problems in cocaine addiction is relapse that can be triggered by craving induced by stress (Wallace 1989), the drug itself (Jaffe et al. 1989) or drug-associated cues

(Ehrman et al. 1992). Despite the urgent need for effective treatment, no medications are currently approved by the Food and Drug Administration (FDA) to treat cocaine addiction. Thus, the development of pharmacological agents that can prevent relapse is a focal point in medicinal chemistry and behavioral pharmacology.

The strong influence of drug cues on addiction-related behaviors cannot be underestimated and - to the contrary- should be appreciated. Drug cues can support compulsive drug taking, elicit physiological arousal, prompt craving and trigger relapse

(Childress et al. 1988a; O'Brien et al. 1990). Cues that are associated with drug taking acquire the capacity to increase DA levels in the NAcc (Gratton and Wise 1994; Kiyatkin 47 et al. 1993) and come to function as conditioned rewards (Davis and Smith 1976). As such, drug cues can maintain responding for drug for long periods of time or when work demands are high (e.g., under a second order schedule of reinforcement) (Arroyo et al.

1998). Conditioned cues can retard extinction of cocaine seeking (Ciccocioppo et al.

2004; Ranaldi and Roberts 1996), reinstate cocaine seeking (de Wit and Stewart 1981;

Grimm et al. 2001; Meil and See 1994), elicit cocaine CPP (Bardo et al. 1986; Spyraki et al. 1982) or conditioned hyperactivity and stereotyped behavior (Barr et al. 1983; Tatum and Seevers 1929). In addition, cocaine cues can activate cortical and mesolimbic structures as indicated by functional imaging in addicts (Childress et al. 1999; Garavan et al. 2000; Grant et al. 1996; Maas et al. 1998; Wang et al. 1999; Wexler et al. 2001), electrochemical measurements (Di Ciano et al. 1998; Duvauchelle et al. 2000; Gratton and Wise 1994; Kiyatkin and Stein 1996; Weiss et al. 2000) and cfos expression (Brown and Fibiger 1992; Le Foll et al. 2002), the latter a biomarker for neuronal activation in animals.

The effects of such cues can be blunted by blockade of D1 and D3 receptors. A vast number of pharmacological studies has demonstrated that D1 and D3 receptor partial agonists and antagonists can reduce cocaine reward (Awasaki et al. 1997; Galaj et al.

2014a; McGregor and Roberts 1993; Xi et al. 2006) and cocaine seeking induced by cues

(Galaj et al. 2014a), stress ( Song et al. 2014; Xi et al. 2004) or drug itself (Gilbert et al.

2005; Peng et al. 2009; Vorel et al. 2002; Xi et al. 2006, Green and Schenk 2002;

Norman et al. 1999). Such agents can diminish the capacity of conditioned cues to elicit the expression of cocaine CPP (Cervo et al. 2005; Hachimine et al. 2014; Nazarian et al.

2004; Sanchez et al. 2003), or to maintain CPP (Ashby et al. 2015a, Galaj et al. 2016, 48

Sabioni et al., 2012).

Although the potential of agents that selectively target D1 or D3 receptors is well recognized as promising therapeutics for cocaine addiction, much less is known about the utility of different compounds that have differential functions at multiple DA receptors.

To my knowledge there have been no preclinical studies attempting to validate a treatment with simultaneous D1 receptor partial agonism and D3 receptor antagonism against cocaine-related behaviors. Thus, Specific Aim 1 of this dissertation was to test the effects of the D1 receptor partial agonist, SKF 77434 and the D3 receptor full antagonist, NGB 2904, individually and in combination, on cue-induced reinstatement of cocaine seeking and on expression of cocaine CPP. Given that D1 and D3 receptors play a strong role in cue-induced cocaine-related behaviors and often form heteromers that modulate the intracellular signaling, the combined treatment with NGB 2904 and SKF

77434 was hypothesized to reduce active lever pressing (in the reinstatement experiment) and time spent in the cocaine-paired compartment (in the CPP experiment) to greater degrees than the individual agents would do so alone.

2.2. Methods

2.2.1. Subjects

Animal housing and care conditions were consistent with those specified by the

Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal

Resources on Life Sciences, National Research Council, 2011). The protocols used in the 49 present experiments were approved by the Queens College Institutional Animal Care and

Use Committee.

Subjects (weighing 350-450 g) were male Long Evans rats bred in our facility from males and females obtained from Charles River Laboratories (Kingston, NY). All animals were housed individually and maintained on a reversed 12:12 hour light/dark cycle (lights turned off at 1 PM). Rats weighed between 350-450 g and had free access to food and water. All experiments were conducted during the animal’s active period (dark cycle). Animals were handled for three days prior to experimentation.

2.2.2. Surgery

Surgeries were conducted under atropine sulfate (0.54︎ µg/0.1 ml of distilled water) and sodium pentobarbital [65 mg/kg, by intraperitoneal (IP) injection] anesthesia.

A small incision was made to the right of the midline of the neck. The jugular vein was externalized and opened and a silastic catheter (Dow Corning, Midland, MI) was inserted so that its tip reached the entrance to the right atrium. The catheter was secured to the vein with silk suture and its free end was fed subcutaneously around the back of the neck to exit at the back of the skull. Next, the catheter was connected to a bent 22-gauge stainless steel tube that served as a connector between it and the fluid line; the connector was mounted to the rat’s skull using four stainless steel screws and dental acrylic. To maintain its patency, the catheter was filled with 0.05 ml of heparin (200 U/ml of saline) immediately after surgery and daily thereafter. The rats were given two or three days of recovery before intravenous self-administration began. 50

2.2.3. Apparatus

2.2.3a. Self-administration chambers

Cocaine self-administration sessions were conducted in eight operant conditioning chambers, each measuring 26 cm x 26 cm x 30 cm (l x w x h) and housed in a sound- attenuating ventilated box with a fan that provided a constant source of masking noise.

Each chamber was equipped with two retractable levers, a white light above each lever and a drug line consisting of a metal tether covering a polyethylene tubing which, through a fluid swivel, was connected to a syringe pump (Razel, 3.33 rpm) loaded with a

20 ml syringe.

2.2.3b. Conditioned place preference chambers

Six conditioned place preference test chambers were used, each measuring 43 cm x 43 cm x 30 cm (l x w x h) and placed in a sound-attenuating ventilated box. Each conditioning chamber was equipped with 16 photo emitters and detectors spaced along opposite walls and 6 cm above the floor. These photo-beam detectors tracked the position of the rats in one compartment or the other. Chambers consisted of two distinct compartments (with striped or non-striped walls and stainless steel mesh or grid floors) and were separated by an opening that could be occluded by a guillotine door.

2.2.4. Drugs

Cocaine (a gift from NIDA) was dissolved in 0.9% physiological saline to achieve a dose of 0.75 mg/kg/injection for the self-administration experiment and a dose of 10 mg/kg for the CPP experiment. The selective DA D3 receptor antagonist, NGB 2904

(Tocris, MO, US), was dissolved in 10% cyclodextrin to achieve the doses of 0.25, 1 and

2 mg/kg and the selective D1 partial agonist, SKF 77434 (Tocris, MO, US), was dissolved in saline to achieve the doses of 0.5, 1 and 2 mg/kg.

