(+)-PHNO to Dopamine D2/D3 Receptors in Rat Brain: Lack of Correspondence to the D2 Receptor Two-Affinity-State Model

Ex vivo binding of the agonist PET radiotracer [11C]-(+)-PHNO to dopamine D2/D3 receptors in rat brain: Lack of correspondence to the D2 receptor two-affinity-state model

Patrick Neil McCormick

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy,

Graduate department of Institute of Medical Science, in the University of Toronto

Ex vivo binding of the agonist PET radiotracer [11C]-(+)-PHNO to dopamine D2/D3 receptors in rat brain: Lack of correspondence to the D2 receptor two-affinity-state model

Patrick Neil McCormick Doctor of Philosophy Institute of Medical Science University of Toronto 2010

Abstract

The dopamine D2 receptor exists in vitro in two states of agonist affinity: a high-affinity state mediating dopamine’s physiological effects, and a physiologically-inert low-affinity state.

Our primary goal was to determine the in vivo relevance of this two-affinity-state model for the agonist PET radiotracer [11C]-(+)-PHNO, developed for measurement of the D2 high-affinity state. Our second goal was to characterize the regional D2 versus D3 pharmacology of [3H]-(+)-

PHNO binding and assess its utility for measuring drug occupancy at both receptor subtypes.

Using ex vivo dual-radiotracer experiments in conscious rats, we showed that, contrary to the two-affinity-state model, the binding of [11C]-(+)-PHNO and the antagonist [3H]-raclopride were indistinguishably inhibited by D2 partial agonist (aripiprazole), indirect agonist

(amphetamine) and full agonist ((-)-NPA) pretreatment. Furthermore, ex vivo [11C]-(+)-PHNO binding was unaffected by treatments that increase in vitro high-affinity state density (chronic amphetamine, ethanol-withdrawal), whereas unilateral 6-OHDA lesion, which increases total

D2 receptor expression, similarly increased the ex vivo binding of [11C]-(+)-PHNO and [3H]- raclopride. These results do not support the in vivo validity of the two-affinity-state model, suggesting instead a single receptor state for [11C]-(+)-PHNO and [3H]-raclopride in conscious rat. Importantly, we also demonstrated that the increased amphetamine-sensitivity of the agonist

ii radiotracers [11C]-(+)-PHNO and [11C]-(-)-NPA, commonly seen in isoflurane-anaesthetized animals and cited as evidence for the two-affinity-state model, is due to the confounding effects of anaesthesia.

Using in vitro and ex vivo autoradiography in rat and the D3 receptor-selective drug

SB277011, we found that [3H]-(+)-PHNO binding in striatum and cerebellum lobes 9 and 10 was due exclusively to D2 and D3 receptor binding, respectively, but in other extra-striatal regions to a mix of the two receptor subtypes. Surprisingly, the D3 contribution to [3H]-(+)-

PHNO binding was greater ex vivo than in vitro. Also surprising, several antipsychotic drugs, at doses producing 80% D2 occupancy, produced insignificant (olanzapine, risperidone, haloperidol) or small (clozapine, ~35%) D3 occupancy, despite similarly occupying both receptor subtypes in vitro. These data reveal a significant discrepancy between in vitro and ex vivo measures of dopamine receptor binding and suggest that the D3 occupancy is not necessary for the therapeutic effect of antipsychotic drugs.

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Acknowledgments

I would first like to thank Dr. Philip Seeman, who co-supervised my MSc degree and a portion of my Ph.D. project, for providing me with the opportunity to come to Toronto and begin my career in brain research. Nearing the end of my PhD, I am convinced that this is the correct path for me, and I owe this realization to the opportunity afforded me by Dr. Seeman.

Had it not been for him, I might have ended up taking the offer to do a Ph.D. studying beetle pheromones, and not met any of my current colleagues, my wife or many of my dearest friends.

I would also have learned nothing of the fascinating field of PET imaging (We’re pushing resolution limits with rodent, not to mention beetle scanning!).

In terms of practical supervision and day-to-day guidance, my gratitude goes to Dr. Alan

Wilson. Thanks, Alan, for providing me with an environment of rigourous, disciplined, logical investigation that has set a high standard in my mind for the way science should be conducted.

Thank you also for your steadfast support throughout my studies, particularly when I found myself in the unenviable position of being part-way through a PhD without official supervision!

On a personal note, your unflinching candour, which at first I found slightly intimidating, has become the characteristic I admire most in you. I doubt that the phrase “what you see is what you get” applies more aptly to any other sentient being in the universe. Thanks for your honesty, support and exceptional training.

I would also like to thank many other people at the CAMH. To the director of the PET centre, Dr. Sylvain Houle, my sincere thanks for providing much-needed salary support when I found myself “out in the cold,” and for allowing me a comfortable space to work and learn.

Thanks to the members of my PhD committee, Drs. Neil Vasdev, Jeff Meyer, Gary Remington,

Shitij Kapur and Nathalie Ginovart, for helpful criticism, intellectual stimulation and continual encouragement. Thanks also, Neil, for involving me in various interesting projects, both at

CAMH and elsewhere. I took it as a vote of confidence from an excellent scientist. Thanks to iv

Armando Garcia, Winston Stableford, and Min Wong for the unfailing and punctual synthesis of

[11C]-(+)-PHNO, without which this work would have been impossible, and to Doug Hussey,

Jun Parkes, Alvina Ng, Greg Reckless, Zoe Rizos, Roger Raymond, Dr. José Nobrega and

Mikael Palner for training me in the various techniques this work required and for their countless hours of help with experiments. Thanks to Lori Dixon for her strict yet flexible approach to animal facility matters, which tended to facilitate, rather than impede, the work in this thesis. Thanks to Isabelle Boileau for creating the parametric maps in the front of this thesis and for letting me participate in her [11C]-(+)-PHNO study (not to mention the decent monetary compensation). Thanks to the Canadian Institutes for Health Research for funding this work

(grant numbers MOP-74702 and MOP-44051). I would also like to offer a line to the animals used in this work who, by virtue of fate, contributed far more to this project than anyone else.

To my parents, thanks for your love and support, which I felt even during my (often spectacular) failures, for instilling me with a love of learning and nurturing my fascination with the world around me. To my brother, Andrew, and my sister, Jenn, thanks for sticking with me and wanting the best for me. Thanks to my late brother, Jeff, whose life was the single most powerful influence in mine. Thanks to Dr. Solomon Shapiro for his guidance and especially for his genuine love of Homo sapiens. Finally, special thanks to my wife, Conny, for continually shining a brilliant, clarifying light on the world, and for being an endless source of fun, humour and warmth I couldn’t have done without.

During my PhD, [11C]-(+)-PHNO often occupied my thoughts, but on a couple of occasions it even occupied my dopamine receptors! Shown above are parametric maps of [11C]-(+)-PHNO binding potential (BPND) in my brain at baseline (left) and after 35 mg oral amphetamine (right). In the top images, the greatest binding is seen in the striatum (here at a level including the caudate, putamen and globus pallidus) and in the bottom images within the substantia nigra.

Table of contents

1. General introduction 1 2. Literature review 3 2.1. The dopaminergic system 4 2.1.1. Overview of the functional anatomy of the dopaminergic system 4 2.1.2. Intracellular effects of dopamine receptor activation 11 2.1.2.1. Intracellular effects of the D1-like receptor family 11 2.1.2.2. Intracellular effects of the D2-like receptor family 13 2.1.3. Brain distribution of dopamine receptors and the dopamine transporter 16 2.1.3.1. Distribution of the dopamine D1 receptor 16 2.1.3.2. Distribution of the dopamine D2 receptor 17 2.1.3.3. Distribution of the dopamine D3 receptor 19 2.1.3.4. Distribution of the dopamine D4 receptor 22 2.1.3.5. Distribution of the dopamine D5 receptor 23 2.1.3.6. Distribution of the dopamine transporter 24 2.1.4. Involvement of the dopaminergic system in substance abuse and schizophrenia 26 2.1.4.1. The dopaminergic system and substance abuse 26 2.1.4.2. The dopaminergic system and schizophrenia 29 2.2. Quantification of in vivo PET and SPECT radiotracer binding 34 2.2.1. Radiotracer binding under equilibrium conditions 34 2.2.2. Radiotracer binding under non-equilibrium conditions 42 2.2.2.1. Kinetic modeling of dynamic time-concentration data 43 2.2.2.2. Graphical analysis of dynamic time-concentration data 48 2.3. PET and SPECT radiotracers for dopaminergic imaging in human brain 53 2.3.1. Aromatic amino acid decarboxylase (AAAD), dopamine synthesis and storage 53 2.3.2. The dopamine transporter (DAT) 54 2.3.3. Vesicular monoamine transporter 2 (VMAT2) 58 2.3.4. Dopamine D1 receptors 58 2.3.5. Dopamine D2/D3 receptors 60 2.3.6. D2/D3 radiotracer-based imaging of endogenous dopamine 62 2.4. The high-affinity state of the dopamine D2 receptor and the development of agonist D2/D3 positron emission tomography radiotracers 66 2.4.1. [11C]-(-)-NPA 68 2.4.2. [11C]-(-)-MNPA 70 2.4.3. [11C]-(+)-PHNO 72 3. Brief introduction and rationale for thesis studies 79 4. Dopamine D2 receptor radiotracers [11C](+)-PHNO and [3H]raclopride are indistinguishably inhibited by D2 agonists and antagonists ex vivo 81 4.1. Abstract 82 4.2. Introduction 83 4.3. Materials and methods 84 4.4. Results 89 4.5. Discussion 90 4.6. Conclusions 98

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5. Ex vivo [11C]-(+)-PHNO binding is unchanged in animal models displaying increased high-affinity states of the D2 receptor in vitro 99 5.1. Abstract 100 5.2. Introduction 101 5.3. Materials and Methods 102 5.4. Results 106 5.5. Discussion 112 5.6. Conclusions 120 6. Isoflurane anaesthesia differentially affects the amphetamine-sensitivity of agonist and antagonist D2/D3 positron emission tomography radiotracers: implications for in vivo imaging of dopamine release 121 6.1 Abstract 122 6.2 Introduction 123 6.3. Materials and methods 124 6.4. Results 127 6.5. Discussion 133 6.6. Conclusions 139 7. The antipsychotics olanzapine, risperidone, clozapine and haloperidol are D2-selective ex vivo but not in vitro 140 7.1. Abstract 141 7.2. Introduction 142 7.3. Materials and methods 144 7.4. Results 149 7.5. Discussion 154 7.6. Conclusions 162 8. Concluding remarks and future directions 163 9. References 170

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List of tables

Table 1. Density of [3H]-PD128907 binding sites in rat brain 21

Table 2. In vivo binding potentials and their definition in terms of volumes of distribution and kinetic rate constants 40

Table 3. Common kinetic analysis methods 46

Table 4. Dose-response parameters for inhibition of [11C]-(+)-PHNO and [3H]-raclopride by dopaminergic drugs 91

Table 5. Striatum and cerebellum %ID/g and SBR values for [11C]-(+)-PHNO and [3H]-raclopride in rats sensitized to AMPH (after acute saline or 4 mg/kg i.v. AMPH pretreatment) and rats withdrawn from chronic ethanol treatment 108

Table 6. Left striatum, right striatum and cerebellum %ID/g values for [11C]-(+)-PHNO and [3H]-raclopride in left-lesioned, right-lesioned and sham-lesioned rats 111

Table 7. Striatum (STR) and cerebellum (CER) standard uptake values (SUV) for conscious (CON) and isoflurane-anaesthetized (ISO) rats after pretreatment with saline (SAL) or 4 mg/kg AMPH 128

Table 8. Antipsychotic drug concentrations in blood plasma 151

Table 9. Regional ex vivo occupancy by antipsychotic drugs or SB277011 153

Table 10. Regional in vitro occupancy in antipsychotic- or SB277011-treated brain sections 154

List of figures

Figure 1. Axonal projection fields of the ventral tier (VT, bottom) and dorsal tier (DT, top) dopaminergic neurons of the SNc/VTA 5

Figure 3. Schematic diagram of the system of feedforward loops connecting the striatal complex and the mecencephalic dopaminergic neurons 9

Figure 4. The 1-TC model containing an arterial plasma compartment, CP, and one tissue compartment, C1 35

Figure 5. The 2-TC model, containing a blood plasma compartment, CP, the non-displaceable binding tissue compartment, CND, the specific binding compartment, CS and four rate constants K1, k2, k3 and k4. 37

Figure 6. Simulated competition between a D2 antagonist radioligand and an agonist ligand 66

Figure 7. The chemical structures of the D2/D3 agonist radiotracers [11C]-(-)-NPA, [11C]-(-)-MNPA and [11C]-(+)-PHNO 68

Figure 8. [11C]-(+)PHNO time-activity curves in human brain demonstrating the different washout rates of [11C]-(+)-PHNO from A) CAU and PUT relative to B) ventral STR and especially GP 76

Figure 9. Inhibition of striatal [11C]-(+)-PHNO (filled circles) and [3H]-raclopride (open circles) SBR by treatment with the D2 ligands (-)-NPA (A), aripiprazole (B), haloperidol (C) and clozapine (D) 90

Figure 10. Effect of the D3-selective antagonist SB277011 on the striatal SBR of [11C]-(+)-PHNO (filled circles) and [3H]-raclopride (open circles) 92

Figure 11. Effect of AMPH pretreatment of [11C]-(+)-PHNO and [3H]-raclopride SBR 92

Figure 12. Locomotor response to i.p. injection of 0.5 mg/kg AMPH in chronic AMPH- and saline-treated rats 107

Figure 13. Striatal SBR of [11C]-(+)-PHNO and [3H]-raclopride in chronic AMPH- and saline-treated rats. 107

Figure 14. Percent decrease in the SBR of [11C]-(+)-PHNO and [3H]-raclopride after i.v. injection of 4 mg/kg AMPH 109

Figure 15. Rotational behaviour of unilaterally 6-OHDA lesioned rats after injection of 0.05 mg/kg apomorphine 110 x

Figure 16. Striatal [11C]-(+)-PHNO and [3H]-raclopride SBR in 6-OHDA lesioned and sham lesioned rats 110

Figure 17. Ratio of [11C]-(+)-PHNO and [3H]-raclopride SBRs in lesioned striatum to that of the intact striatum 111

Figure 19. Percent decrease in [11C]-(+)-PHNO, [3H]-(+)-PHNO, [11C]-(-)-NPA and [3H]-raclopride SBR after pretreatment with 4 mg/kg i.v. AMPH in conscious and isoflurane-anaesthetized rats, expressed as a percent of the average specific binding ratio (SBR) in the respective saline-pretreated control group 130

Figure 20. Ex vivo [11C]-(+)-PHNO and [3H]-raclopride time-activity curves generated by sacrifice at various times after radiotracer injection 131

11 3 Figure 21. Binding potential (BPND) values for [ C]-(+)PHNO and [ H]-raclopride 132

Figure 22. Average metabolite-corrected plasma input curves for [11C]-(+)-PHNO in all treatment groups 136

Figure 23. Affinity (pKi) of various antipsychotic drugs for cloned dopamine D2 and D3 receptors (human and rat) 143

Figure 24. Typical control [3H]-(+)-PHNO autoradiographs in rat brain measured ex vivo (left) and in vitro (right) 150

Abbreviations

6-OHDA 6-hydroxydopamine (+)-PHNO 4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol (-)-MNPA (-)-2-methoxy-N-propylnorapomorphine (-)-NPA (-)-N-propylnorapomorphine α-methyl-Trp α-methyltryptophan AAAD aromatic amino acid decarboxylase AMPH amphetamine AMPT α-methyl-p-tyrosine Bmax binding site density BP binding potential BPF binding potential with respect to free plasma radiotracer concentration BPP binding potential with respect to total plasma radiotracer concentration BPND binding potential with respect to radiotracer concentration in the non- displaceable compartment cAMP 3'-5'-cyclic adenosine monophosphate CAU caudate nucleus Cdk5 cyclin-dependent kinase 5 CER cerebellum COMT catechol-O-methyl transferase DARPP-32 dopamine- and cAMP-regulated phosphoprotein of 32 kDa molecular molecular weight DAT dopamine transporter EP entopeduncular nucleus FRTM full reference tissue model GP globus pallidus GPCR G protein-coupled receptor GPe globus pallidus external GPi globus pallidus internal KD equilibrium dissociation constant MAO monoamine oxidase NACC nucleus accumbens PET positron emission tomography PKA protein kinase A PP-1 protein phophatase 1 PP-2A protein phosphatase 2A PUT putamen ROI region of interest SBR specific binding ratio SERT serotonin transporter SN substantia nigra SNc substantia nigra pars compacta SNr substantia nigra pars reticulata SPECT single photon emission computed tomography SRTM simplified reference tissue model STh subthalamic nucleus STR striatum SUV standard uptake value xii

TC tissue compartment (as in 1-TC, 2-TC, etc.) VMAT2 vesicular monoamine transporter 2 VTA ventral tegmental area

xiii 1 1. General introduction

The studies in this thesis examine the behaviour of the carbon-11-labeled (radioactive half-life = 20.4 min) agonist D2/D3 PET radiotracer, [11C]-(+)-PHNO, using ex vivo radiotracer binding experiments in rat. This radiotracer was originally developed for the purpose of selectively measuring the high-affinity, G protein-coupled form of the D2 receptor, thought to be responsible for the physiological actions of dopamine at the D2 receptor in vivo and to be involved in the pathophysiology of schizophrenia and substance abuse (see section 2.2.3).1-3 The two-affinity state model of the D2 receptor and other G protein-coupled receptors, which states that the receptor exists in separate states of high and low agonist affinity, is based exclusively on evidence from in vitro radioligand binding experiments and thus cannot be uncritically applied to the results of in vivo imaging studies using agonist radiotracers. The first three studies in this thesis address the in vivo validity of the two-affinity state model by examining two major predictions that follow from the model regarding the in vivo binding of an agonist radiotracer (in our case [11C]-(+)-PHNO). The first prediction (sections 3 and 5) is that the binding of an agonist radiotracer should be inhibited to a greater degree than that of an antagonist radiotracer by other agonist ligands, either exogenous (i.e. direct D2 agonist drugs) or endogenous (i.e. extracellular dopamine). As a consequence, an agonist radiotracer should allow more sensitive measurement of changes in extracellular dopamine concentration than an antagonist radiotracer, and therefore be advantageous for the in vivo measurement of dopaminergic activity in the human brain (see section 2.2.2.1.6). The second prediction (section 4) is that the in vivo binding of an agonist radiotracer to the D2 receptor should be increased in animal models for which an in vitro increase in the D2 high-affinity state has been demonstrated. The binding of an antagonist radiotracer, on the other hand, should be unaffected by such a change. This prediction could have major relevance to PET and SPECT brain imaging in schizophrenia and substance abuse, which are thought to be associated with increased D2 receptor high-affinity state.

2 A second major topic of this thesis is the study of the D2 versus D3 receptor contribution to [11C]-(+)-PHNO binding. Since its original ex vivo characterization,4 several studies have demonstrated that [11C]-(+)-PHNO binds to both the D2 and D3 receptors in vivo,5-8 and that the

D2- and D3-related binding signals are largely anatomically segregated, making [11C]-(+)-

PHNO a potentially useful radiotracer for examining drug occupancy at both receptor types. The fourth and final study in this thesis (section 6) examines the D2 versus D3 receptor contribution to regional [11C]-(+)-PHNO binding in rat brain, and addresses the utility of [11C]-(+)-PHNO for simultaneous in vivo measurement of D2 and D3 receptor drug occupancy. This study also critically examines the accepted D2 versus D3 receptor pharmacology of several antipsychotic drugs in the context of their in vivo therapeutic action.

3 2. Literature review

2.1. The dopaminergic system

Though originating from a only small number of mesencephalic neurons (approximately

40,000 in rat and 450,000 in humans),9 the dopaminergic system is heavily involved in the modulation of diverse brain systems including those responsible for control of motor output, motivation and reward, emotion, and cognitive function. Not surprisingly, the dopaminergic system is involved in the aetiology of diseases affecting these brain systems such as Parkinson’s disease (motor),10-12 pathological drug seeking and addiction (motivation and reward),13-15 major depressive disorder (affect)16 and schizophrenia (cognition).17-19 In the following sections

(2.1.1–2.1.4) a basic overview is presented of the functional anatomy of the dopaminergic system (section 2.1.1), the function (section 2.1.2) and distribution (section 2.1.3) of the dopamine receptor subtypes and the dopamine transporter, and the involvement of the dopaminergic system in two major brain disorders, addiction and schizophrenia (sections 2.1.4.1 and 2.1.4.2), with a particular focus on important in vivo functional neuroimaging findings.

4 2.1.1. Overview of the functional anatomy of the mammalian dopaminergic system

Although this section summarizes data from at least four separate species (mouse, rat, non-human primate and human), most of the material presented here can be generalized to all of these species. Four neuronal pathways make up the mammalian brain dopaminergic system, the nigrostriatal, mesocortical, mesolimbic and tuberoinfundibular pathways. The tuberoinfundibular pathway, the most regionally-restricted of the dopaminergic pathways, originates from a group of cell bodies in the mediobasal hypothalamus and projects to the pituitary gland where it regulates the release of the hormone prolactin.20,21 The nigrostriatal, mesocortical and mesolimbic pathways, which are the major dopaminergic pathways innervating large areas of the diencephalons and telencephalon, originate from cell bodies located within two closely related mesencephalic nuclei, the substantia nigra pars compacta

(SNc) and the ventral tegmental area (VTA). Based on the location of their axonal terminal fields, the mesencephalic dopamine cells are often divided into ventral and dorsal groups or

“tiers”, each containing cell bodies from both the SNc and VTA (Figure 1).22,23 The ventral tier neurons form the nigrostriatal pathway, innervating the central and dorsolateral portions of the caudate (CAU) and putamen (PUT).24-27 The dorsal tier neurons give rise to the mesocortical pathway, which innervates the entire cerebral cortex, with especially dense innervation to the prefrontal, anterior cingulate, insular and entorhinal cortices, and to the mesolimbic pathway innervating primarily the nucleus accumbens (NACC), the ventromedial portion of the CAU and

PUT, the hippocampus, amygdala, thalamus and basal forebrain.24-32

The role of dopamine in the striatum (STR; composed of the CAU, PUT and NACC) has been described in more detail than in any other brain region. The STR serves as the input nucleus for a complex network involving many cortical and non-cortical brain areas and influencing a wide array of brain functions. The core or this network is the well-studied basal ganglia circuit (Figure 2).33-35 In basic terms, the role of this circuit is to receive, modulate and

Figure 1. Axonal projection fields of the ventral tier (VT, bottom) and dorsal tier (DT, top) dopaminergic neurons of the SNc/VTA. Grey scale indicates the approximate relative density of afferent dopaminergic projections to each region. In cortex, dark bands indicate the cortical layers receiving heaviest dopaminergic input. Other abbreviations: Amy, amygdala; BF, basal forebrain; BL, basolateral amygdala; Ce, central amygdala; DS, dorsal STR; Ec, entorhinal cortex; F, frontal cortex; GP, globus pallidus; Hipp, hippocampus; MD, mediodorsal thalamus; VS, ventral STR; P, parietal cortex; O, occipital cortex; T, temporal cortex; Th, thalamus. Figure from reference 23 with permission.

Figure 2. Simplified representation of the basal ganglia circuitry showing the direct pathway (A), the indirect pathway (B) and the feedback circuitry between the striatum (STR) and substantia nigra pars compacta/ventral tegmental area (SNc/VTA) (C; see text and Figure 3 for details). Inhibitory, excitatory and dopaminergic connections are shown in red, green and blue, respectively. Other abbreviations: GPe, globus pallidus external; GPi/SNr, globus pallidus internal/substantia nigra pars reticulate; STh, subthalamic nucleus; THAL, thalamus. Figure modified from reference 23 with permission.

integrate glutamatergic signals primarily from the cortex (but also from other regions such as the hippocampus and amygdala) and re-direct the resulting processed information to more functionally- and anatomically-restricted cortical areas where it influences specific cognitive, limbic and motor tasks. The role of the basal ganglia circuit in the control of motor function has been particularly well-studied and is used below to illustrate the circuit’s key features. It should be noted that in addition to motor effects, the basal ganglia circuitry and dopamine release within the STR complex has influences on many other cortex-related brain functions. The basal ganglia circuit is composed of several nuclei: the STR, which serves as the input nucleus of the circuit; the external (or lateral) segment of the globus pallidus (GPe) and the subthalamic

7 nucleus (STh), known as the relay nuclei, which serve intermediate processing roles; the internal

(or medial) segment of the globus pallidus (GPi) and the substantia nigra pars reticulate (SNr) which together constitute the output nucleus (GPi/SNr); the thalamus which conveys the resulting processed signals to the motor cortex; and the SNc and VTA which together provide the dopaminergic modulation of the circuit (SNc/VTA).34,35 Cortical information, in the form of glutamatergic impulses, arrives in the STR and activates either one of two pathways, the direct or indirect pathways. The direct pathway consists of a direct GABAergic connection between the STR input nucleus and the GPi/SNr output nucleus. Via GABAergic efferents from STR to

GPi/SNr and from GPi/SNr to thalamus, glutamatergic activation of direct pathway neurons causes the disinhibition of excitatory thalamic projections to the motor cortex, thus promoting motor output. In the indirect pathway, striatal GABAergic neurons are separated from the

GPi/SNr by the GPe and the STh. The net effect of cortical activation of indirect pathway neurons is the disinhibition of excitatory projections from the STh to the GPi/SNr, in turn resulting in inhibition of thalamic excitatory output to the motor cortex and the inhibition of movement. Thus, the direct and indirect pathways have opposing effects on motor output, the net influence on the motor cortex representing a balance between the two pathways.34,35

The influence of the STR on motor behaviour is mediated mostly by its dorsolateral aspect, which receives heaviest cortical inputs from the premotor and motor cortices.26 However, the STR also receives inputs from the entire cortex and each area of the STR can influence the activity of the dorsolateral STR and therefore indirectly influence motor behaviour.22,26 In terms of the organization of cortical inputs, the STR is often described as a gradient running along the ventromedial to dorsolateral axis, transitioning from limbic-related cortical input in the ventromedial STR, to associative in the central STR, to premotor and motor cortical input in the dorsolateral STR. Starting in the most ventromedial portion of the STR, a series of STR–

SNc/VTA–STR feedforward loops promote the activity of successively more dorsolateral

8 portions of the STR (see Figure 3).26 This system, which is dependent on mesencephalic dopaminergic neurons and striatal dopamine release, facilitates transfer of information through the STR along the ventromedial to dorsolateral axis, thus allowing limbic, associative, premotor and motor cortical information to influence the eventual output of the STR responsible for modulation of motor behaviour.