2.2.5. Procedures

2.2.5. Experiment 1: Cue-induced reinstatement of cocaine seeking

Rats were trained to self-administer cocaine under a FR1 schedule of reinforcement during daily 3-h sessions. Responding on the active lever activated the injection pump for 4.5 s and turned on the light above the active lever for 20 s.

Responding on the inactive lever was counted but had no consequences. Thirteen sessions of stable self-administration were required before beginning the extinction phase during which responding on either lever produced no consequences; cocaine was not delivered and cocaine-related cues (20-s light and 4.5-s syringe pump) were not presented. This phase continued until extinction criteria were met; extinction criteria were defined as 9 or fewer lever presses per hour and less than 21 total lever presses in a

3-h session on the active or inactive levers for 3 consecutive sessions. 52

The cue-induced reinstatement test (120 min long) occurred one day following the last extinction session. Rats were injected with one of the doses of either SKF 77434 (15 min prior to the test session; [vehicle (n=9), 0.5 (n=8), 1 (n=8) or 2 (n=8) mg/kg], NGB

2904 (30 min prior to the test session; [vehicle, 0.25, 1 or 2 mg/kg (n=8 in each)] or a combination of NGB 2904 and SKF 77434 [vehicle/vehicle (n=9), 0.25/0.5 (n=8), 1/0.5

(n=8) or 1/1 (n=8) mg/kg]. Non-contingent cocaine cues (20-s light and 4.5-s syringe pump) were presented twice, 2 min apart, at the beginning of the reinstatement session.

Active lever presses were reinforced with the cocaine cues (light/pump) but cocaine was not delivered. Responding on the inactive lever produced no consequences.

To test the possibility that effects of NGB 2904/SKF 77434 on cue-induced reinstatement were due to motoric deficits we evaluated the effects of NGB 2904/SKF

77434 (1/1 mg/kg) on lever pressing reinforced by food, a procedure that produces many times more lever presses than the reinstatement procedure. Seven rats were trained to lever press for food under a PR schedule of reinforcement, as described in Galaj et al.,

2014a. After stable responding they were treated with NGB 2904/SKF 77434 (1/1 mg/kg) and the number of lever presses during 120 min, the same period of time as in the reinstatement test, was recorded.

2.2.5. Experiment2. Cocaine conditioned place preference

In session 1 – the pre-exposure session – rats were placed in the CPP chamber with free access to both compartments for 15 min and time spent in each compartment was measured. Half of the rats were conditioned with cocaine to their preferred 53 compartment (the compartment in which a rat spent most of its time during the pre- exposure session) and the remaining half to the non-preferred compartment. Prior to sessions 2, 4, 6 and 8 rats were injected with either cocaine or saline (IP) and immediately placed in one of the two compartments. Prior to sessions 3, 5, 7 and 9 rats were injected with the alternate solution and were placed in the other compartment.

Conditioning sessions were 30 min long. The order of cocaine/saline conditioning days was counterbalanced among rats. During session 10 rats were tested for their CPP. Prior to the CPP test rats were injected with a dose of either SKF 77434 (15 min prior to the test [vehicle, 0.5, 1 or 2 mg/kg (n=8 in each)], NGB 2904 (30 min prior to the test

[vehicle, 0.25, 1 or 2 mg/kg (n=8 in each)] or the combination NGB 2904/SKF 77434

[vehicle/vehicle, 0.25/0.5 (n=8), 1/1 (n=8) or 2/2 (n=7) mg/kg]. The dividing partition was removed and rats were placed in the CPP chamber having free access to both compartments for 15 min. Time spent in each compartment was recorded.

2.2.6. Data analysis

For each compound/combination the average number of active and inactive lever presses during the first 120 min of the last three extinction sessions and the number of active and inactive lever presses during the 120-min reinstatement test were compared using a separate three-way analysis of variance (ANOVA) [phase

(extinction/reinstatement) and lever as repeated-measures factors and dose as a between- groups factor]. A significant three-way interaction was followed by dose x phase interaction comparisons at each level of lever and by tests of simple effects of dose at 54 each level of phase. Tests of simple effects of lever at each level of phase were also used to determine reinstatement effect on active lever responding. In the experiment addressing potential motoric deficits, rats treated with the 1/1 mg/kg dose of NGB

2904/SKF 77434 prior to lever pressing for food were compared on lever presses with the vehicle-treated group in the reinstatement experiment using an independent samples t- test.

In Experiment 2, the numbers of seconds spent in the cocaine compartment during the pre-exposure and CPP test sessions were recorded for each dose group in each compound condition (SKF 77434, NGB 2904, and NGB 2904/SKF 77434) and analyzed with three separate two-way ANOVAs with dose as a between-groups factor and phase

(pre-exposure vs preference test) as a repeated-measures factor. A significant interaction was followed by tests of simple effects of phase at each level of dose.

2.3. Results

2.3. Experiment 1: Cue-induced reinstatement of cocaine seeking

During extinction, responding on the active lever was similar among all dose/compound groups with slightly higher pressing on the active than inactive levers in all dose/compound groups (Figures 1 A, B and C, left panels). In all dose/compound groups responding increased in the reinstatement test compared to extinction sessions and the increases on the active lever were greater than on the inactive lever. Rats treated with

0.25, 1 or 2 mg/kg of NGB 2904 showed a slight reduction in responding on the active 55 lever as compared to the NGB 2904 vehicle group (Figure 1 A, right panel). Responding on the inactive lever during the reinstatement test was similar in all NGB 2904 dose groups. Statistical analysis with a three-way ANOVA revealed a significant phase x lever interaction, regardless of the dose group [F(1,28)=39.72; p<.001]. Simple effects of lever at each level of phase revealed a significant lever effect only during the reinstatement test [F(1,28)= 88.7; p<.001].

Similarly, during the reinstatement test rats treated with SKF 77434 showed a slight reduction in active lever presses as compared to the vehicle group (Figure 1B, right panel). Responding on the inactive lever during the reinstatement test was similar in all

SKF 77434 dose groups. A three-way ANOVA revealed a significant phase x lever interaction, regardless of the dose group [F(1,29)=86.39; p<.001]. Simple effects of lever at each phase revealed a significant lever effect during the reinstatement test

[F(1,29)=180.6; p<.001] but not during the extinction phase.

Rats treated with the combination of NGB 2904 and SKF 77434 showed much greater, dose-related reductions in active lever pressing than those treated with either compound alone (Figure 1C right panel). A three-way ANOVA revealed a significant phase x lever x dose interaction [F(3,29)=5.34; p<.01]. Phase x dose interaction comparisons at each level of lever revealed a significant phase x dose interaction for the active lever [F(3,29)=186.89; p<.001] but not for the inactive lever. Tests of simple effect of dose on active lever at each phase revealed a significant dose effect during the reinstatement test [F(3,29)= 44.17; p<.001]. Dunnet’s tests confirmed that the combinations of 1/0.5 and 1/1 mg/kg of NGB 2904/SKF 77434 were significantly different from the NGB 2904/SKF 77434 vehicle group in active lever pressing (ps<.05). 56

Figure 2 shows that rats treated with the 1/1 mg/kg of NGB 2904/SKF 77434 made at least as many, and in fact many times more, lever presses than the vehicle-treated group in the reinstatement experiment during a similar 120-min period. A between- groups t-test showed a significant difference in lever pressing between the groups

[t(14)=-2.96; p<.01].

2.3. Experiment 2: Cocaine conditioned place preference

During the pre-exposure session all groups that would later be treated with one of the doses of SKF 77434, NGB 2904 or the combination of the two spent similar amounts of time in the CPP compartment that would later be paired with cocaine. Rats treated with NGB 2904 prior to the test session spent more time in the cocaine-paired compartment during the test session than they did during the pre-exposure session, regardless of the dose they were treated with (Figure 3 top panel). A similar pattern was observed in rats treated with SKF 77434 such that rats treated with one of the SKF 77434 doses prior to the test session spent more time in the cocaine-paired compartment than they did during the pre-exposure session (Figure 3 middle panel). Two separate two-way

ANOVAs revealed a significant phase effect for NGB 2904 [F(1,28)=85.03; p<.001] and for SKF 77434 [F(1,28)=63.58; p<.05], but no dose x phase interactions for either.

For the combined treatments groups, rats treated with 0/0 or 0.25/0.5 mg/kg of

NGB 2904/SKF 77434 spent more time in the cocaine-paired compartment during the test than they did during the pre-exposure sessions but rats treated with 1/1 or 2/2 mg/kg of NGB 2904/SKF 77434 spent as much time in the cocaine side during the test as they 57 did during pre-exposure (Figure 3 bottom panel). A two-way ANOVA revealed a significant dose x phase interaction [F(3,29)=4.02; p<.05] and tests of simple effects of phase at each level of dose revealed phase effects for only the vehicle/vehicle and

0.25/0.5 mg NGB 2904/SKF 77434 groups [F(1,29)=18.92, p<.01 and F(1,29)=6.81; p<.05, respectively. 58

A 80 Active Lever Inactive Lever 60

20 Figure 1: Mean (± SEM) number of presses on the active presses (120 min) lever Mean and inactive levers averaged 0 across the last three extinction Veh 0.25 1 2 Veh 0.25 1 2 sessions (left panels; in rats B NGB 2904 (mg/kg) grouped by the compound 80 (SKF 77434, NGB 2904 or a Active Lever combination of the two) and Inactive Lever the dose that they would 60 eventually be treated with during the cue-induced reinstatement test) and during the cue-induced reinstatement 40 test (right panels) for all dose and combination groups. A: Animals treated with A: NGB 20 2904, B: SKF 77434 or C: combined NGB 2904/SKF presses (120 min) lever Mean 77434 prior to the start of the 0 Veh 0.5 1 2 reinstatement test. * represents Veh 0.5 1 2 active lever pressing SKF 77434 (mg/kg) significantly different from C 80 vehicle at p < .05. Active Lever Inactive Lever 60

40 * * 20 Mean lever presses (120 min) lever Mean

0 Veh .25 1 1 Veh .25 1 1 Veh .5 .5 1 Veh .5 .5 1 Last 3 days of extinction Reinstatement NGB 2904 SKF 77434 (mg/kg) 59

Vehicle-treated reinstatement group from Figure 1 Food reward group treated with 1 mg NGB 2904/1 mg SKF 77434 250 **

200

150

100

50 Lever presses (120 min) presses (120 Lever

0 Group

Figure 2: Mean (± SEM) number of presses emitted by rats treated with vehicle/vehicle during the reinstatement test and rats responding for food and treated with 1/1 mg/kg of NGB 2904/SKF77434. ** represents a significant difference between groups in lever pressing, p < .01

700

600

500

400

300

200 Time in cocaine- side (s) paired 100

0 Figure 3: Mean (± SEM) Veh .25 1 2 time spent in the cocaine- paired compartment of the NGB 2904 (mg/kg) CPP apparatus during pre- 700 exposure and preference tests for all dose and 600 combination dose groups. 500 Prior to the CPP test animals treated with NGB 400 2904 (Top panel), SKF Pre-exposure 77434 (Middle panel) or 300 Test NGB 2904/SKF 77434 (Bottom panel). * represents 200 the presence of a cocaine CPP. 100 Time in cocaine- (s) chamber paired 0 Veh .5 1 2 SKF 77434 (mg/kg) 700

600 * * 500

400

300

200

Time in cocaine- (s) chamber paired 100

0 Veh .25/.5 1/1 2/2 NGB 2904/SKF 77434 (mg/kg) 61

2.4. Discussion

The present study evaluated the effects of individual and simultaneous D1 receptor partial agonism and D3 receptor antagonism on cue-induced cocaine seeking and cocaine CPP. While NGB 2904 or SKF 77434 alone produced no significant changes in cue-induced reinstatement the simultaneous administration of the compounds caused a dose-dependent reduction in active lever pressing. The combined agents did not significantly affect responding on the inactive lever. Furthermore, rats treated with the

NGB 2904/SKF 77434 combination and responding for food were able to emit at least as many, and in fact emitted significantly more, lever presses than the vehicle-treated group in the reinstatement experiment during a similar 120-min period. This suggests that reductions in lever pressing in NGB 2904/SKF 77434 reinstatement groups were not due to motoric deficits in lever pressing. Thus, the effect of combined NGB

2904/SKF 77434 treatment on cue-induced reinstatement is best understood as a reduction in cocaine seeking. In the CPP experiment, rats were treated with the same compounds alone or combined prior to the CPP test. Again, these doses of NGB 2904 or

SKF 77434 alone failed to significantly affect cocaine CPP (i.e., all groups with NGB

2904 or SKF 77434 alone treatments and their respective vehicle treatments showed significant CPPs). However, when these doses of the compounds were administered simultaneously they abolished the cocaine CPP.

The results from the present study suggest that simultaneous D1 receptor partial agonism and D3 receptor antagonism can reduce the reward value of the conditioned cues resulting further in the reduction of cocaine seeking and the expression of an established 62 cocaine CPP. D3 receptor partial agonists and antagonists can reduce the expression of cocaine (Cervo et al. 2005; Hachimine et al. 2014; Micheli et al. 2007; Song et al. 2013;

Vorel et al. 2002), heroin (Ashby et al. 2003; Galaj et al. 2015), morphine (Hu et al.

2013), nicotine (Micheli et al. 2007; Pak et al. 2006) and methamphetamine CPP (Sun et al. 2016). D1 receptor partial agonists and antagonists produce mixed results: they either reduce the expression of stimulant CPP (Galaj et al. 2014b; Hiroi and White 1991; Liao et al. 1998) or have no effect (Adams et al. 2001; Cervo and Samanin 1995; Liao et al.

1998). In addition, D1 receptor antagonists (Alleweireldt et al. 2002a; Crombag et al.

2002), (Liu et al. 2010) (Bossert et al. 2007; Shaham and Stewart 1996) as well as D3 receptor antagonists (Cervo et al. 2007; Chen et al. 2014; Galaj et al. 2014a; Galaj et al.

2015; Gilbert et al. 2005; Higley et al. 2011a; Roman et al. 2013; Song et al. 2014) can reduce cue-induced drug seeking. Thus, it appears that D1 and D3 receptors are necessary for drug cues to elicit drug seeking or CPP (in the absence of the drug). The results of the present study suggest that the combined treatment of D1 receptor partial agonist and D3 receptor antagonist can block the effects of drug cues.