Dopamine release in the STR plays a modulatory role in the activity of both the direct and indirect basal ganglia pathways. Dopaminergic neurons of the SNc/VTA send projections to all parts of the STR, where they terminate primarily on GABAergic neurons. At the sub-cellular level, dopamine receptors are often located on the shafts of dendritic spines whose heads receive glutamatergic synapses from other regions of the brain.36 This arrangement is ideally suited for the direct dopaminergic modulation of incoming glutamatergic signals. However, in part because of the predominantly extrasynaptic location of the dopamine transporter37,38 which is responsible for removal of dopamine from the extracellular space, it is thought that released dopamine can diffuse to form a sphere or cloud of micrometer diameter, centered around the point of release, within which the dopamine concentration is sufficient to elicit physiological effects.39 The physiological relevance of this extrasynapatic dopamine is strongly suggested by the ubiquity of extrasynaptic dopamine receptors within the STR.40-42 As a result of asynchronous release of dopamine from sites across the STR and the overlap of dopamine diffusion spheres, the extracellular concentration of dopamine is thought to be maintained at relatively constant tonic levels across large areas of the STR, with the exact dopamine concentration presumably being related to the density of dopamine release sites within the particular STR area.39 Furthermore, because each SNc/VTA axon releases dopamine at multiple points spanning the entire area of the STR, increased activity of dopaminergic SNc/VTA cells results in an increase in extracellular dopamine across the STR.39

Figure 3. Schematic diagram of the system of feedforward loops connecting the striatal complex and the mecencephalic dopaminergic neurons. See text for details. Abbreviations: OMPFC, orbitomedial prefrontal cortex; DLPFC, dorsolateral prefrontal cortex; VTA, ventral tegmental area. Figure from reference 23 with permission.

10 Dopaminergic modulation of the direct and indirect basal ganglia pathways are thought to be mediated primarily by the dopamine D1 and D2 receptors, respectively.43 Through activation of intracellular second messenger systems, both of these receptor subtypes elicit a complex array of intracellular effects including changes in the activity and phosphorylation state of other receptors, synthetic enzymes, protein kinases, protein phosphatases and ion channels

(see section 2.1.2). This makes it difficult to formulate general statements about the effect of D1 or D2 receptor activation on basal ganglia network activity. Furthermore, the short-term effects of dopamine receptor activation, mediated primarily through effects on K+ and Ca2+ ion channels and the NMDA receptor, are thought to be dependent on the polarization state of the postsynaptic membrane at the time of dopamine release,44,45 such that D1 receptor activation during a state of membrane depolarization is thought to potentiate further depolarization, whereas the reverse is thought to be the case during membrane hyperpolarization. This has been envisioned as the basis of a so-called “sample and hold” mechanism, in which the influence of

D1 receptor activation is to encourage the network (in this case the direct basal ganglia pathway) to remain in a state corresponding to the onset of increased dopaminergic stimulation.39 Since the D2 receptor, in general, mediates intracellular effects that oppose those of the D1 receptor, it could be envisioned that its activation receptor has similar but reciprocal effects on the indirect pathway. This mechanism could be important in volition as striatal dopamine release (especially in the ventromedial STR) could allow a sustained motor response to salient external stimuli.

Outside the STR, in the terminal fields of the mesolimbic and mesocortical dopaminergic pathways, dopaminergic innervation is less dense than in the STR itself.

Nevertheless, dopaminergic projections to many non-striatal regions, including the frontal cortex, hippocampus and amygdala, have critically important effects on the circuitry regulating motivation, reward, leaning, working memory and attention, and in the communication of these circuits with one another and with motor-related systems.46,47

11 2.1.2. Intracellular effects of dopamine receptor activation

The dopamine receptors can be divided into two families: the D1-like receptor family which consisting of the D1 and D5 receptors and the D2-like family consisting of the D2, D3 and D4 receptors. Within each of these receptor families, receptor subtypes are homologous with one another in terms of their amino acid sequence, pharmacology and intracellular effects.

All of these receptor subtypes are membrane-spanning proteins with seven transmembrane domains, and are coupled to intracellular biochemical pathways via binding to, and activation of trimeric GTP-binding proteins (G protein). Below is presented a survey of the major intracellular effects of the dopamine receptors. Although the discussion is limited to the immediate effects of dopamine receptor activation, these receptors also have long-term effects mediated by changes in gene expression. Such changes are caused by activation of the transcription factors such as CREB and AP-1,48-52 which induce the expression of immediate early genes, such as c-Fos, JunB and c-Jun. Many of these encode other transcription factors that control the expression of still more genes. For example, D1 agonist treatment in 6-OHDA lesioned rats was found to induce the expression of over 30 individual genes.53 The result is a complex system of gene and protein expression changes thought to be responsible for long-term neuronal adaptation and synaptic plasticity. Further discussion of this topic is beyond the scope of the current thesis.

2.1.2.1. Intracellular effects if the dopamine D1-like receptor family

The intracellular effects of D1 and D5 (D1-like) receptors are very similar. They are mediated through the direct activation of heterotrimeric G proteins which contain either the

Gαolf α-subunit or the Gαs α-subunit. Those containing Gαolf are found in dopaminergically-

54,55 innervated GABAergic neurons of the STR, while receptors linked to Gαs-containing G proteins mediate D1-like signaling in non-striatal brain regions.54 These two α-subunits have a

12 stimulatory effect on the enzyme adenylate cyclase (AC), which is responsible for synthesis of the intracellular second messenger 3'-5'-cyclic adenosine monophosphate (cAMP). The consequent increase in cAMP concentration is the primary event that mediates the intracellular response to D1-like receptor activation. The most important and well-studied effect of cAMP is the activation of cAMP-dependent protein kinase (PKA). Through phosphorylation, PKA regulates the activity of a wide variety of intracellular proteins, of which several important examples are discussed below.

DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, 32 kDa molecular weight) is a phosphoprotein that exists in two functionally-important phosphorylation states. When phosphorylated at its position 75 threonine residue (Thr75) DARRP-32 inhibits the action of

PKA,56 whereas when it is phosphorylated at Thr34 it becomes an inhibitor of protein phosphatase 1 (PP-1), which is largely responsible for dephosphorylation of PKA substrates.57-59

Phosphorylation of DARPP-32 at Thr75 is accomplished by cyclin-dependent kinase 5 (Cdk5),56 whereas protein phosphatase 2A (PP-2A) is responsible for the dephosphorylation of this residue and the conversion of DARPP-32 to its PP-1-inhibiting form.60,61 Under baseline conditions, the balance between Cdk5 and PP-2A activity favours the Thr75-phophoryated form of DARPP-32, enabling it to inhibit PKA activity.62 In the presence of a D1-like receptor- mediated increase in cAMP levels, activated PKA has two major effects on the DARPP-32 signaling system; 1) PKA phosphorylates and thereby activates PP-2A, which can then dephosphorylate Thr75, freeing PKA from DARPP-32 inhibition; and 2) PKA phosphorylates

DARPP-32 at Thr34, converting it to the PP-1-inhibiting form. Thus D1-like receptor activation directly increases PKA activity, but also allows PKA to free itself from DARPP-32 inhibition.62

Under conditions of D1-like receptor activation and increased intracellular cAMP, PKA influences the activity of several proteins that are involved in post-synaptic membrane voltage and excitability. PKA phosphorylation decreases voltage-gated Na+ channel currents,63,64

13 increases Ca2+ currents through voltage-gated L-type Ca2+ channels,65 decreases Ca2+ currents through N- and P/Q-type Ca2+ channels,65,66 increases cation currents through glutamatergic

67-69 70-72 - AMPA and NMDA receptor channels, and reduced Cl currents through GABAA receptor channels.73 As well, D1-like activation reduces the activity of the Na+/K+ exchanger responsible for maintenance of resting membrane voltage.74,75 Thus D1-like receptor activation produces wide-ranging, often antagonistic effects, making it difficult to formulate general statements about its short-term effects on post-synaptic neurons. Furthermore, since D1-like receptor signaling is heavily dependent on PKA, which has many protein targets, the ultimate effect of D1-like receptor activation is a function of the protein complement of the post-synaptic cell. However, it is thought that the net effect of D1-like activation depends on initial polarization state of the post-synaptic neuron.45 For example, in a state of membrane depolarization, D1-like receptor activation is thought to increase membrane excitability by potentiating currents through voltage-gated L-type Ca2+ channels and the NMDA receptor channel, whereas when the postsynaptic membrane is hyperpolarized, D1-like receptors are thought to reduce excitability by inhibition of N- and P/Q-type Ca2+ channels.45,71

2.1.2.2. Intracellular effects of the dopamine D2-like receptor family

With respect to intracellular effects, the D2, D3 and D4 (D2-like) receptors behave in very similar ways. The only major difference between D2-like receptor subtypes is the apparently lower efficacy of the D3 receptor, compared to D2 or D4, for activation of virtually all of their shared intracellular responses.76-79 Like the D1-like receptors, the D2-like receptors

(D2, D3 and D4) produce the majority of their intracellular effects through the activation of G proteins. Whereas D1-like receptors activate Gαolf and Gαs, which stimulate the activity of AC, the D2-like receptors activate G proteins containing the pertussis toxin-sensitive Gαi or Gαo subunits, which inhibit AC and thereby reduce the concentration of intracellular cAMP. In brain,

14 the D2-like receptor functions primarily through activation of Gαo, which is much more

80 abundant than Gαi. As expected, the reduced intracellular cAMP concentration resulting from

D2-like receptor activation has effects on PKA activity opposing those of D1-like receptors. In the presence of reduced cAMP, DARPP-32 is maintained in its Thr34-phosphorylated state by the activity of Cdk5, and is therefore exercises a potent inhibitory effect on PKA.81,82 In addition, under these conditions, the activity of PP-1 is the dominant factor in determining the phosphorylation state of PKA protein substrates.

The D2-like receptors also have intracellular effects that are independent of the inhibition of cAMP synthesis. For example D2-like receptors cause the activation of G protein- activated inwardly-rectifying K+ channels (GIRKs) in pituitary cells leading to membrane hyperpolarization and a reduction in membrane excitability.83-85 This GIRK activation is inhibited by pertussis toxin, indicating the involvement of Gαi/o, but is independent of AC activity.86,87 In striatal neurons D2-like receptor regulation of K+ currents is more complicated, with both stimulatory83,86,88 and inhibitory89 effects reported. However, these discrepant findings appear to be the result of two separate effects: the Gαo activation of GIRKs, and the inhibition of

GIRKs by PP-1 under conditions of low cAMP/PKA activity.90 D2-like receptor activation of

GIRKs in the brain is thought to be important in the D2-like autoreceptor inhibition of dopamine release.91-93

Also independent of AC activity are the D2-like receptors’ effects on intracellular Ca2+.

Firstly, D2-like receptors reduce Ca2+ currents through voltage-gated N-type Ca2+ channels,87,94 an effect that, like the D2-like receptor-mediated activation of K+ currents, could be involved in

D2-like autoreceptor function. Though the reduction in Ca2+ current was at first thought to be due to the hyperpolarization resulting from D2-like receptor-mediated K+ currents,95 it has since been linked directly to the action of G proteins.87,94 Secondly, D2-like receptors influence intracellular Ca2+ by reducing currents through L-type Ca2+ channels.96,97 This effect has been

15 found to be coupled to another D2-like receptor-mediated pathway, the activation of the enzyme phospholipase C (PLC). In response to D2-like receptor activation, the G protein βγ-subunit (Gβγ) activates PLC which initiates the phosphoinositol signaling pathway culminating in the release

Ca2+ from the endoplasmic reticulum. Elevated intracellular Ca2+ stimulates protein phophatase

2B (also known as calcineurin) which dephosphorylates and thereby inactivates L-type Ca2+ channels.97 Thus by utilizing a rapid and short-lived increase in intracellular Ca2+, D2-like receptors cause a potentially longer-lived inhibition (by protein desphosphorylation) of Ca2+ entry into the cell through voltage-gated channels.

In conclusion, the effects of D1-like and D2-like receptors on intracellular signaling pathways are complex and may depend on the initial biochemical and electrophysiological state of the postsynaptic neuron. Some of the effects caused by a single receptor can be seemingly antagonistic (e.g. D1-like inhibition of voltage-gated Na+ channels and activation of NMDA receptor channels), as can be the effects of receptors from different families (e.g. D1-like versus

D2-like effects on L-type Ca2+ channels). However, the dopamine receptors can also have synergistic intracellular effects (e.g. both receptor families reduce currents through N-type Ca2+ channels). These complexities provide a challenge to the understanding of the effects of dopamine receptors at the whole-cell and network levels.

16 2.1.3. Brain distribution of dopamine receptor subtypes and the dopamine transporter

As discussed above, the intracellular actions of dopamine are mediated by the five dopamine receptor subtypes – D1, D2, D3, D4 and D5. In addition, the tonic extracellular dopamine concentration and the kinetics of phasic changes in extracellular dopamine are governed by the dopamine transporter. Thus, in terminal fields of SNc and VTA dopaminergic neurons, the net effect of released dopamine on neuronal activity is governed in part by the distribution and relative abundance of each of these protein species. The following sections

(2.1.3.1–2.1.3.6) describe the distribution of the dopamine receptor subtypes and the dopamine transporter in rodent, non-human primate and human brain.

2.1.3.1. Distribution of the dopamine D1 receptor

The D1 receptor is expressed in many areas of the rat, human and non-human primate brain, including all areas to which dopaminergic cells originating in the SNc and VTA project.

Using radioligand autoradiographic and immunohistochemical experiments in rat brain, the highest D1 receptor densities were seen in the olfactory tubercle, STR, SN, and entopeduncular nucleus (EP; rat homolog of the human GPi), medium density was seen in various cortical regions, the major island of Calleja, ventral pallidum, lateral septal nuclei, amygdala, hippocampus, STh, thalamus, hypothalamus, and the molecular layer of the cerebellar cortex, whereas low densities were seen in the VTA and GP (rat homolog of the human GPe).41,42,98-101

Correspondingly, D1 receptor mRNA was found in to be most abundant in the olfactory tubercle and STR, but also present in other regions such as the cortex, lateral septal nuclei, amygdala, hypothalamus and thalamus,102-108 where D1 binding sites or immunoreactivity has been demonstrated. D1 mRNA was not seen, however, in the several other regions rich in D1 receptor protein, such as the VTA, GP, SNr, SNc and EP, a discrepancy likely the result of transport of protein from the site of mRNA transcription and protein synthesis (the cell body) to distant sites

17 within the axonal terminal field. This is most evident within the SN, where D1 receptor binding sites are not associated with SN cell bodies or dendritic processes104 but rather with the terminals of axons originating within the STR and projecting to the SNr.41,109-111

Within the rat STR, the D1 receptor is expressed on medium-sized GABAergic neurons, either on dendrites postsynaptic to excitatory synapses of cortical origin or often extrasynaptically.42,101,107,112 In cortex, D1 receptors are expressed either presynaptically or, more often, on dendrites postsynaptic to either excitatory or inhibitory synapses.41,101 The placement of receptors on dendrites postsynaptic to glutamatergic or GABAergic synapses suggests that the D1 receptor is involved in modulating the response of neurons to these neurotransmitters.

In human and non-human primate brain, the distribution of D1 mRNA,106,113-115 immunoreactivity41,112 and binding sites116-119 is similar to that of the rat, with highest levels seen in CAU, PUT, NACC, SN and olfactory bulb, and an overall higher expression of D1 than

D2-like binding sites (10-20 times higher in cortex).117 Unlike in the rat, however, D1 receptor mRNA is also expressed in the SNr.106 Cortical D1 binding shows a rostrocaudal decreasing gradient similar to that of D2-like receptors with highest binding in prefrontal and lowest binding in occipital cortex.120 As in rat, D1 receptors are primarily located on dendrite spines postsynaptic to glutamatergic112,121 or GABAergic synapses,112 indicting their role in modulation of postsynaptic glutamatergic and GABAergic responses.

2.1.3.2. Distribution of the dopamine D2 receptor

Like the D1 receptor, the D2 receptor is expressed in all of the major dopamine neuron projection areas. In the rat, the highest levels of D2 mRNA,102,113,122,123 immunoreactivity41,124 and D2-like binding sites102,106,125-130 were seen in the STR, NACC, olfactory tubercle and olfactory bulb, whereas moderate levels were seen in many forebrain structures including the

18 cortex (especially prefrontal, anterior cingulate, entorhinal and perirhinal cortices), the islands of

Calleja, ventral pallidum, GP, amygdala, hippocampus, subiculum, lateral habenula, STh and mammilary bodies, as well as midbrain and brainstem nuclei such as the SN, the superior and inferior colliculi, the dorsal raphe nucleus and the locus coeruleus. Throughout the rat brain D2- like receptor binding site density is approximately 10-30% that of the D1 receptor,130,131 with the maximum brain D2-like receptor density (that in STR and NACC) being approximately 25-30 fmol/mg tissue (equivalent to approximately 250-300 fmol/mg protein or 25-30 nM).132-134 It should be noted that because of the D2/D3 non-selectivity of the radioligands used (such as

[3H]-raclopride,102 [125I]-iodosulpiride122,125 and [3H]-spiperone100) the radioligand autoradiographic experiments above more accurately characterize the summed distribution of

D2 and D3 (D2-like) receptors, rather than the distribution of the D2 receptor alone. Many of the common D2 ligands were pharmacologically re-classified as D2-like ligands after it was shown that they had similar affinity for the then newly-discovered D3 receptor.135 This also helps to explain the presence of D2-like binding sites in regions such as the islands of Calleja and lobes 9 and 10 of the cerebellum, which do not express D2 mRNA or immunoreactivity.122

D2-like binding in these regions is instead attributable to the D3 receptor (see section 2.1.3.3).

In human and non-human primate the distribution of the D2 mRNA and D2-like binding sites is generally similar to that seen in the rat. Highest levels of D2 mRNA are seen in the CAU and PUT and within the ventral tier neurons of the SNc.115,136,137 Substantial D2 mRNA expression is also seen various other brain areas including the hippocampus, bed nucleus of the stria terminalis, preoptic area, cerebral cortex, thalamus, amygdala complex and hypothalamus.115,138-142 The distribution of D2-like binding sites is consistent with the distribution of D2 mRNA with the highest D2-like binding seen in the CAU, PUT and

NACC.116,143-146 Lower levels of D2-like sites were seen in the GPe (about 25% of CAU and

PUT), whereas D2-like binding sites were very low in the GPi.147 Unlike D1 binding in CAU

19 and PUT, D2 binding in these regions shows little heterogeneity between patch and matrix compartments.148 D2-like binding site density in cortical regions was found to be 10-20 times lower than the density of D1 receptor binding sites,120 with the highest D2-like binding seen throughout the temporal cortex.147

At the cellular level, striatal D2 receptors, as assessed by immunohistochemical techniques, are expressed in a wide variety of locations – postsynaptically on the cell bodies and dendritic processes of both GABAergic projection neurons and cholinergic interneurons,149,150 pre-synaptically on both dopaminergic42,151 and non-dopaminergic40,41,152 axon terminals, as well as extrasynaptically.42 In the frontal cortex, the main cortical projection area of dopaminergic neurons, D2 receptors are found on neurons whose size is consistent with that of either glutamatergic pyramidal neurons or GABAergic interneurons and appear, based on cell size, to be expressed on a different population of cells than the D1 receptor.153 In the SNr and SNc, D2 receptors are found presynaptically both on cell bodies and on dendritic processes.41,42 In the

NACC and olfactory tubercle, D2 receptors are presynaptically located on dopaminergic axon terminals, as evidenced by their co-expression with tyrosine hydroxylase (the enzyme responsible for the rate-limiting step in dopamine synthesis and a marker for dopaminergic cells), and postsynaptically on dendritic processes.154-157 The data indicate that the D2 receptor is optimally located to mediate three physiological functions of dopamine: 1) postsynaptic modulation of neuronal responses to glutamatergic, GABAergic and synaptic transmission; 2) presynaptic regulation of dopaminergic neuron function; 3) regulation of neuronal function in response to extrasynaptic dopamine (often referred to as volume transmission).

2.1.3.3. Distribution of the dopamine D3 receptor

Although expressed in many of the same regions, the D3 receptor has a more limited distribution, and is expressed at lower levels, than either the D1 or D2 receptor subtypes. In the

20 rat brain, the distribution pattern of D3 receptor mRNA partially overlaps with that of the D1 and D2 receptors, with highest D3 mRNA expression found in the NACC, olfactory tubercle, islands of Calleja, SN and lobes 9 and 10 of the cerebellum.122,158,159 Lower, but appreciable levels of D3 mRNA are seen in the mammillary bodies, hypothalamus, septal area, the bed nucleus of the stria terminalis, the diagonal band of Broca and the lateral geniculate nucleus.122,158,159 A similar pattern of D3 mRNA expression is seen in human brain with the highest levels in the NACC and ventral STR, substantial expression seen also in the primary visual cortex and the dentate gyrus of the hippocampus, and moderate to low expression seen in remaining cortical areas, CAU and PUT, anterior and medial thalamic nuclei, mammillary bodies, amygdala, hippocampal CA region, lateral geniculate body, SNc, locus coeruleus and raphe nuclei.160-162

Mapping of D3 receptor binding sites was first done with the radioligand [3H]-7-OH-

DPAT, although there is now evidence that the D3-selectivity of this ligand is less than originally thought,163 adding the potential complication that a portion of the reported binding signal is due to the D2 receptor. Nevertheless, the distribution of [3H]-7-OH-DPAT binding sites agrees in general with the distribution of D3 binding sites visualized with ligands of greater selectivity, such as [3H]-PD-128907 (see below). In rat brain, the highest density of [3H]-7-OH-

DPAT binding sites is seen in the olfactory tubercle and islands of Calleja, followed by lobes 9 and 10 of the cerebellum, NACC, olfactory bulb and STR.164 A similar regional pattern is seen with the more D3-selective radioligand [3H]-PD-128907.165,166 The regional density of [3H]-PD-

128907 binding sites in rat brain is shown in Table 1. This regional rank order has also been confirmed using autoradiography with single concentrations of [3H]-PD-128907.167,168 Note that the density of the D3 binding sites represents at most a few percent of total D2-like binding sites in STR and NACC (25-30 nM).

21 Table 1. Density of [3H]-PD128907 binding sites in rat brain Binding site density Brain region fmol/mg proteina nMb Islands of Calleja 40 4 NACC 12 1 CER lobes 9 & 10 5 0.5 Hypothalamus 3.4 0.3 STR 2.3 0.2 SN & VTA 2.0 0.2 Amygdala 1.9 0.2 Frontal cortex 1.4 0.1 a data from reference 167 b calculated from fmol/mg protein data assuming ~100 mg of protein per mL of wet tissue weight and a tissue density of 1 g/mL.

The binding of both [3H]-7-OH-DPAT and [3H]-PD-128907 have also been examined in human brain, and for the most part paint a similar picture of the distribution of the D3 receptor.148,169,170 For both radioligands, the highest binding is seen in the Islands of Calleja and

NACC, followed by the ventral CAU, ventral PUT, with binding in the remaining areas similar to that seen in low D3-receptor expression areas, such as the cerebral and cerebellar cortices.148,169,170 The binding of these radioligands is exceedingly low in the globus pallidus (3-

10% of that seen in NACC),170 which is a surprising finding given that this region generates a large in vivo binding signal with the newly-developed D2/D3 agonist radiotracers [11C]-(+)-

PHNO and [11C]-(-)-NPA that can be blocked by treatment with D3-selective drugs. Other, less direct in vitro autoradiographic methods, such as using 7-OH-DPAT treatment to estimate the

D3 component of [125I]-epidepride binding, have indicated the presence of D3 receptors in the globus pallidus, but as yet there is no way to fully reconcile the available in vitro data with the presence of presumably D3 binding in the globus pallidus in vivo.

22 2.1.3.4. Distribution of the dopamine D4 receptor

The distribution of the dopamine D4 receptor is somewhat different from that of the other dopamine receptor subtypes, with mRNA171,172 and immunoreactivity173-175 in rodent brain being more heavily expressed in cortex (especially frontal and piriform cortices) than in any other brain region. Lower levels of expression have also been noted in NACC, STR (especially within the patch compartment), SNc, the medial temporal lobe (including hippocampus, amygdala and entorhinal cortex), thalamus and hypothalamus.172-175 In general, this distribution suggests a preferential involvement of the D4 receptor in emotional and cognitive functions, rather than in motor function as is the case for the D1 and D2-like receptors. Within the STR,

D4 receptor immunoreactivity is found both on cell bodies and within the neuropil, most often associated with dendritic shafts and spines,176 whereas in the NACC it is found associated primarily with axonal terminals.177 These data suggest that the D4 receptor may play a role either as a heteroreceptor (in STR) or as an autoreceptor (in NACC).177

In the human and non-human primate brain, D4 receptor mRNA and immunoreactivity, like in the rodent brain, are found at their highest levels within the frontal cortex, with substantial expression also seen within the amygdala, hippocampus and entorhinal cortex, the hypothalamus and cerebellum.141,178,179 Unlike in rodents, however, the receptor mRNA and immunoreactivity does not appear to be expressed in the STR, VTA or SN (Meador-woodruff et al. 1996).

Quantitation of regional D4 receptor binding sites has been accomplished in both rat and human brain with the D4-selective radioligand [3H]-NGD 94-1.180,181 These studies revealed, in general agreement with in situ hydridization and immunohistochemical experiments, that D4 binding sites in rat brain were highest within the cortex, septum, hippocampus, areas of the amygdala, hypothalamus, with lower levels of binding seen in the medial geniculate nucleus, superior colliculus and STR, whereas no detectable specific binding was seen in the NACC.181

23 3 In the human brain, highest [ H]-NGD 94-1 binding was seen in the dorsomedial thalamus (Bmax

= 20.8 fmol/mg of protein, ~2 nM), entorhinal cortex (19.5 fmol/mg, ~2 nM), hippocampus (8.9 fmol/mg, ~1 nM), hypothalamus (11.8 fmol/mg, ~1 nM), lateral septal nucleus (28.9 fmol/mg,

~3 nM), prefrontal cortex (10.6 fmol/mg, ~1 nM), whereas no quantifiable specific binding sites could be found in NACC, CAU, PUT or cerebellum.180,181 These data not only confirm the general distribution described by D4 mRNA and immunoreactivity, but also indicate that the D4 receptor is expressed overall at very low levels relative to the D1 or D2-like receptors.

2.1.3.5. Distribution of the dopamine D5 receptor

A quantitative description of the distribution of the D5 receptor is severely hampered by the lack of D5-selective radioligands. Therefore D5 receptor distribution has been characterized only using in situ hybridization (for determination of mRNA),182-184 or immunohistochemistry185-188 for semi-quantitative determination of D5 protein. The dopamine

D5 receptor has a pattern of brain distribution distinct from that of the other dopamine receptors.

In the rat brain, the highest levels of D5 receptor mRNA and immunoreactivity are found in the frontal cortex, hippocampus, hypothalamus, thalamus, mammilary nuclei and pretectal area, whereas only very low levels are seen in the STR, olfactory tubercle and cerebellum (within the vermis).184-186 In non-human primate brain a similar, but slightly wider pattern of mRNA distribution was found, with highest levels in hippocampus, cortex, intermediate levels in amygdala, thalamus, CAU, PUT, SN and GP.182 In contrast, the distribution of D5 mRNA in human brain was much more restricted, displaying high levels in hippocampus and cortex, lower levels in SN and entorhinal cortex, low levels in SN and no detectable signal in CAU, PUT or

GP.182,187

At the sub-cellular level, the D5 receptor in rat and human STR is localized on both

GABAergic and cholinergic interneurons, primarily on dendritic spines postsynaptic to

24 presumably excitatory synapses, indicating that D5 receptors likely play a role in mediating dopaminergic modulation of afferent glutamatergic signals.112,186,188 In human hippocampus and cortex, the D5 receptor is also located primarily on dendritic processes of pyramidal neurons, suggesting that in this area as well the D5 receptor mediates post-synaptic effects.187

2.1.3.6. Distribution of the dopamine transporter

The dopamine transporter, unlike the dopamine receptors, is expressed exclusively in dopaminergic neurons originating in the SN and VTA.189-192 DAT protein is associated with cell bodies, axon terminals within dopaminergic projection fields such as STR, NACC, hippocampus, cerebral cortex (prefrontal, entorhinal insular and primary visual), amygdala, the bed nucleus of the stria terminalis and thalamus37,40,193-196 as well as dendritic processes descending from the

SNc to the SNr.37,190,191,197 A quantitative description of the distribution of the DAT is made difficult by the complexity of DAT radioligand binding. Some radioligand binding studies report a single homogenous population of DAT binding sites,198-202 some report the presence of two classes of non-interconverting binding sites,203-205 while still others report a either one or two classes of binding sites depending on the type of binding experiment performed (saturation or competition),206 the radioligand used204,207 or the cell line used to express the DAT protein.208

209,210 Similar problems plague in vivo determinations of DAT Bmax. Although it seems that the two classes of DAT binding sites exist on the same protein molecule, as opposed to separate populations of DAT molecule,203 no precise estimate of the stoichiometry of these sites exists, making it difficult to interpret binding site density in terms of density of DAT protein molecules.

Nevertheless, studies reporting either one or two classes of DAT binding sites generally agree on the total density of STR binding sites in rat, non-human primate and human, which is in the range of 1-3 pmol/mg of protein (100-300 nM)199-201,203,204,211 although how this relates to DAT protein density is uncertain. Across human and non-human primate brain regions, DAT binding

25 is highest in the CAU and PUT, followed by the NACC and SN, whereas binding in remaining regions, including those known to receive dopaminergic innervation such as globus pallidus, frontal cortex, and hippocampus, is extremely low.205,212

26 2.1.4. Involvement of the dopaminergic system in substance abuse and schizophrenia

2.1.4.1. The dopaminergic system and substance abuse

Drug dependence can be defined in general by a pattern of compulsive drug seeking and drug taking behaviours that are undertaken despite their harmful physical, psychological or social consequences for the individual engaging in them. Drug dependence is often also associated with the phenomenon of withdrawal syndrome, which consists of physically or mentally painful symptoms that accompany prolonged cessation of drug taking. Various drugs have different propensities for causing dependence, and, for a given drug, the risk that an individual will develop dependence is a function of other factors including genetics, pre-existing psychiatric illnesses and even route of drug administration. The dopaminergic system is intimately involved in the addictive properties of various drugs of abuse, as well as in the harmful effects that chronic use of these drugs can have on brain function. Many addictive drugs, including the stimulants nicotine,213 cocaine214,215 and amphetamine,214,216 but also the non- stimulant drugs ethanol217 and morphine,214 increase extracellular dopamine concentration in the

STR, especially in the NACC, as measured directly by in vivo microdialysis. With the exception of nicotine, which influences dopamine transmission indirectly through cholinergic mechanisms, the elevation of dopamine levels produced by the above stimulant drugs is mediated trough the

DAT: cocaine (and derivatives) and methylphenidate block the DAT thereby preventing dopamine re-uptake; amphetamine and derivatives such as methamphetamine and MDMA inhibit uptake of dopamine into synaptic vesicles, resulting in increased cytosolic dopamine concentration and reverse transport through the DAT to the extracellular space.218-221

Destruction of the ascending dopaminergic fibers from the SNc/VTA222-224 or treatment with dopamine receptor antagonists225,226 destroys the reinforcing effects of stimulants, demonstrating that increased extracellular dopamine is necessary for the reinforcing effects of these drugs.

27 In vivo, changes in extracellular dopamine concentration can be non-invasively assessed by examining dopamine’s inhibitory effect on the binding of the D2/D3-selective benzamide

PET and SPECT radiotracers such as [11C]-raclopride and [123I]-iodobenzamide ([123I]-IBZM)

(123I radioactive half-life = 13.2 h).227-229 In human and non-human primate, addictive drugs such as cocaine,230-232 amphetamine233-235 and methamphetamine236 decrease the striatal binding potential of these radiotracers in accord with their ability to increase extracellular dopamine concentration. Importantly, in human, the magnitude of extracellular dopamine elevation in the ventral STR and NACC, as indicated by the percent decrease in D2/D3 radiotracer binding, correlates with subjective drug-induced europhoria.13,233,237,238 Furthermore, subjects for whom the stimulant drugs amphetamine and methylphenidate produced the smallest change in extracellular dopamine reported no pleasurable effects,237 suggesting that increased ventral STR and NACC dopamine is necessary for the reinforcing effects of these drugs.

Such individual differences in drug effect are also seen in animal models. In rats, a relatively high tendency for stimulant self-administration is associated with increased baseline extracellular dopamine concentration in the NACC and increased firing rate of SNc/VTA neurons,239 and is behaviourally predicted by the propensity of these animals to explore novel environments.240 This data is paralleled by findings from human subjects showing that the subjectively pleasurable effects of the methylphenidate are predicted by low baseline D2/D3 receptor availability, potentially representing increased extracellular dopamine, whereas displeasurable effects are reported by those with high baseline D2/D3 availability.13 In addition, novelty-seeking traits are an important predictor of drug abuse in human subjects.241 PET experiments have shown that in monkey, the tendency to self-administer cocaine is also linked to low baseline D2/D3 receptor availability.242 Interestingly, in the same monkeys, inter- individual differences in D2/D3 receptor availability were seen only after the monkeys had been switched from individual to group housing conditions, with lowest D2/D3 receptor availability

28 (and highest cocaine self-administration) seen in individuals with lowest social rank.242 These findings indicate a fascinating link between social factors such stress, dopaminergic function and drug self-administration.

Chronic substance abuse is associated with dopaminergic abnormalities including reduced D2/D3 and DAT availability in cocaine,231,243 methamphetamine244 and alcohol abusers.15,245 In methamphetamine abusers, reductions in DAT availability is associated with motor and cognitive deficits,246,247 and although DAT availability approaches control levels over time, it is not necessarily accompanied by a similar restoration of motor and/or cognitive function.247,248 In cocaine-dependent subjects, reduced D2/D3 availability is associated with decreased glucose metabolism243 and grey matter volume249 in orbitofrontal, cingulated and prefrontal cortex, areas involved in limbic function and, importantly, implicated in obsessive- compulsive disorder250,251 suggesting their involvement in compulsive drug-seeking behaviours.243 However, it is not clear whether the differences in D2/D3 receptor binding and cortical function between cocaine users and control subjects represent pathological changes associated with chronic drug use or pre-existing abnormalities that may predispose the individual to drug taking behaviours. In rodents, chronic stimulant administration leads to an in vitro increase in the dopamine D2 receptor high-affinity state.1,252 One of two in vitro states of the dopamine D2 receptor (the high- and low-affinity states) (see section 2.2.3), the high-affinity state has high-affinity for dopamine and other agonists and is thought to mediate the physiological actions of dopamine.253,254 This increase in the high-affinity state is thought to contribute to drug sensitization,252 relapse from abstinence in alcohol abusers,2 and the development of psychosis,1 which is sometimes seen in heavy stimulant abusers.255 However, currently there is no reliable way to measure the D2 high-affinity state in vivo, nor is there any direct evidence that a model with two D2 receptor affinity states is an accurate description of the dopamine D2 receptor in vivo. In vivo measurement of the D2 high-affinity state and the

29 applicability of the two-state model to in vivo D2/D3 radiotracer binding is the subject of much of this thesis, especially sections 3 and 4.

Taken together, human and animal studies demonstrate that the tendency to self- administer stimulant drugs, and thus the potential for harmful drug abuse, is associated with measurable changes in the dopaminergic system, especially D2/D3 receptor availability and the magnitude of stimulant-induced increases in extracellular dopamine. Interestingly, the degree to which an individual experiences pleasurable, reinforcing stimulant drug effects may be predicted by behavioural and/or social factors, such as novelty-seeking and social stress, opening the possibility of identifying groups at high risk for the development of substance abuse.

Finally, stimulant drugs produce long-lasting changes in the dopaminergic system and in the cortex that are associated with functional deficits.

2.1.4.2. The dopaminergic system and schizophrenia

Schizophrenia is a chronic psychotic illness resulting in life-long impairment of emotional, cognitive and social function. The symptoms of schizophrenia, typically first seen during adolescence to early adulthood, are commonly divided into the positive and negative symptoms. The negative symptoms, thus called because they represent functional deficits, consist of anhedonia (the loss of the experience of pleasure), avolition (loss of motivational drive), asociality (social withdrawal), alogia (poverty of speech) and affective flattening

(reduced expression of emotion). The positive symptoms are difficult to envision as a deficit in any particular functional domain, instead representing mental phenomena that are not part of normal experience. The positive symptoms typically consist of bizarre, often persecutory delusions, auditory and visual hallucinations, and disordered thought; often manifesting as disordered or incomprehensible speech. The first line of treatment for schizophrenia is the administration of antipsychotic drugs, some common examples being haloperidol, risperidone,

30 olanzapine and clozapine. These anti-dopaminergic drugs effectively ameliorate hallucinations and delusions, but unfortunately are of little use against (and may even exacerbate) the negative symptoms of schizophrenia.

The original dopaminergic hypothesis of schizophrenia, formulated in the mid 1960s, proposed that the symptoms of psychosis, particularly the so-called positive symptoms i.e. hallucinations and delusions, are caused by hyperactivity of the dopaminergic system.256

Consistent with this hypothesis were the subsequent findings that all antipsychotic drugs inhibit dopaminergic neurotransmission (by blocking dopamine D2/D3 receptors)257,258 and that stimulant drugs, which are indirect dopaminergic agonists, cause psychosis in high doses and exacerbate psychotic symptoms in schizophrenic subjects.259-261 These early findings inspired decades of research, primarily using in vitro binding techniques, probing for causative abnormalities in the dopaminergic system. More recent work using PET and SPECT imaging has allowed the non-invasive measurement of dopamine receptors, dopamine receptor occupancy by antipsychotic drugs, and dopaminergic neurotransmission in the living brain of schizophrenic subjects. This latter work, as discussed below, has yielded important findings that empirically confirm the dopaminergic hyperactivity originally postulated to explain the positive symptoms of schizophrenia.

Many in vitro postmortem studies using various tritiated ligands demonstrated an increase in D2/D3 receptor binding in schizophrenic basal ganglia.262-271 Other studies, however, demonstrated no such increase in D2/D3 binding sites,272-274 and the increases found in previous studies may have been in part due to the effects of antemortem antipsychotic treatment, which is known to cause D2 receptor upregulation.275,276 Postmortem investigations have also examined the binding of the other dopamine receptor subtypes. Indirect measurements of D4 receptor binding (e.g. [3H]-nemonapride binding (D2 + D3 + D4) minus [3H]-raclopride binding (D2 +

D3)) have yielded increases273,277-279 or no change274,280 relative to healthy controls, whereas

31 direct measurement using the D4-selective radioligand [3H]-NG 94-1 indicate increased D4 receptor expression in the entorhinal cortex of schizophrenic patients.180 Binding of the D3- selective radioligand [125I]-trans-7-OH-PIPAT was found to be increased in the basal ganglia of non-medicated schizophrenic subjects but not in patients receiving chronic antipsychotic treatment, suggesting an ameliorative effect of antipsychotic drugs on schizophrenia-related D3 overexpression.281 However, no such increase in D3 receptor binding could be found in vivo using the D3-selective PET radiotracer [11C]-(+)-PHNO (see sections 2.2.3.3 and 6 for discussion of the pharmacology of [11C]-(+)-PHNO). In vitro binding studies have found no change in the expression of the D1 receptor268,270,282,283 or DAT284-286 binding sites in schizophrenic versus healthy brain. Thus, in vitro binding studies reveal no “smoking-gun” receptor changes that can be labeled as the causative dopaminergic abnormalities in schizophrenia.

In vivo PET and SPECT studies paint a similar picture. Many studies have examined radiotracer binding to D2/D3 receptors in antipsychotic-naïve or drug-free schizophrenic patients using the D2/D3 receptor radiotracers [11C]-N-methylspiperone ([11C]-NMSP), [76Br]- bromospiperone ([76Br]-Br-SPIP) (76Br radioactive half-life = 16.2 h), [11C]-raclopride, [123I]-

IBZM, [76Br]-lisuride or [11C]-(+)-PHNO. Although two of these studies using [11C]-NMSP and

[76Br]-Br-SPIP reported increased D2/D3 receptor binding in the STR,287,288 the vast majority reported no difference in D2/D3 receptor binding between schizophrenic patients and control subjects.17,19,289-302 PET studies with high-affinity radiotracers [18F]-fallypride (18F radioactive half-life = 109.8 min) and [11C]-FLB-457 have reported decreased binding in extrastriatal regions such as thalamus, amygdala, cingulated cortex and temporal cortex.303,304 In addition,

D1 PET studies have reported changes in D1 binding in frontal cortex of schizophrenia patients, although these findings are now considered suspect due to the low D1 to 5-HT2 selectivity of the radiotracers used (see section 2.2.2.1.4.).291,305 Thus, functional in vivo imaging studies

32 therefore agree in general with in vitro postmortem studies in that they suggest no dopaminergic abnormality at the receptor level that could fully explain dopaminergic hyperactivity in schizophrenia. Though limited in number, PET studies reporting decreased cortical D2/D3 and

D1 receptor binding may be relevant to cortical dopaminergic hypoactivity thought to underlie the cognitive deficits (i.e. negative symptoms) of schizophrenia. A final consideration for dopamine receptor imaging in schizophrenia is the fact that in vitro radioligand binding studies consistently demonstrate increased D2 receptor high-affinity state binding in animal models of psychosis,306 which has led to the hypothesis that increased high-affinity state mediates dopaminergic hyperactivity in vivo in schizophrenic patients. However, a PET study with the

D2/D3 agonist radiotracer [11C]-(+)-PHNO, which should selectively label the high-affinity state in vivo, revealed no difference in binding between schizophrenic and healthy subjects.302

However the interpretation of in vivo [11C]-(+)-PHNO binding as a measure of high-affinity state receptors is debatable (see sections 3 and 4).

Many PET and SPECT studies have demonstrated the importance of D2/D3 receptor blockade in the therapeutic action of antipsychotic drugs.307 Common antipsychotics including haloperidol, risperidone, and olanzapine, as well as the less commonly used antipsychotic amisulpiride308 produce therapeutic effects at doses producing between 60 and 80% D2/D3 receptor occupancy,309-313 whereas in general the risk of motor and side effects increases above

80% D2/D3 occupancy.310,314-317 These studies have also pointed out important exceptions to this relationship, such as clozapine and quetiapine, which produce significantly lower D2/D3 occupancy at clinically-effective doses than the above antipsychotics.310,314,318 Although debate still remains as to why these antipsychotic are efficacious at lower-than-expected D2/D3 occupancy, it has been suggested that this property is due to either to a high ratio of serotonin 5-

HT2 to D2 (clozapine and quetiapine)319 or D1 to D2 blocking ratio (in the case of clozapine),320 high affinity for D4 receptors,321 or increased dissociation rate from D2/D3 receptors relative to

33 other antipsychotics.322 It does not appear that D1, D4 or 5-HT2 activity contributes in any direct way to the increased efficacy of clozapine or quetiapine, as selective D1323-326 or D4327,328 receptor antagonists do not possess antipsychotic efficacy, and amisulpiride and remoxipride exhibit antipsychotic efficacy without significant 5-HT2 activity.329 At present the low clozapine and quetiapine D2/D3 occupancy appears best accounted for by rapid dissociation kinetics322 such that significant dissociation has occurred by the time of the PET or SPECT measurement.

In agreement with this, Kapur et al. have demonstrated in rat that with appropriately short duration between administration and ex vivo occupancy determination, clozapine and quetiapine can produce high levels of D2/D3 receptor occupancy.330 The above body of work confirms the central role of D2/D3 receptor occupancy in antipsychotic drug efficacy and demonstrates that there exists no clear relationship between therapeutic effect and occupancy of other receptor types, such as the dopamine D1, D4 or serotonin 5-HT2 receptors, to which some antipsychotics also bind.

Recent work utilizing the competition between endogenous dopamine and the benzamide

PET and SPECT radiotracers [11C]-raclopride and [123I]-IBZM has provided strong evidence for dopaminergic hyperactivity in schizophrenia. Treatment with the dopamine-releasing drug amphetamine (AMPH) produces a larger reduction in the striatal binding of these radiotracers in schizophrenic subjects than in healthy controls.17,19,296 Importantly, increased AMPH-induced dopamine release in schizophrenics was associated with a transient increase in positive psychotic symptoms that resolved after drug washout,17,296 indicating the relevance of released dopamine to the severity of psychotic symptoms. Similar studies by the same authors have shown that in response to the dopamine-depleting drug α-methyl-para-tyrosine (α-MPT) the increase in [123I]-IBZM binding was greater in schizophrenic subjects,18 indicating greater baseline occupancy of D2/D3 receptors by dopamine compared to healthy controls. Together these studies indicate that the dopaminergic system in schizophrenic subjects is hyper-

34 responsive to pharmacological stimulations and that it is also hyperactive under normal, non- pharmacological conditions. This work also suggests that schizophrenic subjects have an increased expression level of D2/D3 receptors that is masked from PET and SPECT measurement by elevated baseline dopamine occupancy.331 Together, these functional imaging studies provide a major confirmation of the dopaminergic hyperactivity hypothesis of schizophrenia, and represent a significant inroad to the understanding of the neurochemical basis of schizophrenia.

35 2.2. Quantification of in vivo radiotracer binding

The following section (2.2.1) provides an overview of the basic theory required to understand the in vivo biodistribution of radiotracers, with particular focus on the quantification of reversible radiotracer binding to protein targets. The discussion in these sections assumes that the radioactivity in tissue (CT, CS etc.) is due only to the parent (i.e. non-metabolized) radiotracer.

2.2.1. Radiotracer binding under equilibrium conditions

The theoretical approach to quantification of in vivo radiotracer binding in both PET and

SPECT brain studies has its roots in the analysis of in vitro radioligand binding at equilibrium.332 The specific binding of a radioligand at equilibrium obeys the Michaelis-Menton relationship:

B F B = max (1) K D + F

-1 where B is the concentration of bound radioligand (mol·L ), Bmax is the density of radioligand specific binding sites (i.e. neurotransmitter receptor or transporter proteins, enzymes etc.)

-1 -1 (mol·L ), F is the concentration of free radioligand (mol·L ) and KD is the equilibrium dissociation constant (mol·L-1) representing the radioligand concentration at which fifty percent of binding sites are occupied. At tracer radioligand concentration F << KD and equation (1) reduces to

B F B = max (2) K D

Rearrangement of this equation gives the classical definition of the binding potential (BP):332

B B BP ≡ = max . (3) F K D

36 This relationship indicates that at tracer concentration, the BP is equal to the product of radioligand binding site density (Bmax) and the affinity of the radioligand for these binding sites

(affinity = 1/KD). KD can also be expressed as the ratio of the dissociation rate constant koff

-1 -1 -1 (min ) to the association rate constant kon (mol·L ·min ), and the BP can therefore be written as

B k B BP = = on max (4) F koff

This expression of the BP is the most useful form for the current discussion as it expresses the ratio of B to F in terms of the rate constants koff and kon, quantities that have analogous parameters (or expressions) in vivo (discussed below).

Figure 4. The 1-TC model containing an arterial plasma compartment, CP, and one tissue compartment, C1. Exchange of radiotracer between these two compartments is governed by the first-order rate constants K1 and k2

Radiotracers for PET or SPECT imaging are typically administered intravenously, and through arterial blood are distributed throughout the body. Arterial blood plasma and individual tissues regions of interest (ROIs) represent separate “compartments” in which radiotracer can be distributed and between which radiotracer can exchange. Figure 4 shows a simple compartmental model in which free radiotracer in plasma exchanges with a single tissue compartment (1-TC model). In this model, the transfer of radiotracer from plasma to tissue is

-3 -1 governed by the rate constant K1 (mL·cm ·min ) and transfer in the reverse direction is

-1 governed by the rate constant k2 (min ). If the concentration of radiotracer in blood plasma (CP) is held constant by continuous intravenous infusion of radiotracer,333,334 equilibrium is

37 eventually reached with the tissue compartment (C1). Under these conditions, the flux of radiotracer from the plasma to tissue is given by

J1 = K1CP (5) and the flux in the reverse direction by

J 2 = k2C1 (6)

The net flux, J = J1 + J2, at equilibrium is zero (i.e. J1 = -J2) and we can therefore obtain the relationship between C1 and CP, which is also known as the volume of distribution (V) for radiotracer in the tissue compartment:

C1 K1 V1 = = (7) CP k2

This quantity (cm3·mL-1) is referred to as a “volume” because it can be conceptualized as the ratio of tissue and plasma volumes (cm3 and mL, respectively) containing the same mass of radiotracer.335 The importance of the volume of distribution to radiotracer binding in vivo is that it can be written in terms of the kinetic parameters of the model. CP can be measured by arterial blood sampling, C1 by the imaging modality (PET or SPECT) or in animal studies by counting the radioactivity in excised tissue samples, and V1 calculated as the ratio of C1 to CP. However,

K1 and k2 cannot be resolved with equilibrium measurements alone (see section 2.2.1.2 for description of analysis under non-equilibrium conditions).

We will next consider a more complex model, the two-tissue compartment (2-TC) model, commonly used in PET and SPECT (Figure 5). This model consists of three compartments: an arterial plasma compartment and two tissue compartments, one containing only non- displaceable radiotracer binding (indicated by the concentration CND), the other containing only specific binding (CS). Non-displaceable binding refers to binding that cannot be blocked or displaced by other pharmacological agents. The non-displaceable compartment can be further divided into separate compartments representing free radiotracer in tissue (CFT) and non-

specifically bound radiotracer (CNS). However, to simplify the model and reduce the total number of kinetic parameters, it is commonly assumed that CFT and CNS equilibrate very rapidly, in comparison to the other compartments, such that they collapse to form a single non- displaceable compartment. Importantly, the two tissue compartments in this model (CND and CS) can exist within the same physical space such that a particular tissue volume can contain either

CND, or both CND and CS (however, no tissue can have CS without also having CND).

At equilibrium the flux of radiotracer moving from plasma to the non-displaceable compartment (J1) is equal to that moving in the reverse direction (J2) such that the volume of distribution can be written as

CND K1 VND = = (8) CP k2

The flux of radiotracer moving into the specific binding compartment is given by

J3 = k3CND (9) and, by expressing CND in terms of K1, k2 and CP, can be written as

K1k3 J3 = ⋅CP (10) k2

The flux of radiotracer moving in the reverse direction is given by

J4 = k4CS (11)

Given that the net flux at equilibrium is zero (J3 = J4) the volume of distribution for the specific binding compartment is

CS K1k3 VS = = ≡ BPP (12) CP k2k4

This quantity is especially important because it is proportional to the product of available binding site density (Bavail) to radiotracer affinity (1/KD), and thus represents the in vivo

335 analogue of the in vitro BP defined by equation (4). The subscript P refers to the fact that CS is expressed relative to the total concentration of parent radiotracer in plasma. In a given tissue volume, VS (or BPP) is the difference between the total volume of distribution, VT, and VND.

Thus both VT and VND must be estimated in order to estimate VS. VT is easily calculated as the ratio of total tissue (CND + CS) to plasma concentrations at equilibrium, whereas VND is typically assumed to be equal to the total volume of distribution in a reference region devoid of specific binding.

To see clearly why BPP is proportional to the product of binding site density and affinity, we will next express the in vitro BP (equation (4)) in terms of parameters measurable in vivo.

The in vivo parameter equivalent to the B is the concentration in the specifically bound compartment:

B = CS (13)

F is equivalent to the concentration of free aqueous radiotracer in the tissue compartment (CFT):

F = C FT (14)

At equilibrium, CFT is equal to the concentration of free aqueous radiotracer in plasma (CFP), which in turn is equal to CP multiplied by the fraction of total plasma radiotracer concentration that is free to transfer into the tissue compartment, fP (i.e. not taken up by blood cells or bound to plasma proteins). Thus equation (14) can be written

F = f PCP (15)

40 Using equations (13) and (15), the in vitro BP in equation (4) can be written in terms of in vivo parameters:

C BP = S (16) f PCP

Substituting for CS using equation (12) gives the equation for the in vivo binding potential, BPF, which expresses the ratio of specifically-bound radiotracer to free radiotracer in plasma:

K1k3 VS BPF ≡ = (17) f Pk2k4 f P

3 -1 Note that that BPF (cm ·mL ) is equal to BPP divided by the free fraction of radiotracer in plasma, fP. That is, both BPP and BPF are proportional to Bavail/KD.