CHAPTER 3. THE INTERACTION OF D1 AND D3 RECEPTORS IN COCAINE

REWARD

3.1. Introduction

In the search for efficacious pharmacotherapies to treat cocaine addiction much attention has been given to agents targeting D1 or D3 receptors because of their involvement in cocaine-related behaviors. D1 and D3 receptor antagonists have been shown to reduce cocaine reward (Awasaki et al. 1997; Galaj et al. 2014a; McGregor and

Roberts 1993; Xi et al. 2006), reinstatement of cocaine seeking (Alleweireldt et al.

2002b; Anderson et al. 2003; Capriles et al. 2003; Cervo et al. 2007; Galaj et al. 2014a;

Vorel et al. 2002; Xi et al. 2004) and cocaine induced locomotor activity (Adams et al.

2001; Galaj et al. 2014a). In addition, D1 and D3 receptors play a role in cocaine CPP

(Cervo et al. 2005; Hachimine et al. 2014; Nazarian et al. 2004; Sanchez et al. 2003).

However, translation of these encouraging preclinical results with selective D1 or D3 receptor agents into clinical efficacy has been limited due to a number of factors the include toxicity, poor pharmacokinetic properties and increased prolactin production, high blood pressure and sedative side effects. One of the major challenges in discovering effective anti-cocaine pharmacological therapeutics is to block DA signaling selectively in the mesolimbic reward system. Many traditional DA receptor antagonists that are effective at reducing cocaine-related behaviors often produce undesirable extrapyramidal side effects due to D2 receptor blockade (Lublin et al. 1993; Peacock et al. 1999; Platt et al. 2002). Given the restricted distribution of D3 receptors to the 64 mesolimbic regions, such as the NAcc shell, Islands of Calleja, and olfactory tubercle

(Sokoloff et al., 1990; Diaz et al., 1995, 2000), a search for highly selective D3 receptors antagonists has become a major focus in the development of anti-cocaine pharmacotherapies.

Of clinical relevance is the development of D1 receptor partial agonists as potential therapeutics because of the interesting feature that they can function as D1 receptor antagonists under conditions of high dopamine levels, such as in the presence of cocaine or cocaine cues, and they can reduce cocaine-related behaviors. D1 receptor partial agonists alter cocaine self-administration (Caine et al. 1999; Katz and Witkin

1992; Mutschler and Bergman 2002; Platt et al. 2001; Spealman et al. 1997), cocaine discriminative stimulus effects (Platt et al. 2000) and facilitate the extinction of cocaine

CPP (Sabioni et al. 2012). D1 receptor partial agonists are particularly interesting because, unlike traditional DA receptor antagonists, they do not produce extrapyramidal side effects (Platt et al. 2000) and exhibit low abuse potential [(Grech et al. 1996; Weed and Woolverton 1995) but see (Self and Stein 1992)].

D1 and D3 receptors often form heteromers that have distinct pharmacological and cell-signaling properties (Fiorentini et al. 2008; Marcellino et al. 2008). Given the role of D1 and D3 receptors in cocaine-related behaviors, and the unique location of D1-

D3 heteromers, such heteromers are likely to play a role in drug addiction and might be of pharmacotherapeutic relevance. Perhaps a treatment that combines agents that have differential functions at multiple DA receptors might produce potentiating effects on the capacity to reduce cocaine reward. 65

To my knowledge there have been no studies evaluating simultaneous D1 receptor partial agonism and D3 receptor antagonism on cocaine self-administration.

Specific Aim 2 of this dissertation was to test the effects of the D3 receptor antagonist,

NGB 2904, and the D1 receptor partial agonist, SKF 77434, individually and in combination, on cocaine reward. The combined treatment was hypothesized to reduce

BP in responding for cocaine under a PR schedule of reinforcement to a greater degree than the individual compounds would do so alone.

3.2. Methods

3.2.1. Subjects

Subjects were male Long Evans rats bred in our facility from males and females obtained from Charles River Laboratories (Kingston, NY). The animals were housed individually with free access to food (Purina Rat Chow) and water and maintained on a reverse 12 h light: 12 h dark cycle (lights were turned on at 6 pm). Self-administration sessions were conducted during the animals’ active period (the dark cycle).

3.2.2. Surgery

Prior to jugular vein catheterization each animal, weighing between 350 and 450 g, was injected intraperitoneally (IP) with atropine sulfate and anesthetized with sodium pentobarbital (65 mg/kg, IP). A small incision was made on the neck just to the right of 66 the midline. The right jugular vein was exposed by blunt dissection and a catheter (Dow

Corning, Midland, MI) was inserted into the vein and secured into place by tying sutures around it and the vein. The distal portion of the catheter was passed subcutaneously to the top of the skull, where it exited into a connector (a modified 22-gauge cannula) mounted on the skull with four stainless screws and dental acrylic. The catheter’s patency was maintained by filling it with a heparin saline solution (200 U/ml) immediately after surgery and daily thereafter. Self-administration training began 2 or 3 days after surgery.

3.2.3.Apparatus

Self-administration sessions were conducted in eight operant conditioning chambers, each measuring 26 cm x 26 cm x 30 cm (l x w x h). Each chamber was situated inside a sound-attenuating box and was equipped with two retractable levers, a white light above each lever, and a tether protecting polyethylene tubing that connected the animal, through a fluid swivel, to a syringe in a syringe pump (Razel, 3.33 rpm).

3.2.4. Drugs

Cocaine (a gift from NIDA) was dissolved in 0.9% physiological saline to achieve a dose of 0.75 mg/kg/injection. The selective DA D3 receptor antagonist, NGB 2904

(Tocris, MO, US), was dissolved in 10% cyclodextrin to achieve the doses of 1, 2 and 4 67 mg/kg and the selective D1 receptor partial agonist, SKF 77434 (Tocris, MO, US), was dissolved in saline to achieve the doses of 1, 2 and 4 mg/kg.

3.2.5. Procedure.

Experiment 3: Cocaine self-administration under a PR schedule of reinforcement

Animals were trained to self- administer cocaine (0.75 mg/kg/injection) initially under a FR1 schedule of reinforcement during daily 3-hour sessions. Responding on an active lever activated the syringe pump for 4.5 s causing the intravenous delivery of cocaine and the onset of the white light above the active lever for 20 s. Responses on an inactive lever were counted but had no consequences. The active and inactive levers

(left/right) were counterbalanced across animals and remained constant for the duration of the experiment. After the animals demonstrated a steady rate of self-administration for

3 consecutive sessions they were placed on a PR schedule of reinforcement where all PR sessions–training and tests–were 5 h long. The PR schedule required the rats to emit progressively more lever presses (1, 2, 4, 6, 9, 12, 15, 20…) in order to obtain successive infusions during a session. Under this schedule, the requirement for lever pressing becomes so high that eventually the animal stops responding and reaches a break point

(BP). The BP was operationally defined as the total number of infusions earned within an hour of the previous infusion.

Once rats demonstrated a steady baseline BP (three consecutive sessions during which BPs that did not differ by more than 2 ratio steps without descending or ascending 68 trends), they were tested with a dose of either SKF 77434 [vehicle, 1, 2 or 4 mg/kg (n=9 in each)], NGB 2904 [vehicle, 1, 2 or 4 mg/kg (n=7 in each)] or a combination of NGB

2904/SKF 77434 [vehicle/vehicle, 1/1, 2/2 or 4/4 mg/kg (n=9)]. Each rat assigned to a compound group was tested with all doses of the compound in random order, each test occurring after baseline BP was re-established.