A third, and perhaps the most commonly-used form of the binding potential, expresses specific binding relative to non-displaceable binding, and can be expressed as volumes of distribution or kinetic rate constants as follows:

VS CS k3 BPND = = = (18) VND CNS k4

The major advantage of BPND is that its determination does not require arterial blood sampling, as does the determination of BPP or BPF. Determination of BPF is the most labourious of the three, as it requires not only correction for the presence of radiolabeled metabolites in plasma

(typically by high-performance liquid chromatography or thin-layer chromatography) but also the determination of the plasma free fraction (typically by ultracentrifugation to separate cell- and protein-associated radiotracer).

Table 1 summarizes the three forms of the binding potential and their expression in terms of kinetic rate constants and volumes of distribution. The binding potential (either as BPP,

BPF or BPND) is the primary outcome measure in PET and SPECT studies of radiotracer binding,

41 and can be used as a measure of receptor availability as well as to assess changes in either the

336-338 available binding site density (Bavail) or affinity (1/KD).

Table 2. In vivo binding potentials and their definition in terms of volumes of distribution and kinetic rate constants

Binding Specific binding Volumes of distribution Kinetic rate constants potential relative to

K1k3 BPP Total plasma radiotracer (Cp) VS = VT -VND k2k4

Free plasma radiotracer VS VT -VND K1k3 BP = F (f ·C ) P p f P f P f Pk2k4

Non-displaceable radiotracer VS VT -VND k3 BP = ND in tissue (C ) ND VND VND k4

Although the binding potential represents the ratio of bound to free radiotracer at equilibrium, it can also be estimated under non-equilibrium conditions (i.e. after bolus radiotracer injection) using an approach similar to that described above if certain kinetic criteria are met. After bolus injection of a reversible radiotracer, the concentration in each compartment rises to a maximum value then falls over the course of the experiment. Thus, at some point in the experiment CS(t) (specific binding as a function of time) is maximal and therefore

dC ( t ) S = 0 (19) dt

This condition defines so-called transient equilibrium. Equation (19) can be expressed in terms of the rate constant for the specific binding compartment:

dC (t) S = k C (t*) − k C (t*) = 0 (20) dt 3 ND 4 S

42 where t* is the time of transient equilibrium. In the ROI, specific binding is the difference between total (CT(t)) and non-displaceable (CND(t)) binding. Substituting CT(t) - CND(t) for CS(t) and rearranging yields

CT ( t*) − C ND ( t*) k3 = = BPND (21) C ND ( t*) k4

Thus at transient equilibrium, the ratio of CT(t) to CND(t) can be used to estimate BPND. However, in the ROI, only CT(t) can be directly measured. If a suitable reference region exists (i.e. a region that is practically devoid of specific binding) its concentration (CR(t)) can be used as a surrogate for CND(t) in the ROI:

CT ( t*) − CR ( t*) BPND = (22) CR ( t*)

Theoretically this relationship is strictly correct only at the moment of transient equilibrium.

Also, the assumed equivalence between CR(t*) and CND(t*) requires that the kinetics of the reference region and the non-displaceable compartment in the ROI are identical, which may not be precisely true since in the ROI the CND(t), is contrast to CR(t), is coupled to CS(t) by the rate

339 constants k3 and k4. Nevertheless, the BPND determined using the transient equilibrium method generally provides a good approximation of the BPND determined either by analysis at true equilibrium binding or by kinetic analysis under non-equilibrium conditions (described

333,334,340,341 below). To reduce the error involved with such determinations of BPND, the CT(t) and

CR(t) can be integrated over a period of time surrounding t* and these integrals used in place of

334,342 the single time point concentrations. Similarly precise estimates of BPND can also be made using CT(t) and CR(t) (or their integrals) at time points later than that corresponding to transient

334,341,343,344 equilibrium. However, since CND(t) is fed by CS(t), CR(t) may be a less accurate

344 estimate of CND(t) at late time points when CS(t) is relatively high. Accordingly, the late time

344 point method tends to overestimate BPND. Another potential pitfall with the late time point

43 estimates of BPND is that it they are more sensitive to blood flow than those determined by the transient equilibrium method.334,341,345 However, if average blood flow is similar between

340,344 subject groups, late time point methods provide a measure that is proportional to BPND. In this thesis, the outcome measure using a single late time point and is referred to as the specific binding ratio (SBR).

2.2.2. Radiotracer binding under non-equilibrium conditions

Several methods have been developed for analyzing dynamic time-concentration data in order to determine the underlying kinetic rate constants. These methods are mathematically complex relative to the analysis of equilibrium data and only a basic description of the most common methods is provided here.

In general, two types of analysis are used to analyze dynamic time-concentration data, kinetic modeling and graphical analysis. Kinetic modeling (section 2.2.1.2.1) employs non- linear regression to fit the measured data to an operational equation describing the change in tissue concentration over time in terms of the kinetic rate constants of the applied compartmental model. An iterative fitting process determines the values of the kinetic rate constants that best fit the measured data. Graphical analysis,346-349 on the other hand, mathematically transforms the time-concentration data such that a linear relationship is obtained, relating time and tissue and/or plasma concentrations. Linear regression then allows the determination of physiological parameters from the slope and y-intercept of the best fit line.

Although the graphical methods (described in section 2.2.1.2.2) are formulated for the analysis of dynamic time-concentration data, much like equilibrium analysis they typically allow the estimation of volumes of distribution (and from them the binding potential) rather than individual kinetic rate constants.

44 2.2.2.1. Kinetic modeling of dynamic time-concentration data

Since radiotracer in all tissues is originally derived from blood plasma the concentration of free radiotracer in plasma as a function of time is known as the input function (CP(t)). Here, we will consider the input function to be a measure of free radiotracer concentration (i.e. corrected for the presence of radioactive metabolites and radiotracer bound to plasma proteins).

When the concentration of plasma radiotracer is constant, as in an equilibrium experiment with constant radiotracer infusion, CP(t) has a constant value. After a bolus radiotracer injection, however, the value of CP(t) changes over time, with an initial, rapid rise during injection, followed by a slower decrease due to distribution and elimination processes (i.e. extraction of radiotracer by tissue, radiotracer metabolism in liver, renal excretion, etc.). The time course of the plasma input function depends directly on the kinetics of these distribution processes. To understand the relationship between tissue radiotracer concentration and CP(t), we will consider an idealized scenario involving the 1-TC model.350

For simplicity, consider an input function of magnitude CP and infinitesimally short duration (i.e. an “ideal” bolus). The movement of radiotracer from plasma to tissue is governed by the rate constant K1 and the movement in the reverse direction by the rate constant k2. The tissue response, C1(t), to an ideal bolus CP is given by

C1( t ) = CP K1exp(-k2t) (23)

This equation states that in response to an instantaneous input CP, the radiotracer C1(t) falls from an initial value CPK1 in a mono-exponential fashion according to the rate constant k2. Next consider two consecutive ideal boli, the first of magnitude CP1 occurring at time T1 and the second of magnitude CP2 occurring at time T2. The tissue response after the first bolus is given by

C( t ) = CP1K1 exp[ −k2( t − T1 )] for T1 ≤ t < T2 (24)

45 and after the second bolus by

C1(t) = CP1K1e xp[ −k2( t −T1 )] + CP 2 K1 exp[ −k2( t −T2 )] for t ≥ T2 (25)

Thus, the concentration C1(t) is the sum of the tissue concentrations remaining after the boli at

T1 and T2. In fact, for an arbitrarily large number of ideal boli, C1(t) is equal to the sum of tissue concentrations remaining from all previous boli:

−k2 ( t −Ti ) C1(t) = ∑CPi K1e for i = 1, 2, 3, ... (26) i

If we envision the plasma input function CP(t) as being composed of an infinitely large number of individual ideal boli separated by infinitesimally small time periods, the equation for C1(t) can be written as the integral

t C (t) = C (s)K e−k2 (t −s)ds (27) 1 ∫ P 1 0 which describes the concentration C1(t) as a function of time in response to a dynamic input function CP(t). In the context of the 1-TC model, equation (26) is the operational equation which is optimized, by iteratively varying K1 and k2, to fit the measured time-concentration data. The general form of equation (26) is that of the convolution integral:

f ( t ) ⊗ g( t ) = ∫ f ( t )g( t − s)ds (28)

In kinetic modeling f(t) = CP(t), the input function measured by arterial blood sampling, and g(t)

= IRF(t), the impulse response function of the tissue region of interest (ROI). For a 1-TC model

IRF(t) = K1exp(-k2t) as in equation (26), whereas the complexity of IRF(t) increases greatly for higher order compartment models (i.e. 2-TC and 3-TC models).

The most commonly used compartmental model for analysis of reversible radiotracers binding is the 2-TC model (Figure 5), with the binding potential (BPP, BPF or BPND) generally being the desired outcome measure. The most comprehensive kinetic analysis of this model yields estimates of four rate constants (K1, k2, k3 and k4) in each ROI using a metabolite- and

46 free fraction-corrected arterial input function. Calculation of the binding potentials from individual fitted rate constants is sometimes referred to as the direct method.5,334,341 For many radiotracers, or for noisy time-concentration data, however, full kinetic analysis can result in large errors in these estimates333,351,352 and consequently in the value of the binding potential.334,344 Total distribution volumes can often be fit with greater precision than individual rate constants, thus binding potentials may have less uncertainty when calculated from fitted

5,333,344,353 values of VT and VND.

Simplified kinetic models are often used to decrease the overall complexity of the model and thus improve the precision with which the rate constants and binding potentials can be estimated. Table 2 summarizes the simplified kinetic analysis methods described here, the parameters that can be estimated and the assumptions that are required. One strategy is to approximate the time-concentration data in a given region with a simpler compartmental model.

For example, whereas a 2-TC model may be appropriate for the ROI, a reference region with very low levels of specific binding, may be approximated well by a 1-TC model,339,344,354 thus reducing the total number of fitted rate constants. This, however does not apply to some radiotracers despite the apparent lack of specific binding in the reference region.355 A similar simplification is based on the assumption of rapid exchange between non-displaceable and specific binding, such that the time-concentration data in the ROI can also be approximated by a

1-TC model.5,339 In this case, the transfer from the ROI to plasma is given by:

k k k = 2 = 2 (29) 2a k 1− BP 1+ 3 ND k4 and the total volume of distribution given by

K1 K1 K1 VT = = = (1+ BPND ) = VND (1+ BPND ) (30) k2 a k2 k2

1+ BPND

Table 3. Common kinetic analysis methods. Compartments Fitted parameters Method Assumptions1 Ref. region ROI Ref. region ROI

2-TC direct --- 2-TC --- K1, k2, k3, k ---

2-TC direct K1, k2, k3, k4 --- 2-TC --- All regions same VND common VND (constrained K1/k2)

2-TC direct 2-TC 2-TC K1, k2, k3, k4 k3, k4 VND same as ref. region fixed VND

1-TC/2-TC direct 1-TC ref. region 1-TC 2-TC K1, k2 k3, k4 fixed VND VND equals ref. region VT

1-TC ref. region 1- or 2-TC indirect 1-TC 2-TC VT VT = VS+VND VND equals ref. region VT

1-TC ref. region K1, 1-TC 1-TC 1-TC K1, k2 1-TC ROI k2a = k2/(1+BPND) VND equals ref. region VT

1-TC reference region FRTM 1-TC 2-TC K1, k2 R1, k2, k3, BPND VND equals ref. region VT

1-TC reference region SRTM 1-TC 1-TC K1, k2 R1, k2, BPND 1-TC ROI VND equals ref. region VT

1 All models here also include the assumption of rapid free (CF) to non-specifically bound (CNS) transfer kinetics.

The BPND can be calculated if an estimate of VNS can be obtained, usually from a reference region. This simplification is only appropriate for select radiotracers, such as [11C]-raclopride354 and [11C]-SCH23390,339 for which a 1-TC model provides at least as good a fit to the measured data in the ROI as a 2-TC model. For some radiotracers, such as [11C]-(+)-PHNO,5 the 1-TC

ROI criterion cannot be met and thus this simplification cannot be applied.

A second strategy is based on the assumption of equal VND across all regions. For example, the fitting procedure for the ROI can be simplified by setting K1/k2 to that determined for a reference region in a separate fitting procedure or by simultaneously fitting all regions with

5,356 the constraint of finding K1/k2 ratio common to all regions.

48 Although these simplified methods can increase the precision with which kinetic parameters and BPND are estimated, they are still encumbered by the need for invasive arterial blood sampling and correction for plasma free fraction and radiotracer metabolism. Thus several kinetic analysis methods have been developed that obviate the need for arterial blood sampling.

Probably the most important of these methods are the reference tissue models (RTM). The original full reference tissue model (FRTM)228 is based on two assumptions. First, the model assumes that there exists a reference region, CR, devoid of specific binding that can be represented by the 1-TC model. The second assumption is that the total volume of distribution in the reference region (VR) is equal to the non-displaceable volume of distribution in the ROI

(VND). With these assumptions, the time-concentration data for the ROI can be expressed as a function of that in the reference region, i.e. CR(t) can be used as the input function for CS(t), the concentration of specifically bound radiotracer in the ROI. An operational equation can be derived that contains four parameters; k2, k3, BPND, and R1. The parameter R1 is equal to the ratio

K1’/K1, where K1’ and K1 are the rate constants for plasma to tissue transfer for the reference region and ROI, respectively. Although this method generally provides precise estimates of

BPND, the other parameters are estimated with large errors, the fitting procedure is slow to converge and is sensitive to initial parameter values from which the iterative fitting procedure begins.228,339 In order to increase the precision in determination of the fitted parameters, the simplified reference tissue model (SRTM) was developed. This model involves a further assumption that the transfer between non-displaceable and specific binding compartments is rapid such that binding in the ROI can be approximated by a 1-TC model.339 With this assumption, the number of parameters in the operational equation is reduced from four to three

(R1, k2 and BPND), and the errors associated with k2 and R1 and the convergence time and sensitivity to initial values are decreased relative to the FRTM.339 For noise-reduction in the generation of parametric images of BPND and R1, a simplified voxel-based analysis method has

49 also been developed (STRM2), which applies a preliminary SRTM fitting procedure to estimate

357 k2’, then fixes this value for all voxels in a subsequent SRTM procedure, rather than determining a separate value of k2’ for each voxel. The rationale behind this strategy is that, because there is only a single reference region, there should only be one true value of k2’.

Although the simplified kinetic analysis methods described above are convenient in that they do not require arterial blood sampling, and proved precise parameter estimates, they rely on assumptions that are not present in the full 2-TC model. Violation of these assumptions (VR =

VND for FRTM as well as 1-TC region of interest for SRTM) can result in bias in the estimated

355 BPND. Thus care must be taken to validate these methods for each radiotracer and ROI to be sure that parameter errors relative to a full compartmental analysis and bias due to any violations in model assumptions are acceptably small.

2.2.2.2. Graphical analysis of dynamic time-concentration data

Two main types of graphical analysis have been developed for analysis of dynamic time- concentration data in PET and SPECT brain imaging: the linear regression-based models developed by Logan et al.348,349 and the multilinear regression based models developed by Ichise et al.346,347 For a 1-TC model the operational equation of the Logan method is derived as follows.

The instantaneous rate of change in tissue concentration is given by

dC ( t ) 1 = K C (t) − k C (t) (31) dt 1 p 2 1

Integration of equation (30) gives

C (t) = K C (t)dt− k C (t)dt (32) 1 1 ∫ p 2 ∫ 1

Rearrangement and division by C1(t) and k2 gives the operation equation for the Logan plot method:

C1(t)dt K C p (t)dt 1 ∫ = 1 ∫ − (33) C1(t) k2 C1(t) k2

This linear equation has a slope equal to K1/k2. The plot of ∫Cp(t)dt/C1(t) versus ∫C1(t)dt/C1(t) is not linear at early time points after bolus injection. The time needed to reach linearity must be determined empirically for each radiotracer.349 For a 2-TC model, the efflux of radiotracer from the ROI containing non-displaceable and specific binding compartments is k2/(1+k3/k4) and the

Logan plot equation for a 2-TC model can be written as

C (t)dt C (t) ∫ T K1 ⎛ k3 ⎞ ∫ P 1 ⎛ k3 ⎞ = ⎜1+ ⎟ − ⎜1+ ⎟ (34) CT (t) k2 ⎝ k4 ⎠ CT (t) k2 ⎝ k4 ⎠

Where CT(t) is the total radiotracer concentration in the ROI (i.e. CND(t) + CS(t)). Note that in both equation (32) and (33) the slope is equal to the total volume of distribution (VT) for the ROI.

The general form of the Logan plot equation is given by

C ( t )dt C ( t ) ∫ T ∫ P = VT + b (35) CT ( t ) CT ( t ) and thus provides an estimate of VT that is independent of the compartmental configuration. The determination of VT in the ROI and in a reference region (to approximate VND) allows the calculation of BPP, BPF or BPND as (VT - VND), (VT - VND)/fP and (VT - VND)/VND, respectively. The

Logan plot method has been shown to increasingly underestimate VT with increasing noise in the time-concentration data.347,358 This bias can be rectified by applying linear regression procedures that account for noise in both the dependent (∫C1(t)dt/C1(t)) and independent (∫Cp(t)dt/C1(t))

347,358 variables. The Logan plot method has also been adapted for determination of VT with the use of a reference region, such that arterial blood sampling is not required.348 To derive the model equation for the is method, a 1-TC model is assumed for the reference region and the appropriate Logan plot equation (32) is solved for ∫Cp(t)dt:

k ' ⎛ C (t)⎞ C (t)dt = 2 ⎜ C (t)dt+ R ⎟ (36) ∫∫P ⎜ R ⎟ K1' ⎝ k2' ⎠ where CR(t) is the radiotracer concentration in the reference region, K1’ and k2’ are the influx and efflux rate constants for the reference region. Substituting this expression for ∫Cp(t)dt into equation (33) gives

C (t) C (t)dt+ R C (t)dt ∫ R ∫ T k2' K1 ⎛ k3 ⎞ k2' = ⎜1+ ⎟ + b (37) CT (t) K1' k2 ⎝ k4 ⎠ CT (t)

As in the SRTM, if VND = K1/k2 in the ROI is assumed to be equal to VT = K1’/k2’ in the reference region, equation (36) simplifies to give the operational equation for the reference tissue Logan plot method:

C (t) C (t)dt+ R C (t)dt ∫ R ∫ T ⎛ k3 ⎞ k2' = ⎜1+ ⎟ − b (38) CT (t) ⎝ k4 ⎠ CT (t)

The slope of this equation, 1 + k3/k4, is equal to VT/VND or 1 + BPND. The major limitation of this method, apart from the noise-induced bias mentioned above, is that it cannot determine the value of k2’, requiring it to be entered along with the time-concentration data, as an input

348 parameter. The value of k2’ can be estimated, as was suggested in the original formulation of the model, from the known average k2’ of the subject group or by first fitting the data using another method, such as the SRTM. In the latter case, the Logan reference tissue approach serves more as validation of the BPND value obtained using the SRTM, than as an independent analysis method.

A second common graphical analysis method was developed by Ichise et al.347 and is derivative of the Logan plot method. This method also applies linear regression to the data, but the operational equation includes only the integrated tissue time-concentration data as an independent variable, and is typically much less variable than the time-concentration data itself.

52 This method therefore is less susceptible than the Logan plot method to the noise-induce bias in

347 the determination of VT. The operational equation for the Ichise multilinear analysis (MA) method is obtained by rearrangement of equation (34):

V 1 C ( t ) = − T C ( t )dt + C (t)dt (39) T b ∫∫P b T

Using multilinear regression analysis the values of –VT/b and 1/b can be determined and their ratio used to calculate VT. A reference tissue version of this method, MRTM0, has been developed346 and is derived in much the same way as for the Logan reference tissue method.

First, the expression for ∫Cp(t)dt (equation (35)) is substituted into the Logan plot equation

(equation (34)), and after rearrangement yields

K1 ⎛ k3 ⎞ K1 ⎛ k3 ⎞ ⎛ k3 ⎞ ⎜1+ ⎟ ⎜1+ ⎟ ⎜1+ ⎟ CT ( t )dt k k CR ( t )dt k k k ∫ = 2 ⎝ 4 ⎠ ∫ − 2 ⎝ 4 ⎠ + ⎝ 4 ⎠ (40) K ' K ' CT ( t ) 1 CT ( t ) 1 k2 k2' k2' k2'

With the usual assumption that VT = K1’/k2’ in the reference region equals VND = K1/k2 in the

ROI, and the substitution of 1 + BPND for 1 + K3/k4, equation (39) can be simplified to

C ( t )dt C ( t )dt ∫ T ∫ R 1+ BPND CR ( t ) 1+ BPND = (1+ BPND ) − + (41) CT ( t ) CT ( t ) k2' CT ( t ) k2

Since the dependent variables have CT(t) in their denominator, this equation produces a noise- induced bias in the estimated value of 1 + BPND. In order to reduce this bias, rearrangement of equation (40) yields an operational equation for which this bias is reduced (MRTM method):

k k C ( t ) = 2 C ( t )dt − k C ( t )dt + 2 C ( t ) (42) T ∫ T 2 ∫ R R 1+ BPND k2' with the value of 1 + BPND given by the ratio of estimated regression coefficients k2 and k2/(1 +

BPND). In order to reduce noise in the generation of BPND parametric images an approach similar to that of the SRTM2 is used and is known as MRTM2; the value of k2’ is estimated by a

53 preliminary MRTM analysis of the reference region, then k2’ is fixed to this value in all voxels in a subsequent MRTM analysis. The major advantage of the MRTM and MRTM2 over the

Logan reference tissue approach is that they allow the estimation of BPND without the need for an independent estimate of any kinetic parameters.

54 2.3. PET and SPECT radiotracers for dopaminergic imaging in human brain

PET and SPECT radiotracers have been developed for molecular imaging of many brain proteins including those involved in serotonergic, dopaminergic, cholinergic, opioid and

GABAergic signaling, as well as for measuring the rates of metabolic processes such as glucose metabolism ([18F]-FDG) and neurotransmitter synthesis ([18F]-F-DOPA, [11C]-α- methyltryptophan). The dopaminergic and serotonergic systems have been the most extensively studied with PET and SPECT, primarily because the involvement of these systems in neurological and psychiatric disorders has provided a strong driving force for the development of useful radiotracers. The following section (2.2.2.1) describes the basic properties of the PET and SPECT radiotracers available for studying the dopaminergic system in human subjects.

2.3.1. Aromatic L-amino acid decarboxylase (AAAD), dopamine synthesis and storage

The enzyme aromatic L-amino acid decarboxylase (AAAD) is responsible for synthesis of dopamine by the decarboxylation of its immediate precursor L-3,4-dihydroxyphenylalanine

(L-DOPA). This enzyme can be imaged with PET using several structurally-related 11C- and

18F-labeled radiotracers. The oldest and most widely used of these radiotracers is the 18F-labeled analog of L-DOPA, [18F]-FDOPA, which allows estimation of dopamine synthesis and storage capacity.359-362 [18F]-FDOPA is decarboxlylated by AAAD to give [18F]-F-dopamine, which is then taken up, like dopamine itself, into synaptic vesicles in dopaminergic terminals. [18F]-F- dopamine is further metabolized by catechol-O-methyl transferase (COMT) and monoamine oxidase B (MAO-B) in the brain to give the 18F-labeled analogs of the dopamine metabolites 3- methoxytyramine, 3,4-dihydroxyphenylacetic acid and homovanilic acid.359,360,363 These metabolites leave the brain very slowly, such that on short time scales (less than ~90 min in monkey) their loss from brain can be neglected. The rate of radioactivity accumulation in brain thus represents the total rate of the combined uptake, decarboxylation and vesicular storage

55 processes. [18F]-FDOPA can be quantified using graphical analysis methods for irreversible radiotracer uptake.364 A major drawback of [18F]-FDOPA is that it is a substrate for COMT, resulting in peripheral and central production of the brain-penetrant radioactive metabolite [18F]-

3-O-methyl-6-F-L-DOPA, the presence of which must be either reduced by COMT inhibitor treatment or accounted for in kinetic modeling procedures.365 L-DOPA has also been labeled with 11C and behaves in a similar fashion to [18F]-FDOPA, although COMT methylation seems to be less extensive for the [11C]-L-DOPA.366 The drawback to [11C]-L-DOPA, of course, is the limitation that the short isotope half-life places on scanning times and signal quality at late time points, and the necessity for in-house radioisotope production.

Another important radiotracer for investigating dopamine synthesis and storage capacity is [18F]-6-F-L-m-tyrosine ([18F]-FMT). [18F]-FMT is decarboxylated by AAAD to give [18F]-6-

F-3-hydroxyphenylacetic acid ([18F]-FPAC), which is then stored in synaptic vesicles.362,367-369

[18F]-FMT has two advantages over [18F]-F-L-DOPA or [11C]-L-DOPA. First [18F]-FMT is not a substrate for COMT so there are no problematic radioactive metabolites.362,367,369 Second, brain metabolism of [18F]-FMT ends at its AAAD-mediated decarboxylation to [18F]-FPAC.368,369

Therefore, loss of radiometabolites from brain need not be considered after [18F]-FMT injection.

However, the difficulty in achieving high specific activity and/or high radiochemical yield has prevented the widespread use of [18F]-FMT.

2.3.2. The dopamine transporter (DAT)

Many PET and SPECT radiotracers have been developed for imaging of the DAT. One of the first to be used in human subjects was [11C]-cocaine, although it was originally used more to study the in vivo brain distribution of cocaine, which functions through DAT blockade, than specifically to quantify DAT binding.370,371 [11C]-cocaine has fast kinetics (50% striatal clearance in ~30 min), which is valuable for a 11C labeled radiotracer, but suffers from very low

56 370,371 11 specific binding (striatal BPND = 0.4-0.8). A slight improvement over [ C]-cocaine was achieved with [11C]-methylphenidate ([11C]-MP), which had higher, but still relatively low

372 specific binding (BPND = ~1.5 in STR). Several high-affinity cocaine analogs, including β-

CIT, CFT, β-CIT-FE, β-CIT-FP, β-CPPIT, FECNT, RTI-32, TRODAT and PE2I have been radiolabeled for PET and/or SPECT imaging (depending on the radioisotope) and represent improvements over [11C]-cocaine and [11C]-MP in terms of specific binding, but often at the expense of favourable kinetics and/or selectivity.