3.2.6. Data analysis

Baseline BP was the average of three consecutive sessions (prior to the test session) during which BPs did not differ by more than 2 ratio steps without descending or ascending trends. For each compound condition BPs during the test session were compared to the baseline BPs for all doses using a two-way ANOVA with dose as a between-groups factor and phase (baseline versus test) as a repeated measures factor.

Significant interactions were followed by tests of simple effects of phase at each dose.

3.3. Results

Experiment 3: Cocaine self-administration under a PR schedule of reinforcement

(cocaine reward)

During baseline testing all dose/compound groups reached similar BPs. Rats treated with NGB 2904 showed no change in the BP, regardless of the dose they were treated with (Figure 4 top panel). Rats treated with vehicle or 1 mg/kg of SKF 77434 69 reached similar BPs during the test and baseline sessions whereas rats treated with 2 or 4 mg/kg of SKF 77434 showed reductions in BP compared to baseline (Figure 4 middle panel). Two separate two-way ANOVAs revealed no significant dose x phase interaction or main effects for NGB 2904 but a significant dose x phase interaction for SKF 77434

[F(3,24) 5.30, p<.05]. Tests of simple effects of phase at each SKF 77434 dose revealed a significant phase effect for 2 and 4 mg/kg doses [F(1,24)=5.45 and F(1,24)=32.04, respectively; ps < .05]. Similarly, rats treated with NGB 2904/SKF 77434 vehicle reached a similar BP during the test session as they did during the baseline sessions whereas rats treated with 1/1, 2/2 or 4/4 mg/kg of NGB 2904/SKF 77434 showed reductions in BPs on the test day compared to baseline. A two-way ANOVA revealed a significant NGB 2904/SKF 77434 dose x phase interaction [F(3,24)= 3.32; p<.05]. Tests of simple effect of phase at each NGB 2904/SKF 77434 dose revealed a significant phase effect for the 1/1, 2/2 and 4/4 mg/kg dose groups [F(1,24)=21.67, F(1,24)=7.97 and

F(1,24)=14.15, respectively; ps < .05].

Interestingly, rats treated with 1/1 mg/kg of NGB 2904/SKF 77434 emitted on average 578.22 (± 177.65) lever presses within the first two hours of the PR test session

(data not shown), demonstrating that they could make at least as many, and in fact many times more, lever presses than the vehicle-treated group (63.44 ± 10.54) in the reinstatement experiment during a similar 120-min period.

18 70 16

6 Break Point Break 4

0 Veh 1 2 4 Figure 4: Mean (± SEM) BPs 18 NGB 2904 (mg/kg) reached during the baseline and test sessions for all dose and 16 * * combination dose groups. Prior 14 to the start of the test session animals were treated with NGB 12 2904 (Top panel), SKF 77434 10 (Middle panel) or NGB 2904/SKF 77434 (Bottom 8 Baseline panel). * represents a Test 6 significant reduction in BPs in Point Break the test session compared to 4 baseline (ps < .05). 2 0 Veh 1 2 4 SKF 77434 (mg/kg)

16 * * *

10 8 * 6 Break Point Break

0 Veh 1/1 2/2 4/4 NGB 2904/SKF 77434 (mg/kg) 71

3.4. Discussion

In the current study the D3 receptor antagonist, NGB 2904, failed to disrupt responding for cocaine under a PR schedule of reinforcement whereas the D1 receptor partial agonist, SKF 77434 reduced BPs. The combined treatment of NGB 2904 and

SKF 77434 also reduced BPs. Interestingly, a significant reduction in BP was achieved with combined low doses of SKF 77434 and NGB 2904 that themselves were ineffective.

The present findings are in accord with previous studies demonstrating that D1 receptor antagonists and partial agonists can reduce cocaine reward (Barrett et al. 2004;

Britton et al. 1991; Caine and Koob 1994a; Caine et al. 1999; Corrigall and Coen 1991a;

Depoortere et al. 1993; Hubner and Moreton 1991; Koob et al. 1987; McGregor and

Roberts 1993; 1995; Ranaldi and Wise 2001). There have been inconclusive reports on the role of D3 receptors in reward. D3 receptor antagonists have been shown to block stimulant-enhanced BSR (Pak et al. 2006; Spiller et al. 2008; Vorel et al. 2002; Xi et al.

2006) and can reduce BPs in PR responding for drug (Galaj et al. 2014a; Song et al.

2011; Xi and Gardner 2007; Xi et al. 2005) but generally have had no effect on drug self- administration under a FR schedule of reinforcement (Andreoli et al. 2003; Caine et al.

2012; Gal and Gyertyan 2003; Higley et al. 2011a; Khaled et al. 2010; Xi et al. 2006).

The data from the current study suggest that a D1-D3 receptor interaction plays a role in cocaine reward and a selective D1 receptor partial agonist and a selective D3 receptor antagonist can produce robust anti-cocaine effects in animals (see General Discussion for more discussion of this).

CHAPTER 4. GENERAL DISCUSSION

The aims of this dissertation were to evaluate the effects of individual and simultaneous D1 receptor partial agonism and D3 receptor antagonism on cocaine self- administration and cue-induced cocaine seeking. The tested doses of SKF 77434, a partial D1 receptor agonist, or NGB 2904, a D3 receptor antagonist, alone failed to block cue-induced reinstatement while the simultaneous administration of these compounds caused a dose-dependent reduction in active lever pressing. None of the treatments produced significant effects on inactive lever responding. Furthermore, the reduction in lever pressing cannot be explained as resulting from incapacitation of the lever press response since rats treated with the NGB 2904/SKF 77434 combination and responding for food or cocaine under a PR schedule of reinforcement were able to emit at least as many, and in fact emitted significantly more, lever presses than the vehicle-treated group

(and also much more than the 1/1 mg/kg NGB 2904/SKF 77434 combination group) in the reinstatement experiment during a similar 120-min period. This demonstrates that the animals under this treatment (NGB 2904/SKF 77434 combination) in the reinstatement experiment were motorically capable of emitting many more responses than they did, and actually more than their corresponding vehicle group. Thus, the best explanation for reduced responding during the cue-induced reinstatement test is that combined NGB

2904/SKF 77434 treatment reduced cocaine seeking; that is, it reduced incentive motivation for cocaine. In Experiment 2 NGB 2904 or SKF 77434 when administered individually failed to produce significant effects cocaine CPP. However, co- administration of NGB 2904 and SKF 77434 reduced dose-dependently time spent in the cocaine-paired compartment. In fact, the combination of higher, individually-ineffective 73 doses of the compounds completely abolished cocaine CPP. Again, these results suggest that the combined D1 receptor partial agonist and D3 receptor antagonist treatment reduced the incentive motivational effects of cocaine cues.

Interestingly, in both the reinstatement and CPP experiments, the combination of the two compounds appeared to produce an interactive, synergistic effect. For instance, when the compounds were administered alone the 1 mg/kg doses failed to produce effects

(for both reinstatement and CPP) and the next highest dose – the 2 mg/kg dose – also failed to produce effects. If the effect of combining these 1 mg/kg doses was additive one might expect to observe effects similar to the next higher doses of each compound alone. Instead, the combination of these doses (the 1/1 mg/kg combination) greatly surpassed effects observed at the next higher dose levels and produced highly significant effects. This finding suggests that the effects of the combined agents are synergistic rather than additive. This is a unique finding given the fact that animal research relevant to cocaine addiction has focused primarily on targeting selectively D1 or D3 receptors.

The data from the current study suggest that combining a selective D1 receptor partial agonist and a D3 receptor antagonist may synergize to produce more robust effects on cocaine seeking.

In the self-administration study the co-administration of NGB 2904 and SKF

77434 caused a significant reduction in PR responding for cocaine; something that was also observed with SKF 77434 treatment but not with NGB 2904 alone. All of the combined doses reduced BPs whereas in the case of SKF 77434 alone only the two highest doses produced significant effects. Interestingly, the lowest dose of SKF 77434 alone, which was ineffective, reduced BP by 25% when it was combined with the lowest, 74 and ineffective, dose of NGB 2904. This is noteworthy because it suggests that a minimal amount of D3 receptor antagonism can potentiate the effects of D1 receptor partial agonism and reduce cocaine reward.

D3 receptor antagonists and partial agonists have been shown to have limited effects on stimulant self-administration under FR schedules of reinforcement (Andreoli et al. 2003; Chen et al. 2014; Gal and Gyertyan 2003; Higley et al. 2011a; Higley et al.

2011b; Khaled et al. 2010; Martelle et al. 2007b; Xi et al. 2005) but efficiently reduce PR responding for drug (Chen et al. 2014; Galaj et al. 2014a; Higley et al. 2011a; Higley et al. 2011b; Song et al. 2011; Xi and Gardner 2007; Xi et al. 2005) and drug-enhanced

BSR (Pak et al. 2006; Spiller et al. 2008; Vorel et al. 2002; Xi et al. 2006). Because of seemingly large discrepancies in findings, the role of D3 receptors in drug reward remains inconclusive. Conversely, involvement of D1 receptors in drug reward is well established. D1 receptor partial agonists and antagonists can reduce (Nakajima and

O'Regan 1991) while D1 receptor agonists facilitate (Carr et al. 2001; Gilliss et al. 2002;

Lazenka et al. 2016; Ranaldi and Beninger 1994) BSR. Systemic or intra-mesolimbic injections of D1 receptor partial agonists and antagonists can increase stimulant self- administration under FR schedules of reinforcement (Bari and Pierce 2005; Barrett et al.

2004; Britton et al. 1991; Caine et al. 1995; Caine et al. 1999; Epping-Jordan et al. 1998;

Hubner and Moreton 1991; Koob et al. 1987; Maldonado et al. 1993; McGregor and

Roberts 1993; 1995; Mutschler and Bergman 2002; Quinlan et al. 2004) and reduce BPs in PR responding (Depoortere et al. 1993; Hubner and Moreton 1991; McGregor and

Roberts 1993; 1995; Ranaldi and Wise 2001). All in all, these findings suggest that drug reward is mediated primarily by D1 receptors and less so by D3 receptors. The results of 75 the current self-administration studies indicate that the D1 receptor partial agonist produces more robust reductions in cocaine reward when it is co-administered with a D3 receptor antagonist. Therefore, it is being proposed that the rewarding effects of cocaine are possibly mediated by D1-D3 receptor interactions.

D1-D3 receptor interactions may not only play a role in cocaine reward but also in cocaine seeking. In support of this claim are the findings that D1 and D3 receptor antagonists can reduce reinstatement of drug seeking triggered by stress

(Capriles et al. 2003; See et al. 2001; Song et al. 2014; Tobin et al. 2009; Xi et al. 2004), drug (Gilbert et al. 2005; Green and Schenk 2002; Higley et al. 2011b; Peng et al. 2009;

Schenk et al. 2011; Shaham and Stewart 1996; Vorel et al. 2002; Xi et al. 2006) or cues

(Alleweireldt et al. 2002a; Bossert et al. 2007; Cervo et al. 2007; Crombag et al. 2002;

Galaj et al. 2014a; Galaj et al. 2015; Gilbert et al. 2005; Liu et al. 2010; Roman et al.

2013; Song et al. 2014). In addition, under second order schedules of reinforcement, which measure drug reward as well as the role of cues in drug-seeking, D1 and D3 receptor antagonists and partial agonists reduce responding during a drug-free interval

(i.e., before the drug of abuse is self-administered) and reinstatement of responding in the presence of drug cues (i.e., when extinguished responding under this schedule is restored)

(Claytor et al. 2006; Di Ciano et al. 2003; Khroyan et al. 2000; Khroyan et al. 2003;

Martelle et al. 2007b; Mashhoon et al. 2009). Such findings suggest that D1 and D3 receptors play an important role in drug seeking.

In a number of studies, D1 receptor agents reduced the acquisition of stimulant and opiate-induced CPP (Acquas et al. 1989; Acquas and Di Chiara 1994; Akins et al.

2004; Cervo and Samanin 1995; Hiroi and White 1991; Hoffman and Beninger 1989; 76

Nazarian et al. 2004; Pruitt et al. 1995) but produced differential effects on the expression of CPP: they either blocked (Galaj et al. 2014b; Hiroi and White 1991; Liao et al. 1998;

Rezayof et al. 2003; Zarrindast et al. 2003) or had no effect (Adams et al. 2001; Cervo and Samanin 1995; Fenu et al. 2006; Kramar et al. 2014; Liao et al. 1998). In contrast,

D3 receptor agents either blocked (Ashby et al. 2003; Duarte et al. 2003; Song et al.

2013; Vorel et al. 2002) or failed to block the acquisition of CPP (Gyertyan and Gal

2003; Hu et al. 2013; Sun et al. 2016) but reduced the expression of stimulant- (Aujla and

Beninger 2005; Cervo et al. 2005; Hachimine et al. 2014; Song et al. 2013; Vorel et al.

2002) and opiate-induced CPP (Ashby et al. 2003; Frances et al. 2004a; Galaj et al. 2015;

Hu et al. 2013). In addition, D1 and D3 receptor antagonists reduced reinstatement of

CPP (Ashby et al. 2015b; Hu et al. 2013; Rice et al. 2013; Sanchez et al. 2003; Sun et al.

2016). Thus, it appears that stimulation of D1 or D3 receptors is necessary for the effects of cues to elicit a conditioned response. Here it is reported that co-administration of a D1 receptor partial agonist and a D3 receptor antagonist produces synergistic effects in reduction of cue-induced cocaine seeking and CPP.

It has long been established that reward cues, including drug cues, activate the

DA mesocorticolimbic system (Blackburn and Phillips 1989; Brown and Fibiger 1992;

Duvauchelle et al. 2000; Kiyatkin and Stein 1996). DA neurons fire in response to drug- related cues (Kiyatkin and Rebec 2001), a result of which DA is released in the NAcc and

PFC (Bassareo et al. 2007; Bassareo et al. 2011; Gratton and Wise 1994; Ito et al. 2002;

Kiyatkin and Stein 1996). Cortical and mesolimbic regions are activated by drug cues as evidenced by the expression of immediate-early genes in response to drug cue exposure

(Koya et al. 2006; Le Foll et al. 2002) but inhibited under D1 or D3 receptor partial 77 agonism or antagonism treatment (Ciccocioppo et al. 2001; Le Foll et al. 2002).