β-CIT (a.k.a. RTI-55) has high affinity for the DAT (0.11 and 2.6 nM for the high- and low-affinity DAT binding sites)373 and can be labeled with either 11C for PET or 123I for

SPECT.374-376 Although [123I]-β-CIT reaches a favourable STR/CER ratio of >10, it has extremely slow kinetics, reaching peak concentration in STR only after ~20 hours.374 Thus,

[123I]-β-CIT binding within approximately the first day after injection is delivery-dependent,375 which could result in artifacts due to between-group differences in cerebral blood flow (e.g. resulting from drug treatment or disease pathology). Thus [123I]-β-CIT SPECT scans are typically done the day following radiotracer injection when binding is less dependent on blood flow. For [11C]-β-CIT PET scans, which due to short radioisotope half-life must be conducted immediately after radiotracer injection, radiotracer binding is necessarily blood flow-dependent.

Another potential drawback of [123I]/[11C]-β-CIT is that it also binds in vivo to serotonin transporters (SERT).377-379

[18F]-CFT (a.k.a WIN-35428) has faster in vivo kinetics (peak STR uptake at ~225 min) than [123I]-β-CIT, and is suitable for transient equilibrium ratio method measurements at post- injection times between 3.5 and 4.5 hours,380 but not practical for kinetic analysis in human given the long scanning times necessary (to capture both uptake and washout). Another advantage of [18F]-CFT, relative to [123I]-β-CIT, is its lack of SERT binding.380-382

57 β-CIT-FP, labeled with 11C, 18F or 123I, also has high DAT over SERT selectivity relative to [123I]-β-CIT,383 and although [18F]-β-CIT-FP concentration in STR and CER peak at reasonably early times (~40 and 20 min post-injection), no significant washout is seen over a

480 min post-injection period.384 In addition, a radiolabeled lipophilic metabolite of [11C]- and

[123I]-β-CIT-FP was found in human plasma which may enter the brain and confound measurement of DAT binding.385 This radiometabolite was not found after injection of [18F]-β-

CIT-FP.386

The structurally-related radiotracer [18F]-β-CIT-FE, which can also be labeled with 11C or 123I, has faster kinetics than [18F]/[123I]-β-CIT-FP, with peak uptake (as the 18F-labeled radiotracer) in STR and CER by ~30-40 and 10-20 min,384,387 respectively, and demonstrates

~45% washout from STR in 240 min,384 indicating that it is potentially suitable for kinetic analysis within a scan time appropriate for an 18F-labeled radiotracer.384 Similar to 123I and 11C- labeled β-CIT-FP, lipophilic metabolites of [11C]- and [123I]-β-CIT-FE have been detected in human and monkey plasma,385 but these results have not been replicated by other investigators

(at least in monkey plasma).388 Furthermore, the relevance of these findings to the 18F-labeled β-

CIT-FP is unclear.

[11C]-β-CPPIT is highly selective for DAT over SERT,389 but has only moderate levels of specific binding (relative to other DAT radiotracers) and, similar to β-CIT-FE, β-CIT-FP, suffers from slow kinetics (no STR washout observed by ~90 min). Unfortunately, β-CPPIT has no fluorine within its structure that would allow 18F labeling and extended scanning times.

[11C]-RTI-32 is selective for DAT over SERT390 and shows higher levels of specific binding than [11C]-β-CPPIT (STR/CER ratio of 6 and rising steeply at 90 min post-injection).391

However, this radiotracer, like [11C]-β-CPPIT, suffers from a combination of slow kinetics

(especially in DAT-rich areas) and short radioactive half-life, without the possibility for radiolabeling with longer-lived radioisotopes.390

58 [18F]-FECNT has similarly slow kinetics (washout from STR seen only after ~100 min),392 but because of its 18F label can accommodate long scan durations. It also has high selectivity for DAT over SERT,393 and relatively high specific binding (STR/CER = 9 at 90 min post-injection). Rat studies have shown, however, that [18F]-FECNT is metabolized to a brain- penetrant radiometabolite that represented 87% of brain radioactivity.394 This radiometabolite is also present in human plasma and is likely also present in human brain as the volume of distribution increases in cerebellum over time during [18F]-FECNT PET scans, consistent with brain accumulation of a receptor-inactive radiometabolite.394

The SPECT radiotracer [99mTc]-TRODAT is unique among DAT radioligands in its labeling with the gamma-emitting radioisotope 99mTc (half-life = 6 hours).395 The striatal binding of [99mTc]-TRODAT can be modeled using a three tissue compartment kinetic model, the simplified reference tissue model or a simple STR/CER ratio method.396,397 However, slow radiotracer kinetics mean that the ratio method provides good approximations of the BPND only after at least 3-4 hours have elapsed from the time of injection.397 In addition, [99mTc]-TRODAT

395,398 binds to both DAT and SERT, has relatively low specific binding (BPND = ~1.3), and gives rise to a brain-penetrant radiometabolite in human plasma.397,399 Despite these limitations,

[99mTc]-TRODAT has been extensively used in human SPECT studies.

PE2I, which can be labeled with 123I or 11C, is also DAT selective over SERT400 and reaches a respectable STR/CER of >10 by 90 min post-injection. PE2I has the fastest kinetics of any of the above DAT radiotracers, reaching peak in STR between 20 and 30 min.401 When labeled with 123I, PE2I can be quantified with comparable results using either the STR/CER ratio during bolus plus constant infusion, or after bolus injection using 2 tissue compartment kinetic analysis, Logan non-invasive graphical analysis, the simplified reference tissue model or the peak equilibrium ratio method.401,402 The relatively early peak of specific binding (45-75 min) allows quantification of [11C]-PE2I binding using the peak equilibrium approach if long

59 scanning times of perhaps 90 min are used.401,403,404 Thus, [11C]/[123I]-PE2I is probably the most versatile radiotracer available for PET and SPECT imaging of the DAT.

2.3.3. Vesicular monoamine transporter 2

Located within synaptic vesicle membranes, vesicular monoamine transporter 2

(VMAT2) is a transporter protein responsible for the loading of synaptic vesicles with monoamines, such as dopamine, norepinephrine and serotonin.405 There is only one radiotracer,

[11C]-DTBZ, for the in vivo imaging of VMAT2 in humans, although an 18F-labeled derivatives of this radiotracer are currently in pre-clinical development.406-408 [11C]-DTBZ shows highest binding in the STR, which because of the relatively low abundance of other monoamines, represents uptake sites within dopaminergic terminals. [11C]-DTBZ binding is thought to represent a more stable measure of dopamine terminal density than DAT radiotracer binding,409,410 although a recent ex vivo rodent study has demonstrated that [11C]-DTBZ is altered by pretreatment with drugs that alter vesicular dopamine concentration.411 The binding of [11C]-DTBZ in human brain displays fast kinetics with approximately 50% washout within 50 min after injection, which is appropriate for a 11C-labeled radiotracer.412-414 Striatal [11C]-DTBZ binding can be analyzed by two tissue compartment kinetic analysis or by reference tissue approaches (simplified reference tissue model415 and Logan non-invasive graphical method414)

414 11 using the occipital cortex as a reference region. In the STR, [ C]-DTBZ BPND in healthy subjects is ~2.5.

2.3.4. Dopamine D1 receptors

The first widely-used radiotracer for in vivo imaging of the dopamine D1 receptor was the antagonist ligand [11C]-SCH-23390.316,416-418 This radiotracer has high affinity for the D1

419 receptor (Ki = 0.14 nM) fast kinetics (peak in STR at ~10 min followed by 50% washout by

60 ~60 min post-injection),316,417 is amenable to two tissue compartment kinetic analysis and Logan graphical analysis as well as reference tissue approaches including the ratio method (using data from a 30-60 minute interval), Logan non-invasive graphical analysis and the simplified reference tissue model, using the cerebellum as the reference region.339,420 However, the

420 relatively low binding signal of this radiotracer (STR BPND ~ 0.8-1.0) and its activity at serotonin 5-HT2A receptors421-423 inspired the search for other D1 radiotracers. Three other

11 424 11 high-affinity D1 radiotracers, [ C]-SCH-39166 (Ki = 3.4 nM), [ C]-NNC-756 (Ki = 0.17 nM)419 and [11C]-NNC-112 (Ki = 0.18 nM)419 have also been used in human subjects. [11C]-

SCH-39166 was developed and used in humans for D1 imaging,425-427 but suffered from low levels of striatal binding (STR/CER = 1.5 at time of maximum specific binding).425 [11C]-NNC-

756, on the other hand, has relatively high STR/CER ratio (~5 at 60 min relative to ~2 for [11C]-

SCH-23390),428,429 but like [11C]-SCH-23390, also binds to cortical 5-HT2A receptors.428,430

[11C]-NNC-112 was reported to be ~100 fold selective for D1 versus 5-HT2419 receptors in vitro and was therefore considered a strong candidate to supercede [11C]-SCH23390 as the preferred

D1 radiotracer. In human brain, [11C]-NNC-112 has a higher STR/CER ratio than [11C]-

SCH23390 (~4 in STR, ~2 in prefrontal cortex at 60 min post-injection)301 and kinetics amenable to two tissue compartment kinetic analysis,301 Logan non-invasive graphical analysis301 and the simplified reference tissue model.431 Despite the slower kinetics of [11C]-

NNC-112 (only ~20% washout by 120 min) compared to [11C]-SCH-23390, the quantitative approaches above could be reliably implemented with data from a 90 min scan,18,431 which is, in general, within acceptable limits for a 11C-labeled radiotracer. A recent PET study has indicated that 20-30% of [11C]-NNC-112 binding in human prefrontal cortex could be blocked by the antipsychotic risperidone,432 which has high-affinity for 5-HT2 receptors. Similar results have been reported with the 5-HT2-selective drug MDL-100907 in baboon.430 These data suggest that

61 the D1 over 5-HT2A selectivity of [11C]-NNC-112 may be less than the 100-fold original reported.

It is worth noting that the striatal binding of [11C]-SCH-23390, [11C]-NNC-756 or [11C]-

NNC-112 is not significantly blocked by 5-HT2A receptor-selective drugs,430,432 in agreement with the very low levels of 5-HT2A expression in STR. Thus, although all of these radiotracers apparently bind to cortical 5-HT2A receptors, this does not necessarily limit their use as striatal

D1 radiotracers.

2.3.5. Dopamine D2/D3 receptors

PET and SPECT imaging of dopamine D2/D3 receptors is a well-developed field, with several well-characterized antagonist radiotracers for imaging of both striatal ([11C]-raclopride,

[123I]-iodobenzamide ([123I]-IBZM)) and extrastriatal receptors ([18F]-fallypride, [11C]-FLB-457,

[123I]-epidepride). Recently, the agonist D2/D3 radiotracers [11C]-(-)-NPA, [11C]-(+)-PHNO and

[11C]-(-)-MNPA have been developed and utilized in human PET studies. The current section covers the common D2/D3 antagonist radiotracers, whereas the agonist D2/D3 radiotracers are discussed separately in section 2.2.4. The benzamide antagonist and agonist D2/D3 radiotracers are sensitive to levels of extracellular dopamine and can therefore be used to measure dopaminergic activity in living brain. This important technique is covered in the next section

(2.2.2.1.6).

The first radiotracers developed for in vivo D2/D3 imaging in human were the butyrophenones [11C]-N-methylspiperone ([11C]-NMS)433-435 and [18F]-fluoroethylspiperone

([18F]-FESP),436 which have high D2/D3 receptor affinity (Ki = 250-300 pM).437 These radiotracers provide reasonable contrast between STR and cerebellum,433,436,438 but also bind in vivo to cortical 5-HT2 receptors.439-441 Another drawback of these radiotracers is that they exhibit irreversible striatal binding which is highly dependent on cerebral blood flow. In

62 response to these limitations a lower affinity, more selective benzamide radiotracer, [11C]- raclopride was developed,442 and has become the most widely used radiotracer in PET imaging of D2/D3 receptors. Raclopride has moderately high affinity for D2/D3 receptors (~1 nM) and is highly selective over other brain receptors including the dopamine D1, serotonin 5-HT1 and 5-

HT2, and norepinephrine α and β receptors.443-445 In vivo, [11C]-raclopride displays rapid kinetics in human brain (peak in STR at ~20 min and ~30% washout by 60 min post-injection) and a moderately high STR/CER ratio of 4-5 at 60 min.446 The kinetics of [11C]-raclopride binding in STR are amenable to various quantification methods including full kinetic analysis using a two tissue compartment model, transient equilibrium and late time point ratio methods, as well as the simplified reference tissue and multilinear reference tissue models.334,344,354,447 The high D2/D3 receptor selectivity, reasonably high striatal binding signal (BPND ~3), and quantitative flexibility of [11C]-raclopride have contributed to the wide-spread use of this radiotracer in human PET studies. Similar success has been seen with the related SPECT radiotracer [123I]-IBZM,448-450 which also displays high selectivity,451 comparable kinetics, and can be quantified according to similar methods as [11C]-raclopride (though often using cortex, rather than cerebellum as reference region).452

Although useful for quantification of D2/D3 receptors in STR, the D2/D3 affinity of

[11C]-raclopride and [123I]-IBZM limits their utility for measurement of the much lower levels of

D2/D3 receptors in extrastriatal brain regions. Several high-affinity benzamide PET and SPECT radiotracers have been developed for this purpose. [11C]-FLB-457 has very high affinity for

D2/D3 receptors (~20 pM)453 and can be used to measure D2/D3 receptors in various extrastriatal regions such as the thalamus (BPND ~ 2.9) and cortex (BPND ~1.3, 0.8 and 0.7 in temporal, anterior cingulated and frontal cortices).454,455 Extrastriatal [11C]-FLB-457 time- activity curves can be described using a two tissue compartment model or Logan graphical analysis.454-457 The extrastriatal binding of [11C]-FLB-457 can also be quantified using a

63 transient equilibrium ratio approach or the simplified reference tissue model,455 although the assumption of non-negligible D2/D3 receptor binding in the cerebellar reference region has been questioned for radiotracers of such high affinity.458,459 As a result of the high affinity of [11C]-

FLB-457 and the relatively high D2/D3 expression in STR, the striatal kinetics of [11C]-FLB-

457 binding are very slow. When labeled with 76Br (half-life 16 hours), FLB-457 concentration in STR increased for greater than 5 hours, reaching a STR/CER ratio of >30.460 Thus, with a short half-life radioisotope like 11C (half-life 20.4 min), too little of the time-activity curve is captured to allow reliable, blood flow-independent kinetic modeling of striatal [11C]-FLB-457 binding.454,455

[18F]-fallypride has similarly high D2/D3 affinity (Ki ~50 pM) and can be used to quantify extrastriatal D2/D3 receptors using various kinetic and reference tissue approaches including two tissue compartment kinetic analysis, Logan graphical analysis and the simplified

461-463 11 reference tissue model, resulting in BPND values similar to those seen for [ C]-FLB-457

457 11 (BPND ~2.1 and 0.9 thalamus and temporal cortex). Like [ C]-FLB-457, striatal kinetics of

[18F]-fallypride are very slow, increasing until at least 3 hours post-injection, but because it is labeled with a longer-lived radioisotope (half-life 109.8 min), enough of the time-activity curve can be captured to also allow accurate quantification of striatal binding (BPND ~11-19, depending on striatal region).461-463

[123I]-epidepride, the radioiodinated analogue of FLB-457, also has very high affinity for

464 D2/D3 receptors (Ki ~ 24 pM) and can be used to measure extrastriatal receptors using

SPECT.465,466 Like [11C]-FLB-457, striatal kinetics are extremely slow (<40% washout by 24 hours after bolus injection),467 presenting problems for quantification of striatal D2/D3 receptors.465-467 A further limitation of [123I]-epidepride is that plasma metabolite analysis is required to correct for the presence of a peripherally-generated brain-penetrant lipophilic metabolite.466,468

64 2.3.6. D2/D3 radiotracer-based imaging of endogenous dopamine

The binding of dopamine D2/D3 receptor benzamide such as [11C]-raclopride, [123I]-

IBZM, [18F]-fallypride and [11C]-FLB-457)227,235,457,469 and the agonist radiotracers [11C]-(-)-

NPA, [11C]-(-)-MNPA and [11C]-(+)-PHNO343,447,470 are sensitive to treatments that alter the concentration of extracellular dopamine. Drugs that increase extracellular dopamine, such as the dopamine-releasing drug AMPH or the DAT inhibitor cocaine, result in decreased benzamide radiotracer binding,227,230 whereas an increase in binding is observed for dopamine-depleting drugs such as α-MTP or reserpine.18,336 Non-pharmacological manipulations, such as electrical brain stimulation338 and even video game playing in humans,471 have also been shown to decrease [11C]-raclopride binding, presumably through increased extracellular dopamine.

Importantly, the change in extracellular dopamine concentration, as measured by in vivo microdialysis, is directly proportional to the change in radiotracer binding.19,216,472 This correlation has permitted the use of the benzamide radiotracers as in vivo indicators of extracellular dopamine levels in human subjects, leading to important discoveries regarding the role of dopaminergic neurotransmission in psychiatric disease (see section 2.1.4). For example, treatment with AMPH (dopamine release) or α-MTP (dopamine depletion) has been shown to result in larger alterations of [11C]-raclopride BP in schizophrenic subjects than in healthy controls, indicating pharmacological hyper-responsiveness of dopamine release and increased baseline D2/D3 receptor occupancy by dopamine, respectively, in this illness.17,18,473,474

The effect of dopamine-releasing or dopamine-depleting treatments on D2/D3 radiotracer binding is often rationalized in terms of competition between extracellular dopamine and the radiotracer. That is, increased extracellular dopamine is thought to reduce D2/D3 radiotracer binding by competitive inhibition, whereas decreased extracellular dopamine results in a lower level of competition and thus more receptors free for radiotracer binding. The competition model accounts for the inverse correlation between extracellular dopamine and

65 benzamide radiotracer binding. This is further supported by experiments demonstrating that the

11 apparent KD of [ C]-raclopride is increased by AMPH treatment, but decreased by treatment with the dopamine-depleting drug reserpine, consistent with competitive inhibition.336,337

However, despite its general acceptance, the competition model fails to account for several relevant phenomena (for full review see ref. 475). First, as demonstrated by Laruelle et al., there is a considerable temporal discrepancy between AMPH-induced elevation in extracellular dopamine concentration, which after i.v. injection in non-human primates decreases to ~20% of maximum by 2 hours post-treatment, and the time course of [11C]-raclopride BP reduction, which persists for greater than 4 hours post-treatment.216 Other investigators have shown that

[11C]-raclopride BP remains reduced for as long as 24 hours after AMPH treatment.476 This prolonged effect has also been demonstrated for the agonist D2/D3 radiotracers [11C]-(-)-NPA and [11C]-(+)-PHNO.343,476 Second, non-benzamide radiotracers such as the butyrophenone

D2/D3 radiotracer [11C]-NMS and the benzazepine D1 radiotracers [11C]-SCH-23390 and [11C]-

NNC-112 are either unaffected by alteration of extracellular dopamine, or respond in a manner opposite to that predicted by the competition model (i.e. increased extracellular dopamine gives rise to increased radiotracer binding).477-479

In response to these problems, an alternative model, known as the internalization model, was developed. This model relies upon the well-known phenomenon of agonist-induced receptor internalization,480,481 rather than competition with extracellular dopamine, as the mediator of changes in radiotracer binding.475,482 According to this model, the proportion of receptors in the internalized state is a function of the extracellular concentration of dopamine, such that an increase in dopamine results in net receptor internalization, whereas reduced extracellular dopamine causes net movement of receptors to the cell surface. Changes in radiotracer binding are conceptualized as the result of the difference in radiotracer affinity for internalized versus cell surface receptors, possibly because of differences in ionic concentration

66 or pH between the intracellular and extracellular compartments.475,482 As a result, radiotracers with lower affinity for internalized versus surface receptors will respond to dopamine-releasing treatments with lowered binding (i.e. hypothetically, the benzamides), whereas radiotracers with similar or higher affinity for internalized receptors will display no change or an increase in binding (e.g. [11C]-NMS or [11C]-SCH-23390). The converse would be expected for treatments that lower extracellular dopamine. This model has two major advantages over the competition model. First, because the rate-limiting process is receptor internalization rather than direct competition with dopamine, the internalization model allows for a temporal disconnection between radiotracer binding changes and extracellular dopamine concentration. Second, unlike the competition model, the internalization model allows for increased, decreased, or unchanged radiotracer binding in response to dopamine-modulating treatments depending on the relative affinity of the radiotracer for internalized versus cell surface receptors. A recent report indicates that the benzamides raclopride, FLB 457, epidepride, fallypride and IBZM as well as the D2/D3 agonists (+)-PHNO and (-)-NPA have 2-3 fold lower affinity for internalized versus cell surface receptors as predicted by the model.483 However, the butyrophenone NMS, the binding of which is unaffected or decreased by increases in extracellular dopamine, unexpectedly also displayed lower affinity for internalized receptors,483 indicating that the internalization model cannot fully explain the effects of extracellular dopamine on PET and SPECT radiotracer binding.

Neither the competition nor the internalization model can explain other peculiar characteristics of dopamine-induced changes in radiotracer binding. For example, several drugs cause similar reductions in [11C]-raclopride BP despite causing very different elevations in extracellular dopamine.472 Ketanserin (0.3 mg/kg), GBR-12909 (2 mg/kg) and methamphetamine (0.1 mg/kg) all result in similar 17% decreases in [11C]-raclopride despite causing 120%, 350% and 850% elevations in extracellular dopamine, respectively.472

67 2.4. The D2 high-affinity state and the development of agonist D2/D3 PET radiotracers

In vitro, agonist ligands compete with antagonist radioligands for the D2 receptor (and other G protein-coupled receptors) in a biphasic manner (Figure 6). The high- and low-affinity phases of the agonist/antagonist competition curve are typically modeled as separate, non- interconvertible receptor states with differential agonist affinity. The high-affinity phase of the curve can be abolished by high concentrations of GTP,484-486 indicating that the corresponding receptor state (known as the high-affinity state) represents the G protein-coupled form of the

Figure 6. A) Simulated competition between a D2 antagonist radioligand and an agonist ligand. In the absence of GTP, an agonist ligand competes in a biphasic fashion with the antagonist radioligand for binding to the D2 receptor, with separate IC50 values for the high- and low-affinity states. In the presence of sufficient GTP concentration the competition curve becomes monophasic with an IC50 similar to the low affinity phase in the High absence of GTP. B) Two-affinity state model used to assign dissociation constants to the high- (KD ) and low- Low affinity (KD ) phases of the competition curve. According to this simplification of the ternary complex model,(Ref. 485) the receptor couples effectively to the GDP-bound form of the G protein but not to the GTP- bound form. Agonists (A) have higher affinity for the G protein-coupled form of the receptor (RGGDP) than for the High Low uncoupled form (R) (i.e. KD < KD ). The addition of GTP converts the G protein to its GTP-bound form, preventing receptor-G protein coupling and thus eliminating high-affinity agonist binding sites. This is seen as a disappearance of the high-affinity phase of the competition curve in the presence of GTP.

receptor. The affinity of agonists for this high-affinity receptor state corresponds to their in vitro potency in eliciting functional responses such as inhibition of prolactin release254 or adenylate cyclase activity,253 indicating that the high-affinity state represents the functional form of the receptor. The high-affinity state is also of potential clinical relevance in psychosis and substance abuse as it has been shown, using in vitro competitive binding experiments, to be up-regulated in several animal models of these disorders.1,306 The functional relevance of the high-affinity

68 state and its suspected involvement in brain pathophysiology make it an important target for in vivo molecular imaging techniques such as PET. Insofar as the two-affinity-state model applies in vivo, an agonist PET radiotracer should allow the selective in vivo measurement of the high- affinity, functional form of the D2 receptor. This is in contrast to antagonist PET radiotracers, such as [11C]-raclopride and [18F]-fallypride, which should bind non-selectively to both the high- and low-affinity states.

The use of an agonist radiotracer for in vivo imaging of the D2 receptor has three predicted advantages over common antagonist radiotracers. First, as mentioned above, an agonist radiotracer should allow the selective measurement of the G protein-coupled, function state of the D2 receptor responsible for D2 receptor-mediated intracellular responses such as inhibition of cAMP production,487,488 regulation of membrane excitability489,490 and changes in gene expression.491,492 Second, an agonist radiotracer, unlike common antagonist radiotracers, should allow the direct investigation of the involvement of the high-affinity state in brain disorders such as psychosis and substance abuse, in which it is implicated by in vitro studies using animal models.1 Third, an agonist radiotracer should be more sensitive to competition with extracellular dopamine than an antagonist radiotracer. This is because dopamine, being an agonist, should selectively inhibit radiotracer binding to the high-affinity state, which represents the entire population agonist radiotracer binding sites, but only a fraction of antagonist radiotracer binding sites (high- plus low-affinity states). Consequently, an agonist radiotracer should allow more sensitive in vivo measurement of changes in the concentration of extracellular dopamine. This is important for investigation of the role of dopaminergic neurotransmission in normal brain function and in pathological conditions such as psychosis, where changes in baseline dopamine occupancy18 of the D2 receptor and dopamine release17,19,296 have been demonstrated.

69 Several candidate D2 agonist ligands have been evaluated for use as in vivo PET radiotracers. However, the majority of these ligands, often despite favourable in vitro properties, failed as in vivo radiotracers, most often because of a lack of in vivo specific binding493,494 or low contrast between receptor-rich and receptor-poor brain areas.495-501 Three D2/D3 agonist radiotracers, [11C]-(-)-NPA, [11C]-(+)-PHNO and [11C]-(-)-MNPA (Figure 7), have advanced past the pre-clinical stage and are now in use in human subjects. Sections 2.2.3.1-2.2.3.3 describe the basic in vitro and in vivo properties of these three radiotracers.

Figure 7. The chemical structures of the D2/D3 agonist radiotracers [11C]-(-)-NPA, [11C]-(-)-MNPA and [11C]-(+)-PHNO.

2.4.1. [11C]-(-)-NPA

(-)-NPA is an apomorphine derivative that acts as a selective, high-affinity, full agonist at D2 receptors502,503 and similar to other agonists, binds with differential in vitro affinity to the

G protein-coupled and uncoupled states of the receptor.504,505 Like most other D2-like receptor ligands, (-)-NPA also has high in vitro affinity for the D3 receptor subtype.506-509 The first pre-

70 clinical data for [11C]-(-)-NPA were reported in 2000,510 but the ex vivo biodistribution and D2- like pharmacology of the tritiated isotopologue were described nearly 20 years earlier by Köhler et al.511 In this early report, thin layer chromatographic analysis demonstrated that >95% of brain radioactivity after injection of [3H]-(-)-NPA was due to unmetabolized parent compound.