Functional imaging studies with heroin or cocaine addicts also show the activation of these regions in response to drug cues (Childress et al. 1999; Garavan et al. 2000; Grant et al. 1996; Li et al. 2012; Maas et al. 1998; Sell et al. 1999; Wang et al. 1999; Wexler et al. 2001). As mentioned previously, drug cues are major inducers of craving and relapse in addicts (Childress et al. 1988a; Childress et al. 1988b; O'Brien et al. 1990; O'Brien et al. 1984; Sherman et al. 1989) and their effects can be diminished by D1 or D3 receptor agents. In clinical trials, smokers reported decreased subjective craving under the treatment of GSK598809, a D3 receptor antagonist; though these effects were not maintained at later time points (i.e., after overnight abstinence) (Mugnaini et al. 2013).

Another compound, l-THP that functions as an antagonist to D1, D2 and D3 receptors, has been reported to reduce craving and relapse rates in heroin addicts (Yang et al. 2008).

Likewise, the selective DA D1 receptor agonist, ABT-431, can decrease subjective evaluation of smoked cocaine effects and to some extent craving; though its effects on cocaine self-administration appear limited (Haney et al. 1999).

Drug cues can produce rewarding and motivational effects that drive drug-related behaviors and such effects appear to be mediated through D1 and D3 receptors. In a number of animal models of drug abuse and addiction drug cues have been shown to maintain responding for drug for prolonged periods (Arroyo et al. 1998); induce reinstatement of drug seeking, CPP, hyperactive and stereotyped behaviors (Barr et al.

1983; Tatum and Seevers 1929). The effects of cues are limited under treatment with D1 or D3 receptor antagonism.

The findings of the present study are significant and warrant further investigation 78 because of several reasons. First, it is suggested that the observed effects of NGB

2904/SKF 77434 on cue-induced cocaine seeking and CPP might occur through inhibition of drug-cue induced enhancement of mesolimbic DA activity. This adds support for the idea that cocaine cues, just like natural rewards (e.g., food, sex)

(Ettenberg and Camp 1986; Gerber et al. 1981; Hernandez and Hoebel 1988; Pfaus et al.

1995) and drugs of abuse, mediate at least some of their behavioral effects through enhancement of DA neurotransmission in the mesocorticolimbic DA system. Second, the present results suggest that simultaneous stimulation of D1 and D3 receptors may act together to potentiate cocaine’s rewarding effect and cues to induce drug seeking. These unique interactions should be explored further to help us understand the underlying neural mechanisms of addiction. Chronic exposure to cocaine can lead to neuroplastic changes in the brain such as dendritic spine formation in D1- and D2-receptor-containing

MSNs of the NAcc (Lee et al. 2006), an increase in D1 receptor excitability (Pascoli et al.

2011) and the enhanced expression of D1 receptor mRNA in mesocorticolimbic regions

(Laurier et al. 1994). In addition, the upregulation of D3 receptors is often present in cocaine-exposed brains of addicts (Segal et al. 1997; Staley and Mash 1996) and animals

(Le Foll et al. 2002; Neisewander et al. 2004). For example, postmortem studies show that cocaine-overdosed individuals have an enhanced expression of DA D3 receptor mRNA in the NAcc by a 6-fold magnitude (Segal et al. 1997; Staley and Mash 1996).

Individuals with cocaine dependence have a higher level of D3 receptor availability in the substantia nigra, amygdala and hypothalamus than healthy individuals with no history of substance abuse as indicated in PET imaging studies with the D3 receptor-preferring radioligand [(11)C](+)PHNO (Matuskey et al. 2014; Payer et al. 2014). Perhaps then the 79 upregulation of D3 receptors related to chronic cocaine exposure enhances D1 receptor- mediated signaling that would promote motivation to work for drug reward in a progressive ratio task and associative learning about drug cues as seen in a CPP experiment. DA has lower affinity for D1 receptors than D3 receptors but D1- and D2- receptor-containing neurons in the NAcc show supersensitivity to low doses of DA ligands after chronic exposure (Henry et al. 1989). Exposure to drug cues will enhance

DA neurotransmission in the NAcc that consequently will lead to stimulation of supersensitive D1 receptors as well as occupancy of D3 receptors that are overexpressed.

Thus, cocaine-related neuroadaptations in the dopaminergic system may explain why drug cues become powerful inducers of drug seeking.

Third, the present study suggests that simultaneous D1 receptor partial agonism and D3 receptor antagonism produce robust effects on cocaine-related behaviors and might be of therapeutic relevance. This novel approach might be an effective strategy to prevent relapse and reduce cocaine self-administration in addicts.

It is not clear whether the individual compounds of the combined treatment begin their effects through separate mechanisms producing cascades of events that converge at a later point in the neural circuitry or whether they initiate their effects through a common starting point. Perhaps these robust effects, as seen in the present study, are related to the physical D1-D3 receptor interaction occurring within striatal MSNs where

D1 and D3 receptors are expressed. D1 and D3 receptors can form heteromers that promote changes in the ability of ligands to bind to the specific receptors and modulate intracellular signaling cascades (Fiorentini et al. 2008; Guitart et al. 2014; Marcellino et al. 2008). Their unique location and biochemical properties suggest that D1-D3 receptor 80 heteromers might play an important role in addiction and reward-related learning.

Surprisingly, only a few studies have investigated the role of D1-D3 receptor interactions in behavior. D3 receptor stimulation results in the potentiation of D1 receptor-mediated locomotor activity, an effect that was counteracted by a D3 receptor antagonist

(Marcellino et al. 2008). Deletion of D3 receptors increases sensitivity to D1 and D2 receptor agonists in mice (Xu et al. 1997) whereas deletion of both D1 and D3 receptors impairs CPP with low doses of cocaine and alters locomotion (Karasinska et al. 2005). In addition, with chronic L-dopa treatment there is an increase in the expression of D1 and

D3 receptor heteromers that potentiate the striatal D1 receptor sensitivity to DA (Farre et al. 2015). Supersensitivity of D1 receptors has been linked to an increase in D1 receptor- mediated cAMP signaling and emergence of dyskinesia (Feyder et al. 2011), symptoms which can be attenuated by D3 receptor partial agonists and antagonists (Bezard et al.

2003; Visanji et al. 2009). It appears then that heteromerization with D3 receptors changes D1 receptors’ ability to bind ligands and facilitates intracellular signaling mediated through these receptors. As such, D1-D3 receptor interactions play an important role in behaviors controlled by the striatum.

The development of L-dopa-induced dyskinesia and its already expressed symptoms can be blocked by L-stepholidine, a D1 receptor partial agonist and D2 receptor antagonist that can also function as a D1/D2/D3 receptor antagonist. In fact, L- stepholidine has been shown to reduce stimulant-induced locomotor activity (Natesan et al. 2008), heroin seeking (Ma et al. 2014; Yue et al. 2014) and to block the acquisition, but not the expression, of morphine CPP (Wang et al. 2007). Its analogue, L- tetrahydropalmatine (l-THP), that functions as a D1 and D2/3 receptor antagonist, has the 81 ability to reduce cocaine reward as demonstrated in self-administration and BSR studies

(Mantsch et al. 2010; Xi et al. 2007) as well as reduce cocaine (Figueroa-Guzman et al.