Furthermore, the ex vivo binding of [3H]-(-)-NPA binding in brain could be blocked or displaced by D2 ligands such as raclopride, haloperidol, (+)-butaclamol, apomorphine and bromocriptine, but not by non-D2 ligands such as SCH-23390 (dopamine D1), mianserin (serotonin 5-HT2), phenoxybenzamide (α-adrenergic), or propranolol (β-adrenergic).511,512 Both [11C]-(-)-NPA and

[3H]-(-)-NPA preferentially accumulate in the D2/D3-rich basal ganglia with STR/CER ratios for [11C]-(-)-NPA reaching 4.4 at 60 min post-injection in rat and 2.8 at 45 min post-injection in baboon.510 Although these STR/CER ratios are only approximately half that seen for the common antagonist radiotracer [11C]-raclopride,513 they were, at the time of publication, the highest reported for an agonist D2/D3 receptor radiotracer. In isoflurane-anaesthetized baboon, the kinetics of [11C]-(-)-NPA in brain were sufficiently rapid (peak in CER and STR at ~2 and

~8 min post-injection, followed by 50% washout by ~20 and ~50 min post-injection, respectively) to allow precise quantification of kinetic parameters using both full kinetic analysis and simplified non-invasive methods (SRTM, Logan graphical analysis) within a 60 min scanning time.514 Such rapid kinetics are necessary for a radiotracer labeled with a short-

11 11 half life isotope such as C. Depending on the analysis method, [ C]-(-)-NPA BPND in baboon

514 striatum ranged from 1.29 with full kinetic analysis to 1.41 with the SRTM. The BPND calculated with the SRTM correlated well with that determined by full kinetic analysis,514 indicating the appropriateness of a less invasive reference tissue modeling approach. The striatal binding of [11C]-(-)-NPA was substantially lower than that seen for [11C]-raclopride in the same

470 animals (average BPND = 3.0) but nonetheless sufficiently large to measure the occupancy of

D2/D3 receptors by antipsychotic drugs such as haloperidol510 or by endogenous dopamine after

71 470 11 11 amphetamine challenge. The lower BPND of [ C]-(-)-NPA versus [ C]-raclopride is largely the result of relatively higher volume of distribution in the non-displaceable binding

470,515 compartment [VND, estimated by the total volume of distribution (VT) in cerebellum]. In human brain, [11C]-(-)-NPA showed a regional biodistribution pattern very similar to that in baboon, with highest uptake and preferential retention in D2/D3-rich caudate and putamen.6

[11C]-(-)-NPA was metabolized more rapidly in human than in baboon (13% of plasma radioactivity corresponded to parent radiotracer at 30 min post-injection, relative to 30% in baboon), and radioactivity was also cleared from plasma more rapidly (119 L/h in human versus

6 11 29 L/h in baboon). The [ C]-(-)-NPA BPND of 0.9 in human striatum was lower than that of

11 6 [ C]-raclopride in the same subjects (BPND = 2.6), and also lower than in baboon STR, potentially making [11C]-(-)-NPA a less desirable radiotracer than [11C]-raclopride or [11C]-(+)-

PHNO (vida infra) for clinical studies. A final important observation from human [11C]-(-)-NPA

11 experiments is the prominent binding of [ C]-(-)-NPA in globus pallidus (BPND = 0.82 relative to 0.9 in striatum), a region thought to be rich in D3-receptor expression,160,516 relative to that in

6 11 the D2-rich dorsal STR (BPND = 0.87). In contrast, higher [ C]-raclopride binding is seen in

6,517 (human and baboon) dorsal STR (BPND = ~2.6) than in the globus pallidus (BPND = ~1.5), suggesting that the ratio of D3 to D2 affinity of [11C]-(-)-NPA is higher than for [11C]-raclopride and indicating that both D2 and D3 receptor binding must be considered in the interpretation of

[11C]-(-)-NPA PET data. Further studies are needed to clarify the relative contributions of the

D2 and D3 receptor to regional [11C]-(-)-NPA binding.

2.4.2. [11C]-(-)-MNPA

The apomorphine derivative (-)-MNPA is a selective, high-affinity agonist at the D2 receptor518,519 and distinguishes in vitro between G protein-coupled and -uncoupled receptor states.505 Like (-)-NPA and other D2-like receptor ligands, (-)-MNPA has similar in vitro

72 affinities for the D2 and D3 receptor subtypes.505 The 11C radiolabeling of (-)-MNPA, accomplished by 11C-methylation and first reported by Halldin et al.,520 has the advantage of chemical simplicity over the 11C-propylation required for radiolabeling of [11C]-(-)-NPA or

[11C]-(+)-PHNO.510,521 In preclinical studies in ketamine-anaesthetized cynomolgous monkey,

[11C]-(-)-MNPA accumulated and was preferentially retained in the D2/D3-rich striatum and to a lesser extent in thalamus (THA).447,522,523 Peripheral metabolism of [11C]-(-)-MNPA in monkey was comparable to that of [11C]-(-)-NPA in baboon, with ~20% of plasma radioactivity corresponding to parent radiotracer at 30 min post-injection, the remaining plasma radioactivity corresponding to polar metabolites.522,523 In rat, radioactive metabolites were responsible for only ~8-10% or total brain radioactivity,524 which includes ~5% brain vascular volume rich in polar metabolites. [11C]-(-)-MNPA displayed rapid brain kinetics in monkey similar to that of

[11C]-(-)-NPA (in baboon), with peak uptake reached in 5-10 min and 50% washout from STR and CER by ~60 and ~30 min, respectively.522,523 The STR/CER ratio was also similar to that seen for [11C]-(-)-NPA, reaching a maximal value of 2.2-3.0 at 70-80 min post-injection.447,522 In pretreatment studies STR uptake of [11C]-(-)-MNPA could be reduced nearly to CER levels by pretreatment with the D2/D3-selective drug raclopride, demonstrating the D2/D3 specificity of in vivo [11C]-(-)-MNPA striatal binding.522 Studies with AMPH pretreatment and α-MPT- induced dopamine depletion have shown that the striatal binding of [11C]-(-)-MNPA, like that

[11C]-(-)-NPA, is sensitive to changes in endogenous dopamine concentration.447,524 [11C]-(-)-

MNPA time-activity curves in both STR and CER were better fit by a 2 TC than by a 1 TC model, and despite some problems with kinetic parameter identifiability (especially of k3 and k4), total volumes of distribution in both regions (and therefore the BPND) could be precisely

523 11 determined. As with [ C]-(-)-NPA, the BPND calculated using the SRTM (1.05) or MRTM

(1.37) was somewhat higher than that determined using full kinetic analysis (0.8).447,523 In human, [11C]-(-)-MNPA brain distribution was similar to that seen in monkey with preferential

73 accumulation in D2/D3 receptor-rich caudate, putamen and thalamus.525 STR and THA time- activity data were fit better by a 2 TC model than a 1 TC model, whereas the opposite was true

525 for the CER. Very similar BPND values (PUT, 0.8; CAU, 0.6; THAL, 0.3) were obtained in human brain using full kinetic analysis (2 TC in STR and 1 TC in CER), the SRTM or a transient equilibrium ratio method within a 60 min scan time.525 Thus simplified non-invasive

11 methods are appropriate for measurement of [ C]-(-)-MNPA BPND in human brain within scan times acceptable for a 11C-labeled radiotracer.

2.4.3. [11C]-(+)-PHNO

(+)-PHNO is a potent D2-like receptor full agonist that is structurally unrelated to (-)-

NPA or (-)-MNPA. First synthesized in 1984 along with a series of 4-substituted naphthoxazines, (+)-PHNO was shown to possess dopaminergic activity in vitro (IC50 = 23 nM

3 against [ H]-apomorphine) and to be an extremely potent dopaminergic agonist in vivo (EC50 =

5 µg/kg for eliciting turning behaviour in unilaterally 6-OHDA lesioned rats).526 Subsequent in vitro pharmacological characterization indicated that (+)-PHNO bound selectively to D2-like

3 527,528 receptors (IC50 = 55-67 nM against [ H]-spiperone) over various other brain receptor

529,530 targets such as the dopamine D1 (IC50 = 22-35 µM), and the serotonin 5-HT2 (IC50 = 277

528 nM). Early reports indicated that (+)-PHNO also had high affinity for α2 adrenergic receptors

526,528 (IC50 = 77-85 nM), but this was disputed by a later reports showing that the α2 adrenergic receptor ligand clonidine had very low potency for inhibiting [3H]-(+)-PHNO binding to striatal

531 membranes (Ki = 10.3 µM) which is inconsistent with clonidine’s relatively high affinity for

α2 adrenergic receptor (in the 10-30 nM range; NIMH PDSP Ki database, http://pdsp.cwru.edu/pdsp.asp and references therein), and by our ex vivo blocking experiments with clonidine and [11C]-(+)-PHNO in rat (see below). In vitro estimates of the D2-like receptor affinity of (+)-PHNO range from as low as 0.5 nM determined by saturation experiments (with

74 [3H]-(+)-PHNO)531 to values in the 5-67 nM range using competition experiments (against [3H]- apomorphine, [3H]-spiperone or [125I-iodosulpiride).506,526-529,532 In agreement with this high affinity, (+)-PHNO has an in vitro IC50 of 0.5 nM for inhibition of adenylate cyclase activity and prolactin release from anterior pituitary cells.533 This in vitro functional potency likely corresponds to binding to the high-affinity state of the D2 receptor for which (+)-PHNO has affinity in the 0.07-0.6 nM range,509,529,534 greater than 60-fold higher than its affinity for the low-affinity state.509,529 Like (-)-NPA and (-)-MNPA, (+)-PHNO also has high in vitro affinity for the D3 receptor and may even be D3-selective, with a affinities of 0.21 and 0.16 nM reported.506,532 In vitro autoradiographic studies showed that the distribution of [3H]-(+)-PHNO binding was appropriate for a D2/D3 receptor ligand and suggested that a portion of the [3H]-

(+)-PHNO binding signal, that remaining after treatment with the guanine nucleotide GppNHp

(a non-hydrolyzable analogue of GTP), was due to D3 receptor binding,535 particularly in the islands of Calleja which are known to be rich in D3 receptor expression.167,168

In vivo, (+)-PHNO displayed a pharmacological profile consistent with action as a D2- like receptor agonist and was shown to be remarkably potent in several behavioural tests of

526,527,536 dopaminergic activity. Specifically, (+)-PHNO induced hypothermia in mice (ED50 = 3 and 13 µg/kg in two studies),527,536 postural asymmetry in unilaterally caudectomized mice

527 (ED50 = 4 µg/kg), stereotypy and turning behaviour in normal and unilaterally 6-OHDA-

527,537 lesioned rats (ED50 = 10 and 5 µg/kg, respectively) and emesis in dogs (ED50 = 0.05

µg/kg).527 The behavioural effects of (+)-PHNO could be blocked by the D2/D3 antagonist haloperidol and (+)-PHNO did not induce adrenergic or serotonergic responses (mydriasis or reduction in brain serotonin levels, respectively), further supporting its selectivity for D2/D3 receptors.528 Its high in vivo potency and oral availability made (+)-PHNO an attractive candidate drug for treatment of Parkinson’s disease, and it was subsequently shown to produce therapeutic benefits in both animal models538 of the disease and human Parkinson’s patients.539-

75 541 However, despite initially promising clinically data (+)-PHNO was eventually abandoned as an anti-Parkinsonian drugs because of a combination of adverse side effects (nausea, vomiting, orthostatic hypotension),540-542 progressive development of tolerance to its therapeutic effects543 and the superior effectiveness of other pharmacotherapies.544

(+)-PHNO was first labeled with 11C by Brown et al. in 1997,521 but no further data were reported on [11C]-(+)-PHNO as a radiopharmaceutical until our group began development of

[11C]-(+)-PHNO as a PET radiotracer. Using the same radiosynthetic strategy (propylation with

[11C]-propionyl chloride, followed by reduction), we synthesized [11C]-(+)-PHNO and conducted the first ex vivo evaluation of its binding properties in rat.4 In accordance with its

D2/D3 agonist activity, radioactivity after i.v. [11C]-(+)-PHNO injection was preferentially retained in the D2-rich striatum,4,493 whereas radioactivity concentrations in non-striatal regions were similar to that in CER.4 HPLC analysis indicated that 26% of plasma radioactivity at 40 min post-injection represented parent radiotracer, the remaining radioactivity corresponding to polar metabolites that did not enter the brain (<2% of radioactivity in brain was due to polar metabolites).4 [11C]-(+)-PHNO displayed rapid brain kinetics with peak radioactivity in STR and CER at 4.5 and 2 min, respectively, followed by 50% washout from STR and CER in ~40 and ~20 min, respectively.4 The STR/CER ratio of [11C]-(+)-PHNO in conscious rat increased over time reaching 5.6 at 60 min post-injection,4 the highest ratio demonstrated for a D2/D3 agonist radiotracer, and could be reduced by >90% by pretreatment with unlabeled (+)-PHNO and the D2/D3 ligands raclopride or haloperidol, but not by pretreatment ligands for dopamine

D1, σ opioid, serotonin 5-HT1A or adrenergic α2 receptors, demonstrating the saturability and

D2/D3 specificity of [11C]-(+)-PHNO striatal binding.4 Striatal [11C]-(+)-PHNO binding could also be dose-dependently reduced by pretreatment with AMPH (up to 40% at 4 mg/kg) and the

DAT inhibitor RTI-32 (up to 73% at 10 mg/kg), and increased by treatment with the dopamine depleting drugs α-MPT (31% at 250 mg/kg) and reserpine (30% at 2 mg/kg), demonstrating the

76 sensitivity of [11C]-(+)-PHNO binding to changes in the concentration of extracellular dopamine.4 The D2/D3 specificity of [11C]-(+)-PHNO striatal binding (raclopride, haloperidol and (-)-NPA pretreatment) and its sensitivity to endogenous dopamine (AMPH pretreatment) were confirmed in ketamine-anaesthetized rat and isoflurane-anaesthetized cat using intracerebral β-sensitive microprobe and PET experiments, respectively.343,493 As in rat, the kinetics of [11C]-(+)-PHNO in cat brain were appropriately rapid for a 11C-labeled radiotracer, with peak radioactivity in CER and STR reached in ~2 and 8-10 min, respectively, and 50% washout from these regions in ~10 and ~60 min.343

In human brain [11C]-(+)-PHNO showed prominent uptake and retention in regions expressing D2 and/or D3 receptors, with the highest binding seen in globus pallidus (BPND

(SRTM) = 3.6, 3.2-4.2 range over six studies) followed by ventral STR (3.3, range 3.1-3.5), putamen (2.7, range 2.2-3.1), caudate (2.2, range 2.0-3.0) and substantia nigra (1.7, range 1.4-

5,302,545-548 11 547 548 2.1). The BPND of [ C]-(+)-PHNO could be reduced by AMPH or haloperidol pretreatment confirming that in vivo [11C]-(+)-PHNO binding in human brain is sensitive to changes in extracellular dopamine and specific to D2/D3 receptors. Peripheral metabolism of

[11C]-(+)-PHNO in human was generally similar to that of [11C]-(-)-NPA with 30, 19 and 11% of plasma radioactivity due to parent radiotracer at 15, 30 and 75 min post-injection.5 [11C]-(+)-

PHNO binding to the D3 receptor was first suggested by its prominent binding in D3-rich GP and ventral STR and by the slower washout (Figure 8) of [11C]-(+)-PHNO from these regions

(50% washout in ~80 and >80 min, respectively) than from the D2-rich CAU and PUT (~57 min

5 11 in both regions). PET studies in baboon confirmed that [ C]-(+)-PHNO BPND in some brain regions can be reduced by pretreatment with the D3-selective drugs BP-897 and SB277011, with the greatest reduction seen in GP and substantia nigra, followed by ventral STR, but little

7,517 reduction seen in CAU and PUT. Furthermore, BPND in CAU and PUT could be reduced to a greater extent than in GP or ventral STR by pretreatment with the reportedly D2-selective drug

Figure 8. Regional [11C]-(+)PHNO time-activity curves in human brain demonstrating the different washout rates of [11C]-(+)-PHNO from A) CAU and PUT relative to B) ventral STR and especially GP. Image from reference 5 with permission.

7 11 SV-156. In human as well, [ C]-(+)-PHNO BPND in GP and ventral STR could be reduced by pretreatment with D3-selective drug (pramipexole and ABT-925).549,550 These data indicate that

11 the portion of [ C]-(+)-PHNO BPND due to D3 binding follows the rank order GP and SN > VS

> CAU and PUT. This rank order agrees generally with the regional trend in [11C]-(+)-PHNO washout rate, confirming that the kinetic differences between regions, as suggested in the first human [11C]-(+)-PHNO PET studies,5,548 are due to different regional proportions of D3 versus

11 D2 binding. Comparing the regional rank order of BPND for [ C]-(+)-PHNO (GP > ventral STR

> CAU/PUT), [11C]-(-)-NPA (GP ~ ventral STR ~ CAU/PUT) and [11C]-raclopride (CAU/PUT

> ventral STR > GP) suggests that of these three radiotracers the ratio of D3 to D2 affinity is greatest for [11C]-(+)-PHNO, intermediate for [11C]-(-)-NPA and lowest for [11C]-raclopride.

The binding of [11C]-(+)-PHNO to both D2 and D3 receptors in combination with the anatomical separation between D3-rich (GP, ventral STR and SN) and D2-rich (CAU and PUT)

78 permits the simultaneous measurement of drug occupancy at both receptor types and is the subject of section 6 in this thesis.

The kinetics of [11C]-(+)-PHNO were described in detail by Ginovart et al. in 2006.

[11C]-(+)-PHNO time-activity curves in all regions (including cerebellum) were better fit by a 2

TC than a 1 TC model. Although an unconstrained 2 TC model provided precise estimates of

5 regional total distribution volumes, k3/k4 ratios (BPND) were poorly identified. The precision of k3/k4 estimates could be increased by constraining the 2 TC model such that the non- displaceable distribution volume (VND) was the same in each region of interest, either by coupling K1/k2 ratios across regions or by setting the K1/k2 ratio to the total distribution volume

5 obtained in cerebellum (the reference region). BPND values were slightly underestimated (~10%) using the SRTM due to violation of the assumptions of the model (presence of two tissue compartments in the reference region) but were highly correlated with those determined using either of the constrained 2 TC models (r2 > 0.97), and were stable in all regions within a

11 5 scanning time appropriate for a C-labeled radiotracer (80 min). Thus, despite a small BPND underestimate, the SRTM has become the method of choice for determination of [11C]-(+)-

545-547,550,551 PHNO BPND in human.

79 3. Brief introduction and rationale for thesis studies

The four studies in this thesis (sections 4-7) provide a detailed pre-clinical characterization of the agonist radiotracer [11C]-(+)-PHNO. These studies explore three main themes: 1) the in vivo validity of the two-affinity-state model; 2) the influence of isoflurane anaesthesia on the amphetamine sensitivity of [11C]-(+)-PHNO binding; and 3) the utility of

[11C]-(+)-PHNO for measurement of D2 and D3 receptor subtypes.

The first two studies (sections 4 and 5) test the in vivo validity of the two-affinity-state model as it applies to ex vivo [11C]-(+)-PHNO binding. This work has major implications for the interpretation of the in vivo binding of [11C]-(+)-PHNO and other agonist radiotracers (e.g.

[11C]-(-)-NPA and [11C]-(-)-MNPA), whose proposed advantages over the antagonist D2/D3 radiotracers are based solely on the in vivo validity of the two-affinity-state model. In particular, the first study tests the hypothesis that, because [11C]-(+)-PHNO should bind selectively to the high affinity state of the D2 receptor, its ex vivo binding should be more sensitive than that of the antagonist radiotracer [3H]-raclopride to treatment with either indirect and direct D2 agonist drugs. This is particularly relevant to addressing the claim that [11C]-(+)-PHNO and other agonist radiotracers will provide more sensitive measurement of extracellular dopamine (the endogenous agonist) than antagonist radiotracers. The second study tests the hypothesis that

[11C]-(+)-PHNO ex vivo binding should be increased in animal models that have increased high- affinity state as measured in vitro. The results of this study also have direct bearing on the proposed advantages of the agonist D2/D3 radiotracers as they directly address the hypothesis that [11C]-(+)-PHNO binds ex vivo to a state of the receptor analogous to the in vitro high- affinity state.

The third study (section 6) examines the influence of isoflurane anaesthesia on the sensitivity of [11C]-(+)-PHNO and another agonist radiotracer, [11C]-(-)-NPA, to treatment with the dopamine-releasing drug amphetamine. In this study we test the hypothesis that the

80 increased amphetamine-sensitivity of [11C]-(+)-PHNO and [11C]-(-)-NPA relative to [11C]- raclopride is the result of the effects of isoflurane anaesthesia, and not to selective high-affinity state binding of the agonist radiotracers as has been claimed by other investigators.

The final study in this thesis (section 7) examines the utility of [3H]-(+)-PHNO for measurement of both D2 and D3 receptor subtypes. Using both ex vivo and in vitro autoradiographic techniques, this study provides a detailed characterization of the D2 and D3 receptor contributions to [3H]-(+)-PHNO binding in all major dopaminergically innervated regions of rat brain. This study also examines the utility of radiolabelled (+)-PHNO for simultaneous measurement of D2 and D3 receptors as well as occupancy of these receptors by dopaminergic drugs.

81 4. Dopamine D2 receptor radiotracers, [11C]-(+)-PHNO and [3H]-raclopride, are indistinguishably inhibited by D2 agonists and antagonists ex vivo*

Patrick N. McCormick,1 Shitij Kapur,2,3 Philip Seeman,2,4 Eugenii A. Rabiner5 and Alan A.

Wilson2,3

1 Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada M5S 1A8

2 Department of Psychiatry, University of Toronto, Toronto, Ontario, Canada M5S 1A8

3 PET Centre, Centre for Addiction and Mental Health, 250 College Street, Toronto, Ontario,

Canada M5T 1R8

4 Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada M5S 1A8

5 Clinical Imaging Applications, GlaxoSmithKline Clinical Imaging Centre, London, UK

* Reproduced with permission from Nuclear Medicine and Biology 2008; 35: 11-17.

82 4.1. Abstract

Introduction. In vitro, the dopamine D2 receptor exists in two states, with high and low affinity for agonists. The high-affinity state is the physiologically active state thought to be involved in dopaminergic illnesses such as schizophrenia. The PET radiotracer [11C]-(+)-PHNO, being a D2 agonist, should selectively label the high-affinity state at tracer dose and therefore be more susceptible to competition by agonist as compared to the antagonist [3H]-raclopride, which binds to both affinity states.

Methods. We tested this prediction using ex vivo dual-radiotracer experiments in conscious rat. D2 antagonists (haloperidol or clozapine), a partial agonist (aripiprazole), a full agonist ((-)-NPA) or the dopamine-releasing drug amphetamine (AMPH) were administered to rats prior to a co-injection of [11C]-(+)-PHNO and [3H]-raclopride (i.v.). Rats were sacrificed 60 min post radiotracer injection. Striatum, cerebellum and plasma samples were counted for 11C and 3H. The specific binding ratio (SBR) i.e. %ID/g(striatum)-

%ID/g(cerebellum)/(%ID/g(cerebellum) was used as the outcome measure.

Results. In response to D2 antagonists, partial agonist, or full agonist, [11C]-(+)-PHNO

3 and [ H]-raclopride responded indistinguishably in terms of both ED50 and Hill slope (e.g. (-)-

11 3 NPA ED50 0.027 and 0.023 mg/kg for [ C]-(+)-PHNO and [ H]-raclopride, respectively). In response to AMPH challenge, [11C]-(+)-PHNO and [3H]-raclopride binding were inhibited to the same degree.

Conclusions. We have shown that [11C]-(+)-PHNO and [3H]-raclopride specific binding do not differ in their response to agonist challenge. These results do not support predictions of in vivo D2 agonist radiotracer binding behaviour, and cast some doubt on the in vivo applicability of the D2 two state model as described by in vitro binding experiments.

83 4.2. Introduction

In brain tissue homogenates, the dopamine D2 receptor exists in two states of differing affinity for agonists.552 The state with high-affinity for agonists (so-called D2High) is the state coupled to the G-protein and is responsible, in vivo, for the physiological action of agonists.254

The D2 high-affinity state is thought to be involved in the pathophysiology of schizophrenia and other diseases in which the dopaminergic system is implicated.1 As such, the high-affinity state of the brain D2 receptor is an important target for human positron emission tomography (PET) imaging. Since antagonist ligands do not distinguish between affinity states of the D2 receptor, the only way to directly measure the high-affinity state in vivo using PET is through the use of an agonist radiotracer.

The search for an agonist D2 radiotracer suitable for human PET studies has been underway for many years but, until recently with the development of [11C]-(-)-NPA510 and [11C]-

(-)-MNPA,447 had met with limited success. Over the past few years our group developed a novel agonist radiotracer, [11C]-(+)-PHNO, for PET imaging of dopamine D2/D3 receptors. Ex vivo in rat [11C]-(+)-PHNO selectively accumulates in the D2-rich striatum (striatum-to- cerebellum ratio 5.6 at 60 min post injection), displays pharmacology appropriate for binding to the D2 receptor (blocking studies), and is susceptible to pharmacological treatments that alter extracellular dopamine concentration.4 Subsequent investigations have confirmed these findings in vivo in both cat343 and rat493 using PET and intracerebral β-sensitive microprobe studies, respectively. Following this pre-clinical animal work, [11C]-(+)-PHNO has recently been used to successfully image the D2/D3 receptor in human PET studies.548,553

The in vitro binding behaviour of D2 agonists provides a basis for the prediction of the in vivo behaviour of a D2 agonist PET radiotracer. It has been argued that an agonist radiotracer, which labels the high-affinity subset of the receptor population, should be more susceptible to competition by agonists (both endogenous and exogenous) than should an antagonist

84 radiotracer.4,447,470,554 For example, pretreatment with an agonist drug (exogenous agonist) or stimulation of dopamine release (endogenous agonist) should result in higher receptor occupancy when measured with an agonist radiotracer than when measured with an antagonist radiotracer. Conversely, agonist and antagonist radiotracers should be equally sensitive to competition by antagonist drugs, since these drugs do not distinguish between affinity states.

The primary aim of this study was to test these predictions by comparing the effect of exogenous compounds, both agonist and antagonist, and the dopamine-releasing drug amphetamine (AMPH), on the striatal specific binding of [11C]-(+)-PHNO and [3H]-raclopride, using ex vivo dual-radiotracer experiments in rat.

As we were conducting these studies, new observations showed that the [11C]-(+)-PHNO gives rise to a particularly high signal in the in the globus pallidus of human subjects548 and baboons.517 Furthermore, in the baboon the globus pallidus, binding of [11C]-(+)-PHNO was inhibited by the D3-selective drug BP-897 to a greater extent than was the binding of [11C]- raclopride.517 These data suggest that the [11C]-(+)-PHNO binding signal, at least in some parts of the primate brain, is more D3- than D2-dependent. To examine what implication these findings may have for our work, a secondary aim of this study was to define more precisely the dopaminergic receptor types that are responsible for our ex vivo [11C]-(+)-PHNO striatal binding signal in rat.

4.3. Materials and methods

4.3.1. General

Male Sprague-Dawley rats, weighing 334 ± 43 g on the day of the experiment, were housed two per cage under a 12 h light 12 h dark photocycle and were allowed unlimited access to food and water. All rats were housed in the animal facility at the Centre for Addiction and

Mental Health for at least one week prior to experiments). High specific activity (1400 ± 400

85 mCi/µmol at end of synthesis) [11C]-(+)-PHNO ([11C]-(+)-4-propyl-3,4,4a,5,6,10b-hexahydro-

2H-naphtho[1,2-b][1,4]oxazin-9-ol) was synthesized by N-11C-propylation of despropyl-(+)-

PHNO as previously described (see Scheme 1).4 [3H]-Raclopride (60.1 Ci/mmol) was purchased from PerkinElmer Life Sciences. (-)-NPA, haloperidol and clozapine were purchased from

Sigma-Aldrich. Aripiprazole and SB277011 were gifts from Eli Lilly (USA) and

GlaxoSmithKline (UK), respectively. Amphetamine sulphate was purchased from U.S.

Pharmacopeia (USA). All animal experiments were conducted with approval of the Animal

Ethics Committee at the Centre for Addiction and Mental Health and in accordance with the

Canadian Council on Animal Care.

4.3.2. Ex vivo competition studies

Groups of rats were injected (1 mL/kg body weight) 4 mg/kg AMPH (i.v., n = 9) or one of various doses of the following dopaminergic ligands (s.c., n = 6 per group): the D2 full agonist (-)-NPA, the D2 partial agonist aripiprazole, the D2 antagonists haloperidol and clozapine or the D3-selective antagonist SB277011. Drug vehicle solutions were saline for

AMPH, 0.1% ascorbic acid in saline for (-)-NPA, 30% DMF + 1% acetic acid in saline for aripiprazole, 1% acetic acid in saline for haloperidol, 2% acetic acid in saline for clozapine and

20% DMSO in saline for SB277011. For each of the above drugs, a separate group of vehicle- treated animals (s.c., n = 6 for antagonists and direct agonists and i.v., n = 9 for AMPH) served as controls. With the exception of AMPH, all drugs were administered 30 min prior to i.v. co- injection of high specific activity [11C]-(+)-PHNO (1.1 ± 0.3 nmol per rat) and [3H]-raclopride

(0.1 nmol per rat). AMPH was injected i.v. 50 min prior to radiotracer co-injection.

86 4.3.2.1. Dual-radiotracer biodistribution

Regional radiotracer biodistribution was determined as previously described,4,390 with modifications for determination of [3H]-raclopride biodistribution in the same tissue samples, as follows. Rats were weighed and numbered with marker on the base of their tail and brought from the animal facility in opaque transfer cages with 4 rats per cage. [11C]-(+)-PHNO, formulated in 8.4% aqueous sodium bicarbonate, was transported from the radiochemistry lab in a lead pig and placed behind lead shielding in the rat lab fume hood. [3H]-raclopride (1 µCi/µL) was added to the [11C]-(+)-PHNO solution to give approximately 7.5 µCi of [3H]-raclopride per

300 µL of final radiotracer solution (i.e. one i.v. injection volume). Individual numbered radiotracer injection syringes were loaded with 300 µL of radiotracer solution and the 11C radioactivity in each syringe (µCi, using a dose calibrator) and the exact time of measurement were recorded. All times, including radiotracer injection times (below) were recorded to the second (i.e. hh:mm:ss). All manipulation of the radiotracer dose vial and radiotracer syringes was done behind leaded glass. An additional dose syringe was loaded with a similar volume of the radiotracer solution to serve as a standard of the injected dose (see below). The 11C radioactivity in this syringe was also determined in the dose calibrator and the time of measurement recorded.

Just before radiotracer injection, each rat was placed in a Plexiglas rat restrainer and its tail immersed in a beaker of warm water (~45 ºC) for ~30 seconds to dilate the tail vein. The radiotracer injection was injected i.v. in a lateral tail vein over an approximately 4 s interval, after which the rat was quickly returned to its home cage. The next rat was then quickly loaded into a restrainer and brought to the injection area, such that subsequent radiotracer injections were done 2 min apart (typically with an error of less than 30 s). The exact time of each radiotracer injection was recorded. The residual 11C radioactivity remaining in each injection syringe was determined in the dose calibrator and recorded along with the exact time of

87 measurement. The contents of the standard syringe were injected into a 100 mL volumetric flask containing ~80 mL of saline and few milliliters of 95% ethanol (to prevent adsorption of radiotracer to the wall of the flask). The flask was then filled to the 100 mL mark with saline, inverted several times to mix the contents and three 1 mL aliquots of the resulting radiotracer standard solution were pipetted into 7 mL plastic tubes. The radioactivity in these three tubes represented ~1% of the dose injected into each rat – comparable to levels of radioactivity in brain tissue (see results).

60 min after dual-radiotracer injection, each rat was sacrificed by decapitation and its brain removed onto ice. A blood sample was collected at the time of sacrifice (from the trunk of the animal) into a heparinized glass tube and centrifuged (5 min, 1200 RPM) to isolate blood plasma. Brain regions of interest (striatum and cerebellum) were excised and placed, as were blood plasma samples (~100 µL), into pre-weighed, labeled 7 mL plastic tubes and capped

(tubes were pre-weighed with caps on). The tail of each rat was removed and counted in the dose-calibrator to determine the amount of radioactivity that remained near the site of injection.

The capped plastic tubes with tissue or plasma samples were weighed (to 10 µg accuracy) and the 11C radioactivity in each sample, along with the three 1 mL aliquots of the diluted injected dose standard, was determined in a γ-counter. The γ-counter was programmed to back-correct tissue radioactivity to the time when counting was initiated. All data were then entered into a specially-designed Microsoft Excel spreadsheet and analyzed using macros written in Microsoft

Visual Basic, as follows. The macros first determined the relationship between γ-counts and radioactivity (in µCi) using the fact that the standard tubes (counted using the γ-counter) contained 1% of the standard injected dose (measured in the dose calibrator). Using this relationship, the γ-counts in the tissue samples were then converted to µCi, back-corrected to the time of first radiotracer injection, and expressed as a percent of the corresponding injected dose

(also previously measured in the dose calibrator) for each rat (%ID). Each injected dose was

88 reduced by the sum of the radioactivities (back-corrected to first injection) remaining in the injection syringe and the tail of the rat. The %ID values were then divided by the weight of the corresponding tissue sample (g) to give the percent injected dose per gram of wet tissue weight

(%ID/g). The tissue, plasma and standard tubes were placed in the refrigerator until they were processed to determine 3H radioactivity content (see below).

For determination of 3H radioactivity the same tissue and standard samples were digested for 24 h in 3 mL of SolvableTM after which 6 mL of Aquassure scintillation fluid was added and the samples mixed on a rotary sample shaker for 24 h. 3H radioactivity was quantified using a liquid scintillation counter. [3H]-raclopride data were analyzed as described for [11C]-

(+)-PHNO, with two exceptions. First, because exact 3H injected doses could not be measured at the time of injection, the injected [3H]-raclopride doses were entered into the Excel spreadsheet as 7.5 µCi and multiplied by a correction factor expressing the ratio of injected to standard dose determined for [11C]-(+)-PHNO. This correction factor takes into account the radioactivity remaining in the injection syringe and the tail of the rat, which are assumed to represent the same proportion of [11C]-(+)-PHNO and [3H]-raclopride radioactivities. The specific [11C]-(+)-

PHNO and [3H]-raclopride binding in striatal samples was estimated by the specific binding ratio (SBR), defined as:

%ID/g Striatum - %ID/g Cerebellum SBR = . %ID/g Cerebellum

4.3.3. Data analysis and statistics

Inhibition curves were fit using a sigmoidal dose-response relationship using GraphPad

Prism software. The curve-fitting process was completely unrestrained and the fitting model allowed for variable Hill slope. Individual fitting parameters (ED50 and Hill slope) for inhibition of [11C]-(+)-PHNO SBR were compared to those for inhibition of [3H]-raclopride SBR by

Student’s t-test. ED50 and Hill slope values for one drug versus another were compared by

ANOVA followed by Bonferroni’s multiple-comparison test. The average SBR for the

SB277011 and AMPH-treated groups were compared to the corresponding vehicle-treated groups by Student’s t-test. The inhibition of [11C]-(+)-PHNO and [3H]-raclopride SBR by

AMPH pretreatment were also compared by Student’s t-test. Statistical comparisons were considered significant when p < 0.05.

4.4. Results

At 60 min post radiotracer injection the accumulation of 11C radioactivity in the striatum and cerebellum of vehicle pretreated rats was 0.46 ± 0.10 %ID/g and 0.081 ± 0.019 %ID/g, respectively, yielding an average SBR ratio of 4.7. These values are consistent with our previous studies of [11C]-(+)-PHNO biodistribution in rat.4 In the same animals, the accumulation of 3H radioactivity was 0.41 ± 0.11 and 0.036 ± 0.011 %ID/g in striatum and cerebellum, respectively.

The resultant SBR of 11.4 is also consistent with previous reports.340

Pretreatment with the D2 full agonist (-)-NPA, the D2 partial agonist aripiprazole and the D2 antagonists haloperidol and clozapine all caused dose-dependent inhibition of [11C]-(+)-

PHNO and [3H]-raclopride SBR (Figure 9). For all of these drugs, inhibition data were well

2 fitted (R > 0.99) by a sigmoidal dose-response curve. The ED50 and Hill slope values for inhibition of [11C]-(+)-PHNO were not significantly different from those for inhibition of [3H]- raclopride (Table 3) for any of the drugs tested. Hill slope values for inhibition of [11C]-(+)-

PHNO by haloperidol were significantly greater than those for inhibition by (-)-NPA (p < 0.05) and aripiprazole (p < 0.01). There were no other significant differences in Hill slope between drugs. The maximum inhibition of [11C]-(+)-PHNO and [3H]-raclopride SBRs (derived from the fitting process) were greater than 90% for (-)-NPA, aripiprazole and haloperidol. For clozapine the fitted maximum inhibition was 84% and 78% for inhibition of [11C]-(+)-PHNO and [3H]-

90 raclopride, respectively. The D3-selective antagonist SB277011 did not, at any of the doses tested, significantly inhibit [11C]-(+)-PHNO or [3H]-raclopride SBR (Figure 10). AMPH (4 mg/kg, i.v.) caused statistically significant 30 ± 7 (p < 0.0001) and 26 ± 14 % (p < 0.005) decreases in [11C]-(+)-PHNO and [3H]-raclopride SBR, respectively (Figure 11). There was no significant difference in the effect of AMPH on the two radiotracers (Student’s t-test; p > 0.05).

4.5. Discussion

In vitro, it is clear that the dopamine D2 receptor, like other G-protein coupled receptors, exists in two states of affinity for agonists. Competition curves between agonist ligands and radiolabeled antagonists show Hill slopes less than unity, and with certain radioligands, such as

[3H]-domperidone, the competition curves are clearly biphasic.555 Were these same affinity

Table 4. Dose-response parameters for inhibition of [11C]-(+)-PHNO and [3H]-raclopride by dopaminergic drugs.

[11C]-(+)-PHNO [3H]-Raclopride 95% 95% 95% 95% ED Hill ED Hill Drug Drug type 50 Confidence Confidence 50 Confidence Confidence (mg/kg) slope (mg/kg) slope interval interval interval interval D2 full (-)-NPA 0.027 0.015, 0.050 -0.92 -1.40, -0.45 0.023 0.019, 0.028 -1.04 -1.25, -0.85 agonist D2 partial Aripiprazole 0.33 0.13, 0.88 -0.79 -1.2, -0.35 0.15 0.069, 0.34 -0.89 -1.48, -0.33 agonist D2 Haloperidol 0.018 0.014, 0.025 -2.1a,b -3.0, -1.10 0.016 0.013, 0.020 -1.8 -2.39, -1.15 antagonist D2 Clozapine 4.6 3.5, 6.2 -1.4 -2.02, -0.74 6.2 4.4, 8.6 -1.7 -2.64, -0.79 antagonist a Significantly different from Hill slope of (-)-NPA vs. [11C]-(+)-PHNO; Bonferroni’s multiple comparison (p<0.05). b 11 Significantly different from Hill slope of aripiprazole vs. [ C]-(+)-PHNO; Bonferroni’s multiple comparison (p<0.01).

Figure 10. Effect of the D3-selective antagonist SB277011 on the striatal SBR of [11C]-(+)-PHNO (filled circles) and [3H]-raclopride (open circles). Error bars represent the SD of the means. No SB277011-treated group displays an average SBR significantly different from the vehicle-treated group; Dunnett’s multiple comparison (p > 0.05).

Figure 11. Effect of AMPH pretreatment of [11C]-(+)-PHNO and [3H]-raclopride SBR. AMPH pretreatment did not differentially affect the two radiotracers; Student’s t-test (p > 0.05).

states to exist to the same extent in vivo, one would expect that an agonist drug would inhibit the

SBR of an antagonist radiotracer with a Hill slope less than unity, and that an agonist drug should more potently inhibit the SBR of an agonist radiotracer than that of an antagonist radiotracer. Antagonist drugs, on the other hand, should not differentially inhibit the SBR of agonist and antagonist radiotracers, with respect to either Hill slope or ED50.

93 In the present study we used the powerful technique of dual-radiotracer ex vivo biodistribution studies in order to compare, in the same animals and at the same time, the effect of drug pretreatment on the SBRs of [11C]-(+)-PHNO and [3H]-raclopride. Since, in this technique, binding data for two radiotracers can be obtained from a single animal, the noise in the SBR associated with inter-subject differences in animal handling, drug response, radiotracer delivery, etc. can be reduced relative to separate single-radiotracer experiments. The results of this study show that for the antagonist drugs, haloperidol and clozapine, the ED50 and Hill slope values are the same for inhibition of [11C]-(+)-PHNO and [3H]-raclopride binding to the D2

11 receptor. The ED50 values reported here for haloperidol (0.018 and 0.016 mg/kg for [ C]-(+)-

PHNO and [3H]-raclopride, respectively) and clozapine (4.6 and 6.2 mg/kg, respectively) are in agreement with those reported by Kapur et al. (0.02 and 7 mg/kg s.c. for haloperidol and clozapine, respectively) for inhibition of [3H]-raclopride SBR.330 The observation that haloperidol and clozapine do not differ with respect to inhibition of [11C]-(+)-PHNO and [3H]- raclopride agrees with predictions based on in vitro competition experiments in that it suggests that antagonist drugs bind with equal affinity to both the high- and low-affinity states of the D2 receptor. However, this is the only prediction based on in vitro results that is supported by the present study.

The D2 full agonist (-)-NPA inhibited the SBR of [11C]-(+)-PHNO and [3H]-raclopride in indistinguishable fashion. The Hill slopes for inhibition of [11C]-(+)-PHNO and [3H]-raclopride

SBR by (-)-NPA were 1.04 and 0.93, respectively. These Hill slope values, being very close to unity, are indicative of a one-site model. Similarly, for the partial agonist aripiprazole, even though the Hill slope values were less than for inhibition by (-)-NPA, there were no differences in Hill slope between inhibition of [11C]-(+)-PHNO and [3H]-raclopride SBRs, suggesting that that aripiprazole inhibits agonist and antagonist radiotracer binding at identical receptor sites.

11 3 The ED50 values for aripiprazole (0.33 and 0.15 mg/kg for [ C]-(+)-PHNO and [ H]-raclopride,

94 respectively) are in general agreement with that reported by Natesan et al. (0.7 mg/kg, s.c.) for inhibition of [3H]-raclopride SBR.556 Despite numerous differences in methods (species, route of injection, time of drug administration, time of sacrifice after radiotracer injection, outcome measure) the ED50’s reported here for (-)-NPA, haloperidol and clozapine also parallel the results reported by Anderson.557 Neither (-)-NPA nor aripiprazole inhibited [11C]-(+)-PHNO

SBR more potently than [3H]-raclopride SBR.

In agreement with our results from inhibition by exogenous ligands, AMPH-induced dopamine release was associated with the same inhibition of [11C]-(+)-PHNO and [3H]- raclopride SBR. This runs contrary to PET evidence in baboon showing that [11C]-(-)-NPA

11 specific binding (V3”) is more potently inhibited by AMPH treatment than that of [ C]- raclopride.470 Differences between agonist radiotracers and the antagonist radiotracer [11C]- raclopride have been demonstrated by our group with [11C]-(+)-PHNO in cat PET experiments343 and by Seneca et al. using [11C]-(-)-MNPA in cynomolgus monkeys.447 The above three studies were conducted in anaesthetized animals (isoflurane anaesthesia in the cases of [11C]-(-)-NPA and [11C]-(+)-PHNO and ketamine anaesthesia in the case of [11C]-(-)-MNPA), whereas the present data are from conscious animals. Our group has demonstrated that isoflurane anaesthesia increases the susceptibility of [11C]-(+)-PHNO, but not [3H]-raclopride, to inhibition by AMPH.558 We have also observed this phenomenon when [11C]-(-)-NPA is used as the agonist D2 radiotracer (section 5). Furthermore, a recent β-microprobe study by Galineau et al.493 demonstrated that 2 mg/kg (i.v.) AMPH reduced the striatal binding potential (BP) of

[11C]-(+)-PHNO by 69% in ketamine anaesthetized rats, with ~65% decrease in the SBR at the

60 min time point, an effect much greater than the 30% decrease in [11C]-(+)-PHNO SBR we observe in conscious rats after pretreatment with 4 mg/kg (i.v.) AMPH. Thus, the increased susceptibility of agonist radiotracers to endogenous dopamine release, when compared to [11C]- raclopride, may be due more to the confounding effects of anaesthesia than to any inherently

95 greater dopamine sensitivity. However, in one report using a dual-radiotracer design similar to that used in the present study, AMPH treatment was indeed found to decrease the BP of the agonist radiotracer [3H]-(-)-NPA to a greater extent than that of [11C]-raclopride, despite the lack of anaesthetic use.559

The results of the present ex vivo experiments in awake rats are consistent with a one-site model both for [11C]-(+)-PHNO and [3H]-raclopride binding. However, this does not necessarily rule out the existence of high- and low-affinity D2 states in vivo. Fitting of in vitro competitive binding curves to a two-site model implicitly assumes that the high- and low-affinity states of the receptor do not interconvert, at least on the time scale of the competition experiment. In the alternative case of rapidly interconvertible affinity states, agonist would, as in the non- interconvertible affinity states model, compete preferentially with radiotracer bound to the high- affinity state. However, the remaining unoccupied receptors would re-establish the equilibrium mixture of high- and low-affinity state concentrations. Consequently, no matter what the concentration of competing agonist, competition would always be for high-affinity state receptors. Under these circumstances, competition or inhibition curves for agonist and antagonist radiotracers would be identical. Indeed, Sibley et al. found that in intact cells preparations (as opposed cell membrane preparations) competition experiments between apomorphine or (-)-NPA and the antagonist radiotracer [3H]-spiperone revealed only a single affinity state.560 Another possibility is that, in vivo, all of the D2 receptors are configured in the high-affinity state. As such there would be, by definition, no difference in susceptibility to agonist challenge between agonist and antagonist radiotracers, since both would label exactly the same receptor population. Kenakin has pointed out that when the concentration of G protein is much greater than the concentration of receptor, which is the case with many native receptor systems, all receptors can form the high-affinity, G protein-coupled receptor state, resulting in only one observable affinity state.561

96 The present data do not favour, per se, either of these alternative descriptions of the behaviour of an agonist radiotracer. They do show, however, that in terms of susceptibility to inhibition by antagonist or agonist drugs, the behaviours of [11C]-(+)-PHNO and [3H]-raclopride are basically indistinguishable. The similarity in ex vivo binding behaviour between the agonist radiotracer [3H]-(-)-NPA and [3H]-raclopride has been documented previously by Kohler and

Karlsson-Boethius, who showed that the ED50 for inhibition of specific binding by raclopride pretreatment was identical for the two radiotracers.562 Evidence for the similarity of [11C]-(+)-

PHNO and [11C]-raclopride striatal binding sites is offered by Ginovart et al., who found using

PET that the Bmax of the two radiotracers in cat striatum were equivalent (31 pmol/mL and 32

343 3 pmol/mL, respectively). Similar striatal Bmax values for the agonist radiotracer [ H]-(-)-NPA and [3H]-raclopride have also been shown ex vivo in mouse.512,563 Thus, our work here and data gathered from the literature support the pharmacological similarity of [11C]-(+)-PHNO and [3H]- or [11C]-raclopride binding sites in vivo.

One surprising finding in this work is that the Hill slope of the curve describing the inhibition [11C]-(+)-PHNO and [3H]-raclopride SBR by haloperidol are greater than unity. The

Hill slope for inhibition of [11C]-(+)-PHNO and [3H]-raclopride SBR by haloperidol were 2.1 and 1.8, respectively. For inhibition of [11C]-(+)-PHNO, this Hill slope is significantly greater than for inhibition by the full agonist (-)-NPA or the partial agonist aripiprazole. The Hill slope for inhibition of [3H]-raclopride did not differ significantly for these drugs, although even for

[3H]-raclopride the Hill slope for inhibition by haloperidol is the highest seen for any of the drugs tested. Although we cannot say for certain why the Hill slope for haloperidol is so high, one possibility is that haloperidol binding to a portion of the D2 population reduces the affinity of the radiotracer for the remaining receptor population. Indeed, this cooperativity argument is a classic explanation for Hill slopes greater than unity. Since this effect is seen both for [11C]-(+)-

97 PHNO and [3H]-raclopride, it provides further evidence for the similarity in behaviour of the receptor sites labeled by the two radiotracers.

Both the observation of high [11C]-(+)-PHNO BP in the globus pallidus of human subjects548 and the work of Narendran et al. showing that [11C]-(+)-PHNO is blocked to a greater extent than [11C]-raclopride by the D3-selective ligand BP-897517 suggest that [11C]-(+)-

PHNO has high affinity for the dopamine D3 receptor in vivo. This prompted us to examine the dopamine receptor types that are responsible for the ex vivo striatal [11C]-(+)-PHNO signal in rat.

The ex vivo rank order of potency of the drugs tested (haloperidol ~ (-)-NPA > aripiprazole > clozapine) is in general agreement with in vitro and ex vivo D2 pharmacological literature.330,531,556 The D3-selective antagonist SB277011 was unable, at any of the doses tested, to inhibit [11C]-(+)-PHNO or [3H]-raclopride SBR (Figure 10). SB277011 has been shown using in vivo microdialysis to be highly brain penetrant.531,564 Furthermore, at doses lower than used in the present study, SB277011 selectively blocks agonist-induced decreases in dopamine release in the D3-rich nucleus accumbens but not in the D2-rich striatum,565 which is consistent with the

D3-selectivity of this antagonist. Thus, we conclude that in our striatal tissue samples, the SBR of [11C]-(+)-PHNO and [3H]-raclopride reflect D2 receptor binding. It is known that the D2-like receptors in the dorsal striatum are primarily of the D2 type, while those in the ventral striatum and globus pallidus are to a greater extent of the D3 type.122 In our striatal dissection technique, we excise a tissue sample whose bulk mass corresponds to the dorsal striatum. Thus in our rat model, [11C]-(+)-PHNO can be used to study the D2 receptor without contamination with D3 signal. It should be noted that a recent PET study by Ginovart et al. has shown that SB277011 pretreatment did not decrease the BP of [11C]-(+)-PHNO in the cat striatum.

A limitation of the present work is that the binding of [11C]-(+)-PHNO and [3H]- raclopride were measured at only one time point (60 min) following administration of the radiotracers. This presents a possible confound since it could be argued that changes in the SBR

98 in the present study may not necessarily correlate well with more rigorous estimates of radiotracer specific binding, such as the BP, which are derived by kinetic modeling of full regional time-radioactivity curves. However, Ginovart et al. have shown in a β-microprobe study that the ex vivo striatal SBR of [11C]-raclopride at 60 min post-radiotracer injection (or the

340 BPratio-ex vivo, in their nomenclature) correlates well with the kinetically modeled BP. In the same study, the receptor occupancies calculated using either the SBR or the kinetically modeled

BP were also highly correlated. There is little reason to assume that the correlation between SBR and BP, or between SBR- and BP-based calculations of receptor occupancy do not also hold true for [11C]-(+)-PHNO binding. In fact, recent β-microprobe and PET studies in rat and cat, respectively, have shown that in response to SB277011, (-)-NPA and haldol, [11C]-(+)-PHNO striatal BP responds in the same fashion as does our striatal SBR in rat.343,493

3.6. Conclusions

Taken together, the results of the present study indicate that [11C]-(+)-PHNO and [3H]- raclopride label sites in vivo that behave in pharmacologically indistinguishable fashion, with respect to inhibition by exogenous agonist, partial agonist, and antagonist drugs as well as the dopamine-releasing drug AMPH. This finding fails to support one of the fundamental predictions the two state theory makes about the differential behaviour of D2 agonist versus antagonist radiotracers. We have also demonstrated that, in rat striatum, [11C]-(+)-PHNO and

[3H]-raclopride SBRs are due to D2 receptor binding without contamination with D3-related signal. These results help to further characterize the ex vivo binding of [11C]-(+)-PHNO and support its use in the investigation of D2 receptor pharmacology, dopamine release, and in the determination of therapeutic D2 receptor occupancies. However, these results do cast some doubt on the applicability of the high- and low-affinity state model of D2 receptor function as it applies to ex vivo radiotracer and in vivo PET experiments.