2011; Mantsch et al. 2007) or heroin seeking (Yue et al. 2012). In addition, l-THP has been reported to block psychostimulant CPP (Liu et al. 2009; Su et al. 2013) and the substitution of 7-OH-DPAT for cocaine (Mantsch et al. 2007). Together, these studies suggest that simultaneous D1 receptor partial agonism and D2/D3 receptor antagonism

(or antagonism of D1/D2/D3 receptors) reduce drug seeking and reward. The results of the present studies suggest that simultaneous D1 receptor partial agonism and D3 receptor antagonism produce synergistic effects in reduction of cocaine seeking and potentiate the reduction of cocaine reward by D1 receptor partial agonism. It is now necessary to delineate the neurophysiological and neurochemical mechanisms whereby the two receptors do interact to produce the synergistic and potentiating effects on cocaine seeking and reward observed in the present studies.

4.1. Conclusions

In conclusion, the present studies demonstrated that the simultaneous administration of a D1 receptor partial agonist and D3 receptor antagonist reduces cue- induced reinstatement, cocaine CPP and response BP to a significantly greater degree than the individual agents alone. The mechanisms underlying this D1-D3 receptor synergism are unknown, begging further investigation. The results of the present studies support the notion of complex functional interactions between D1 and D3 receptors and suggest that treatments that simultaneously target D1 receptors with partial agonism and 82

D3 receptors with antagonism might be highly effective for cocaine addiction. Given the urgent need for effective anti-cocaine treatments, it is important that we continue to try innovative strategies, like the one reported here. The present results indicate that polypharmacotherapy simultaneously targeting D1 and D3 receptors might be a very efficacious treatment for cocaine addition.

4.2.Implications and future directions

The complexities of the behavioral and neurobiological factors underlying cocaine addiction create enormous challenges in efforts to develop effective pharmacological treatments. Traditional approaches that have focused on blocking selective DA receptors provide encouraging results in animal models of addiction but the clinical utility of these agents is limited due to the side effects they produce. Some of these side effects include liver toxicity, poor bioavailability, sedation, anhedonia, motoric incapacitation, prolactin hypoproductivity and hypertension. Clearly, new approaches are needed.

A novel polypharmacological approach of combining a D1 receptor partial agonist and D3 receptor antagonist, as proposed and studied here, provides distinct therapeutic advantages. First, the goals of treatments for cocaine addiction are to control impulsive drug taking, attenuate the rewarding (and reinforcing) effects of cocaine, devalue drug cues, and, very importantly, prevent craving and relapse. The current study suggests that a D1 receptor partial agonist when combined with a D3 receptor antagonist can attenuate cocaine reward, block the effects of cues and reduce cocaine seeking. Thus, this 83 combined treatment appears to target many aspects of addiction and, importantly, produces robust effects. Second, in our animal models of cocaine addiction, NGB

2904/SKF 77434 was effective at doses that failed to produce motoric impairment. This is especially encouraging given the fact that traditional D1 and D2 receptor antagonists in doses that reduce cocaine-related behaviors also produce undesirable extrapyramidal side effects mostly due to blockade of D2 receptors (Lublin et al. 1993; Peacock et al. 1999;

Woolverton 1986). Third, D3 receptor antagonists have limited usefulness in clinical settings because of their propensity to increase blood pressure, especially when combined with psychostimulants (Appel et al. 2015). However, D1 receptor partial agonists have been known to decrease high blood pressure (Ventura et al. 1984) and some are FDA- approved (e.g., Fenoldopam) to treat hypertension. For that reason, the combined treatment of a D1 receptor partial agonist and D3 receptor antagonist might have counteracting effects in regard to blood pressure, which would make this novel approach safer and more advantageous in the clinical setting. Future studies should directly address concerns of blood pressure, liver toxicity and sedation with this polypharmacological approach.

Although the results from these experiments suggest that the combination of a D1 receptor partial agonist and a D3 receptor antagonist has potential for the treatment of cocaine addiction, additional testing is essential. For example, it is important to evaluate the abuse liability of combined treatment with NGB 2905/SKF 77434. Selective D1 receptor partial agonists and D3 receptor antagonists tend to have low abuse potential as they are not readily self-administered by rats or monkeys when substituted for cocaine

[(Grech et al. 1996; Martelle et al. 2007b; Weed and Woolverton 1995) but see (Self and 84

Stein 1992)], something that is often observed with D1 receptor agonists (Self et al.

1996b; Weed et al. 1993). For that reason, substitution of NGB 2905/SKF 77434 for cocaine is not expected to produce cocaine-like effects and the study could therefore provide additional evidence supporting the utility of this or similar combined treatments for cocaine addiction.

It would be also interesting to learn whether the synergistic effects of NGB

2904/SKF 77434 observed in the current studies are applicable to heroin-like behaviors.

Much focus in D1 and D3 receptor research has been in relation to psychostimulant- related behaviors. To the best of my knowledge no studies have evaluated D3 receptor antagonists or D1 receptor partial agonists against heroin self-administration. Given that other DA agents have been shown to produce effects on heroin reward it would be logical to test the effects of the combined treatment studied here. Some agents such as L- stepholidine (D1 receptor partial agonist and D2/D3 receptor antagonist), THP

(D1/D2/D3 receptor antagonist) and D3 receptor antagonists have been shown to reduce heroin seeking (Galaj et al. 2015; Ma et al. 2014; Yue et al. 2014) and opiate CPP (Ashby et al. 2003; Frances et al. 2004a; Hu et al. 2013). Hence, it remains to be seen whether a combined treatment of a D3 receptor antagonist and D1 receptor partial agonist can synergize to effectively reduce heroin reward and seeking.

Of particular interest might be to evaluate changes in neuronal signaling of the striatal MSNs in animals self-administering cocaine under the combined treatment of a

D1 receptor partial agonist and D3 receptor antagonist. Given that NGB 2904/SKF

77434 produces potentiating effects on cocaine reward reduction and synergistic effects on cocaine seeking reduction, proportional changes in the biochemical signal of these 85 neurons are likely to be observed. As such one may wonder how the effects of NGB

2904/SKF 77434 on cocaine seeking and CPP affect the activation of immune early genes (i.e., cfos expression) in the mesocorticolimbic DA system in response to drug cues.

Discerning the cellular processes that underlie the unique synergistic effects of

NGB 2904/SKF 77434 on cocaine behavior could lead to significant advances in our understanding of addiction and development of anti-cocaine therapeutics. Given that D1-

D3 receptor heteromers exhibit unique ligand binding and signaling characteristics and are restricted to the ventral striatum - a brain region deemed critical for reward and addiction - the existence of these D1-D3 receptor heteromers encourages the consideration of how they may contribute to the synergistic and potentiating effects of

NGB 2904/SKF 77434 observed in the present study. In fact, the D1-D3 receptor heteromers could represent a potential and promising target for future drug development.

A single compound with differential actions at multiple receptors would be more desirable than the combination of two selective agents in regard to pharmacotherapy.

Patients find it easier to take one pill instead of two and potential drug-drug interactions could be avoided. More importantly, the discovery of D1-D3 receptor heteromers opens new avenues to improve anti-cocaine compounds that have limited clinical utility due to a number of side effects. The findings of the present research makes it now conceivable to target D1-D3 receptor heteromers in the development of drug treatments for addiction and other disorders related to the dopaminergic system such as Parkinson disease and schizophrenia.

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