99 5. Ex vivo [11C]-(+)-PHNO binding is unchanged in animal models displaying increased high-affinity states of the D2 receptor in vitro*

Patrick N. McCormick1,2, Shitij Kapur2,3, Greg Reckless2 and Alan A. Wilson1,2,3

1 Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada M5S 1A8

2 PET Centre, Centre for Addiction and Mental Health, 250 College Street, Toronto, Ontario,

Canada M5T 1R8

3 Department of Psychiatry, University of Toronto, Toronto, Ontario, Canada M5S 1A8

* Reproduced with permission from Synapse 2009; 63: 998-1009

100 5.1. Abstract

Dopamine D2 receptor supersensitivity has been linked to an increase in the density of the D2 high-affinity state as measured in vitro. The two-affinity-state model of the D2 receptor predicts that the ex vivo specific binding of [11C]-(+)-PHNO, an agonist radiotracer thought to bind selectively to the high-affinity state in vivo, should be increased in animal models that display in vitro increases in the proportion of receptors in the D2 high-affinity state. Here, we test this hypotheses by comparing the ex vivo SBR of [11C]-(+)-PHNO with that of the antagonist radiotracer [3H]-raclopride in three dopaminergically supersensitive rat models – AMPH- sensitized rats, rats withdrawn from chronic ethanol, and unilaterally 6-OHDA-lesioned rats – using ex vivo dual-radiotracer biodistribution studies. We find that in AMPH-sensitized rats and rats withdrawn from chronic ethanol treatment, models which exhibited ~4-fold increases in the

D2 high-affinity state in vitro, the SBRs of [11C]-(+)-PHNO and [3H]-raclopride are unchanged relative to control rats. In unilaterally 6-OHDA-lesioned rats, we find that the increase in [11C]-

(+)-PHNO SBR is no different than that observed for the antagonist radiotracer [3H]-raclopride

(54 ± 16% and 52 ± 14%, respectively). In addition, the effect of acute AMPH pretreatment (4 mg/kg, i.v.) on the SBRs of [11C]-(+)-PHNO and [3H]-raclopride is equivalent in AMPH- sensitized (-38 ± 12% and -36 ± 8%, respectively) and in control rats (-40 ± 11% and -38 ± 7%).

These data emphasize a significant discrepancy between in vitro and in vivo measures of D2 agonist binding, indicting that the two-affinity-state model of the D2 receptor may not apply veridically to living systems. The potential implications of this discrepancy are discussed.

101 5.2. Introduction

Competitive binding experiments demonstrate that, in vitro, the D2 receptor exists in two states of affinity for agonists.254,484,485,552 The high-affinity state is of special interest because it is the functional form of the receptor in vitro,253,254 and has been implicated in the pathophysiology of psychosis,306 stimulant3,252,306,566 and alcohol abuse.2 Much effort has been invested over the past decade in the development of D2 agonist positron emission tomography

(PET) radiotracers with the aim of selectively measuring the D2 high-affinity state in living brain. To date, three such agonist radiotracers have been developed: the apomorphine derivatives [11C]-(-)NPA470,510,514,515 and [11C]-(-)-MNPA;447,522 and the napthoxazine derivative

[11C]-(+)-PHNO.4,5,343,493,513,547,548,551

[11C]-(+)-PHNO, developed by our group, is a full agonist at D2/D3 receptors526-528,531,567 and distinguishes between high- and low-affinity states of the D2 receptor in vitro.509 We have extensively characterized [11C]-(+)-PHNO as a PET radiotracer in animal models343,493,513,568 and in human subjects.5,547,548,551 The current study focuses on [11C]-(+)-PHNO specific binding ratio (SBR, an estimate of radiotracer specific binding) which, in rat striatum, represents exclusively D2 receptor specific binding.513

The two-affinity-state model of the D2 receptor predicts that, in vivo, a PET radiotracer that is a D2 full agonist should selectively label the high-affinity state of the receptor, as opposed to antagonist radiotracers (e.g. [11C]-raclopride, [18F]-fallypride, [18F]-FLB 457, etc.) which should label non-selectively both high- and low-affinity states. However, although the two-affinity state model is a well-validated description of agonist ligand binding in tissue homogenates and cell membrane preparations,502,569-572 there is no direct evidence that the D2 receptor exists in two distinguishable agonist affinity states in living tissue.

Here we offer a simple and direct ex vivo examination of the two-affinity-state model.

The basis for this examination stems from in vitro work showing that in animal models,

102 dopaminergic supersensitivity is associated with a large increase in the proportion of D2 receptors in the high-affinity state.2,3,252,306,566,573 The current study examines the SBR of the agonist radiotracer [11C]-(+)-PHNO and the antagonist radiotracer [3H]-raclopride in three such animal models: amphetamine-sensitized rats,255,574-578 rats withdrawn from chronic ethanol,579-581 and unilaterally 6-OHDA-lesioned rats.582-586 All of these animal models display dopaminergic supersensitivity and in two of them - amphetamine-sensitized and ethanol-withdrawn rats - the in vitro increase in high-affinity state has been directly measured using competitive binding between dopamine and various radioligands ([3H]-raclopride, [3H]-domperidone).2,3,252,306 The two-affinity-state model predicts that this increase in high-affinity state should be seen as an ex vivo increase in the SBR of [11C]-(+)-PHNO. By contrast, the SBR of [3H]-raclopride, representing binding to both high- and low-affinity states, should be insensitive to changes in the proportion of high-affinity state receptors – but should reflect changes in total D2 receptor expression. Thus, any selective increase in high-affinity states should show up as a larger increase in [11C]-(+)-PHNO SBR than that of [3H]-raclopride. To ensure the most valid comparison we used a simultaneous-injection dual-radiotracer approach such that both tracers were used at the same time in the same animal, and in one of the models (6-OHDA) the unilateral lesion provided a further within-subject control.

5.3. Materials and Methods:

5.3.1. General

Male Sprague–Dawley rats were housed two per cage under a 12-h light/12-h dark photocycle and were allowed unlimited access to food and water. All rats were housed in the animal facility of the Center for Addiction and Mental Health for at least 1 week prior to experiments. High specific-activity [11C]-(+)-PHNO (1400 ± 800 mCi/µmol at the end of synthesis) was synthesized by N-[11C]-propylation of the despropyl precursor, as previously

103 described 4. [3H]-raclopride (60.1 Ci/mmol) was purchased from Perkin-Elmer Life Sciences.

AMPH sulfate was purchased from US Pharmacopoeia (USA). All animal experiments were conducted with the approval of the Animal Ethics Committee at the Center for Addiction and

Mental Health and in accordance with the Canadian Council on Animal Care.

5.3.2. AMPH sensitization

Rats were sensitized to AMPH using previously described methods shown to result in increased in vitro D2 high-affinity state3,578 Thirty-six rats with an average weight of 230 g were divided into two equal groups. One group received intraperitoneal injections of AMPH three times per week (Monday, Wednesday and Friday), starting at a dose of 1 mg/kg in week one and increasing by 1 mg/kg per week to 5 mg/kg in week five. The control group received saline injections on the same days. After this five-week treatment period, animals were left drug-free during weeks six to nine. During week ten, six chronically AMPH-treated rats and six saline- treated rats were administered a test AMPH dose of 0.5 mg/kg i.p. and their behavioural responses to this treatment were monitored in locomotor behaviour boxes (Med Associates Inc,

USA). For three days leading up to behavioural testing rats were habituated to the locomotor boxes for 30 min per day. On the day of testing, rats were again allowed a 30 min period of habituation prior to the AMPH challenge. The locomotor response to the test dose was quantified as the number of infrared beam breaks per five-minute interval for a total of 60 minutes. The total amount of beam breaks over the 60 min test period for chronically saline- and

AMPH-treated rats was compared by the two tailed Student’s t test. Rats that participated in the locomotor testing were not included in the later radiotracer binding experiment which was also performed in week ten. On the day of the radiotracer experiment rats were pretreated with either saline (n=12, 6 AMPH sensitized and 6 chronic saline-treated controls) or 4 mg/kg, i.v. AMPH

(n=12, same distribution as for acute saline treatment) 50 min prior to radiotracer injection.

104 5.3.3. Withdrawal from chronic ethanol

Rats were withdrawn from chronic ethanol treatment according to previously described methods shown to result in a large increase in the D2 high-affinity state.2 Twenty-four rats with an average weight of 270 g were divided into two groups and received twice-daily i.p. injections of either saline or 2 g/kg ethanol (14 mL/kg of 18% ethanol in saline) for ten days. After the final day of chronic ethanol treatment rats were left ethanol-free for five days after which time the dual-radiotracer binding experiment was performed, as described in section 4.3.5 Two rats did not survive the ethanol treatment regime and one additional rat was removed from analysis due to anomalous radiotracer binding data (see results section). Nine ethanol- and 12 saline- treated rats participated in the dual-radiotracer biodistribution experiment.

5.3.4. Unilateral 6-OHDA lesions

Twenty-two rats with an average weight of 337 g at the beginning of the experiment were divided into four groups: left side lesion, n = 8; right side lesion, n = 8; left side sham lesion, n = 3; right side sham lesion, n = 3. Thirty minutes prior to injection of 6-OHDA (or vehicle) rats were injected intraperitoneally with 15 mg/kg desipramine to block uptake of the toxin by norepinephrine transporters. 6-OHDA (11 µg in a total volume of 4 µL) or vehicle

(0.2% ascorbic acid, for sham lesions) was injected under stereotaxic guidance over a period of five minutes into the medial forebrain bundle according to the rat brain atlas of Paxinos and

Watson (coordinates: 4.2 anterior to bregma; 1.8 mm lateral to midline; 7.8 mm below dura mater). After delivery of the toxin (or vehicle), the needle was left in place for 5 minutes to prevent diffusion of the toxin outward along the needle track. After removal of the needle, the hole in the skull was filled with bone wax and the wound sutured. Animals were monitored daily for signs of pain or stress and their weight monitored daily. The procedure was well tolerated and all rats were included in the following behavioural experiment. Two weeks after 6-

105 OHDA injection rats were monitored for rotational behaviour after injection of apomorphine

(0.05 mg/kg s.c.). Rotational behaviour was monitored by RotoRat apparatus and software (Med

Associates Inc., USA) and quantified as the number of full contralateral rotations per one- minute interval for 60 minutes. The total number of full rotations over the 60 minute period for lesioned and sham lesioned animals was compared by two tailed Student’s t test. Four lesioned animals were excluded from further analysis: three were excluded because of complications in the behavioural testing procedure. The radiotracer binding experiment, as described in section

4.3.5, was performed during the same week as the quantification of rotational behaviour. Six sham-lesioned and 13 lesioned rats participated in the dual-radiotracer biodistribution experiment.

5.3.5. Ex vivo dual-radiotracer binding studies

Regional radiotracer biodistribution was determined as previously described (see section

3.3.2.1 for full details).4,390,513 Briefly, rats were co-injected (tail vein) with [11C]-(+)-PHNO

(0.9 ± 0.5 nmol per rat) and [3H]-raclopride (~0.1 nmol per rat), and sacrificed by decapitation

60 min later. Brains were quickly removed onto ice and blood samples were collected from the trunk directly after decapitation and centrifuged to obtain plasma. Striatum samples, whole cerebellum and blood plasma samples were placed in pre-weighed sample tubes. For rats in the

6-OHDA lesion study, left and right striata were placed in separate sample tubes. Tissue samples were weighed and the radioactivity in the samples due to 11C determined using a gamma counter and back-corrected to the time of first radiotracer injection, using diluted aliquots of the original injected dose as standards. For determination of radioactivity due to 3H, the same tissue samples counted for 11C were treated with 3 mL of 0.6 N NaOH, shaken for 24h, and 6 mL of AquassureTM scintillation fluid then added. After a further 24 hours of mixing, the samples were counted in a liquid scintillation counter. Radioactivity in tissue samples was

106 expressed as a percentage of the injected dose per gram of wet tissue weight (%ID/g). The specific binding ratio (SBR) was calculated from the %ID/g as:

%ID/g − %ID/g SBR = STR CER %ID/gCER

5.4. Results

5.4.1. AMPH-sensitized rats

Rats chronically treated with AMPH displayed a heightened locomotor response to the

0.5 mg/kg AMPH test dose relative to chronically saline-treated control rats (Figure 12). The striatum and cerebellum %ID/g values for [11C]-(+)-PHNO and [3H]-raclopride in chronic saline- and AMPH-treated rats are given in Table 4. Chronic AMPH treatment had no effect on the regional %ID/g of either [11C]-(+)-PHNO or [3H]-raclopride (ANOVA, Bonferroni’s multiple comparison test, p > 0.05). Pretreatment with i.v. 4mg/kg AMPH decreased the %ID/g of [11C]-(+)-PHNO and [3H]-raclopride in striatum (Table 4) in both chronic AMPH- and saline- treated rats, but had no effect on the cerebellum %ID/g of either radiotracer (ANOVA,

Bonferroni’s multiple comparison test, p > 0.05). The SBRs of [11C]-(+)-PHNO and [3H]- raclopride in striatum of chronic AMPH-treated rats were no different (ANOVA, Bonferroni’s multiple comparison test, p > 0.05) than in striatum of chronic saline-treated rats (Table 4,

Figure 13). Challenge with i.v. 4 mg/kg AMPH prior to radiotracer co-injection produced the same decrease in the SBR of [11C]-(+)-PHNO and [3H]-raclopride in chronic saline-treated (40 ±

11% and 38 ± 7%, respectively) and AMPH-treated rats (38 ± 12% and 36 ± 8%) (Table 4,

Figure 14).

107

Figure 12. Locomotor response to i.p. injection of 0.5 mg/kg AMPH in chronic AMPH- and saline-treated rats. A) Time course of locomotor behaviour before (10-30 min) and after (30-90 min) AMPH injection. B) Chronic AMPH-treated rats showed significantly more beam breaks in the 60 minute period after AMPH injection than did chronic saline-treated rats (two-tailed Student’s t test).

Figure 13. Striatal SBR of [11C]-(+)-PHNO and [3H]-raclopride in chronic AMPH- and saline-treated rats. No difference between SBRs in the control and AMPH-sensitized rats were found for either radiotracer (two tailed Student’s t test).

4.4.2. Rats withdrawn from chronic ethanol treatment

Withdrawal from chronic ethanol treatment had no effect on the regional %ID/g values or striatum SBR values of neither [11C]-(+)-PHNO nor [3H]-raclopride (Table 4; two-tailed

Student’s t test, p > 0.05). One rat was removed from analysis because of anomalous radiotracer binding data ([11C]-(+)-PHNO SBR or 9.58 compared to a group average of 3.86 ± 0.52).

[11C]-(+)-PHNO [3H]-Raclopride Sensitization Chronic Acute regime treatment pretreatment STR %ID/g CER %ID/g SBR STR %ID/g CER %ID/g SBR Chronic Saline Saline 0.44 ± 0.03 0.09 ± 0.01 4.2 ± 0.4 0.39 ± 0.03 0.052 ± 0.005 6.5 ± 0.3 AMPH 4 mg/kg 0.31 ± 0.08a 0.09 ± 0.03 2.5 ± 0.5d 0.32 ± 0.08b 0.07 ± 0.02 4.1 ± 0.5d AMPH AMPH Saline 0.42 ± 0.03 0.081 ± 0.006 4.2 ± 0.5 0.39 ± 0.02 0.048 ± 0.001 7.0 ± 0.7 4 mg/kg 0.27 ± 0.04a 0.074 ± 0.009 2.6 ± 0.5d 0.27 ± 0.03c 0.053 ± 0.005 4.2 ± 0.5d AMPH Ethanol Saline none 0.4 ± 0.1 0.08 ± 0.02 4.3 ± 0.5 0.32 ± 0.07 0.030 ± 0.008 10 ± 1 withdrawal Ethanol none 0.4 ± 0.1 0.08 ± 0.01 4.7 ± 0.7 0.33 ± 0.06 0.029 ± 0.005 10 ± 1

a Significantly different from saline-pretreated group; ANOVA, Bonferroni’s multiple comparison test (p < 0.001). b Significantly different from saline-pretreated group, ANOVA, Bonferroni’s multiple comparison test, (p < 0.05). c Significantly different from saline-pretreated group, ANOVA, Bonferroni’s multiple comparison test (p < 0.01). d Significantly different from saline-pretreated group, ANOVA, Bonferroni’s multiple comparison test (p < 0.001)

108

109

Figure 14. Percent decrease in the SBR of [11C]-(+)-PHNO and [3H]-raclopride after i.v. injection of 4 mg/kg AMPH. No difference in SBR decrease was found between radiotracers or between chronic saline- and AMPH- treated rats for each radiotracer (ANOVA, Bonferroni’s multiple comparison test).

5.4.3. 6-OHDA-lesioned rats

Rats that received unilateral injection of 6-OHDA showed marked contralateral rotational behaviour in response to s.c. injection of 0.05 mg/kg apomorphine (Figure 15), whereas sham-lesioned rats displayed no rotational response (apart from one sham lesioned rat that displayed 24 rotations over the course of the testing period). Apart from the rats excluded because of complications in the behavioural testing one further rat was excluded because of anomalous [3H]-raclopride binding values (SBR of 1.8 compared to a group mean of 17.4 ± 2.6).

There was a large range of rotational behaviour in the lesioned rats in response to apomorphine challenge, with the total number of rotations over the 60 minute testing period ranging from 118 to 414 (Figure 15, B). The striatum and cerebellum %ID/g values for sham, left and right lesioned animals are given in Table 5. The %ID/g of both [11C]-(+)-PHNO and [3H]-raclopride in lesioned striatum were increased as compared to either intact striatum (striatum contralateral to toxin injection) or sham lesioned striatum (ANOVA, Bonferroni’s multiple comparison test, p

< 0.001). No difference in the %ID/g was seen between intact striatum and sham lesioned

110

Figure 15. Rotational behaviour of unilaterally 6-OHDA lesioned rats after injection of 0.05 mg/kg apomorphine. A) Typical time course of rotational behaviour after apomorphine injection. B) Total rotations over the 60 min testing period for sham and unilaterally-lesioned rats. Lesioned rats showed rotational behaviour whereas sham animals, apart from one animal that displayed 24 rotations, showed no rotational behaviour.

Figure 16. Striatal [11C]-(+)-PHNO and [3H]-raclopride SBR in 6-OHDA lesioned and sham lesioned rats. The SBR of [11C]-(+)-PHNO and [3H]-raclopride were increased in lesioned striatum relative to sham lesioned striatum (ANOVA, post hoc Dunnett’s test, p < 0.01).

Table 6. Left striatum, right striatum and cerebellum %ID/g values for [11C]-(+)-PHNO and [3H]-raclopride in left-lesioned, right-lesioned and sham-lesioned rats. [11C]-(+)-PHNO [3H]-Raclopride

Left striatum Right striatum Cerebellum Left striatum Right striatum Cerebellum

Sham 0.58 ± 0.05 0.55 ± 0.06 0.10 ± 0.01 0.52 ± 0.05 0.52 ± 0.05 0.045 ± 0.009

Left-lesioned 0.8 ± 0.1a 0.52 ± 0.07 0.10 ± 0.01 0.77 ± 0.08a 0.51 ± 0.02 0.042 ± 0.005

a a Right-lesioned 0.58 ± 0.09 0.8 ± 0.1 0.10 ± 0.01 0.54 ± 0.07 0.8 ± 0.1 0.044 ± 0.004 a Significantly different than %ID/g in intact striatum and sham-lesioned striatum; ANOVA, Bonferroni’s multiple

comparison test, p > 0.001.

Figure 17. Ratio of [11C]-(+)-PHNO and [3H]-raclopride SBRs in lesioned striatum to that of the intact striatum. No difference in lesioned / intact ratio was found between radiotracers (Student’s t test).

111

112 striatum for either radiotracer. Relative to the intact striatum, the SBR in the lesioned striatum was increased to the same extent for [11C]-(+)-PHNO and [3H]-raclopride (54 ± 16% and 52 ±

14%, respectively) (Figure 16). The ratio of striatal SBR in intact striatum to that in the lesioned striatum is shown in Figure 17.

5.5. Discussion

Many recent PET studies have described the in vivo specific binding of the newly- developed D2 agonist radiotracers [11C]-(-)-NPA,470,476,510,515 [11C]-(-)-MNPA447,522-524 and

[11C]-(+)-PHNO.4,5,343,493,513,517,547,548,551,558 Some of these studies, conducted in anaesthetized animals, have exploited the greater AMPH-induced reductions of agonist versus antagonist radiotracer BP in order to infer the proportion of receptors in the high-affinity state.343,447,470,515

However, this inference relies on a major theoretical assumption: that the two-affinity-state model of the D2 agonist binding is valid in vivo, and by extension, that the BP of agonist radiotracers represents selective specific binding to the D2 high-affinity state. While this assumption has seemed reasonable, there has yet been no direct demonstration that the two- affinity-state model is a valid description of in vivo D2 agonist specific binding or that the increased AMPH sensitivity of agonist versus antagonist radiotracers is due to selective high- affinity state binding. Indeed, differences in response to AMPH treatment are not unique to comparisons of agonist versus antagonist D2 radiotracers. Major differences in AMPH response are also seen, for example, between D2 antagonist radiotracers (e.g. benzamide versus butyrophenone derivatives), and between D1 and D2 antagonist radiotracers (for review see reference 475).

Several lines of evidence challenge the simple translation of the in vitro two-affinity- state model to the in vivo situation. First, since high-affinity states are only a subset of the total

11 number of receptors, one would expect that the density of [ C]-(+)-PHNO binding sites (Bmax)

113 would be smaller than that measured with the antagonist radiotracer [11C]-raclopride. However,

11 11 343 the Bmax measured with [ C]-(+)-PHNO is identical to that measured by [ C]-raclopride.

Second, although there is greater AMPH-induced inhibition of agonist versus [11C]-raclopride

BP in anaesthetized animals,343,447,470,515 this difference in AMPH effect is not observed when comparing the ex vivo SBR of [11C]- or [3H]-raclopride with that of [11C]-(+)-PHNO in unanaesthetized animals (section 5).513 [11C]-(-)-MNPA as well displays greater BP reductions in ketamine-anaesthetized than in non-anaesthetized monkeys.587 Third, in non-anaesthetized rats, pretreatment with full D2 agonist ((-)-NPA), partial agonist (aripiprazole) or antagonist

(haloperidol, clozapine) results in inhibition curves for [11C]-(+)-PHNO and [3H]-raclopride

SBR that are indistinguishable from one another,513 consistent with the presence of a single affinity state. Thus, experiments performed in anaesthetized and non-anaesthetized animals offer contradictory evidence as to whether the two-affinity-state model is a valid description of the D2 receptor in vivo.

Here we provide a direct ex vivo examination of the two-affinity-state model. We examine [11C]-(+)-PHNO and [3H]-raclopride SBR in AMPH-sensitized, ethanol-withdrawn and unilaterally 6-OHDA-lesioned rats. Dopaminergic supersensitivity, common to all three of these animal models,255,575-586,588 is associated, in vitro, with large increases in the proportion of D2 receptors in the high-affinity state.306 In two of the models examined here – AMPH-sensitized and ethanol-withdrawn rats – 360% increases in the high-affinity state has been directly measured using in vitro competitive binding experiments in striatal membrane preparations.2,3,252,306 According to the two-affinity-state model, increases in the proportion of high-affinity state receptors should be detected ex vivo as an increase of similar magnitude in the

SBR of an agonist D2 radiotracer, whereas that of an antagonist radiotracer, such as [3H]- raclopride, should be insensitive to such receptor changes.

114 The ex vivo dual-radiotracer methodology of the current study allows the examination of the SBR of [11C]-(+)-PHNO and [3H]-raclopride, at the same time and in the same animals, and thus eliminates much of the intersubject variability from the comparison of changes in [11C]-(+)-

PHNO and [3H]-raclopride SBR. The simultaneous measurement of agonist and antagonist specific binding is of great utility in interpreting changes in the SBR of [11C]-(+)-PHNO. For example, although [11C]-(+)-PHNO is predicted to selectively label the high-affinity state, elevated [11C]-(+)-PHNO SBR resulting from an increased in the proportion of high-affinity states could theoretically be obscured by a concurrent decrease in total D2 receptor expression.

Similarly, without the SBR of [3H]-raclopride for comparison, an increase in [11C]-(+)-PHNO

SBR resulting from increased D2 receptor expression could be mistakenly interpreted as an increase in the proportion of high-affinity states. Thus, in the current study, the SBR of [3H]- raclopride, measured at the same time and in the same animals, provides an internal control for changes in D2 receptor expression that might otherwise obscure the interpretation of changes in

[11C]-(+)-PHNO SBR.

It is important to note that in our tissue samples, which correspond predominantly to dorsal striatum, the SBRs of [11C]-(+)-PHNO and [3H]-raclopride are not blocked by pretreatment with the D3-selective antagonist SB277011 (10 mg/kg, i.p.) and thus represent binding solely to D2 receptors.513 The lack of effect of SB277011 on in vivo striatal [11C]-(+)-

PHNO specific binding (BP) has also been found in cat343 and is consistent with several reports on the D3 receptor distribution in rat brain.122,164,167,168,589 A recent study by Rabiner et al. reports a decrease in striatal [3H]-(+)-PHNO total binding following SB277011 treatment, but as no reference region was used to normalize their signal with respect to non-specific binding this result is difficult to compare with the current data.7 Importantly, the report by Rabiner et al. also demonstrates that in D2 receptor knock-out mice, [3H]-(+)-PHNO binding in the striatum is almost completely abolished. This finding is in complete accord with our assertion that we have

115 no D3 signal in our measurements. Since the main hypothesis of this study deals with the two- affinity-state model of the D2 receptor, we have not attempted to examine effects on the SBRs of

[11C]-(+)-PHNO or [3H]-raclopride in brain regions of mixed D2 and D3 expression.

The main finding of this study is that in animal models with documented in vitro increases in the proportion of high-affinity state receptors, no evidence of this receptor change is reflected in the SBR of the agonist radiotracer, [11C]-(+)-PHNO. Relative to control rats, the striatal SBR of the antagonist radiotracer [3H]-raclopride, representing total available D2 receptors (high- plus low-affinity), is unchanged in AMPH-sensitized and ethanol withdrawn rats, whereas in unilaterally 6-OHDA-lesioned rats it is increased by ~50% in the hemisphere ipsilateral to the lesion site. These data are consistent with in vitro studies reporting that withdrawal from chronic AMPH590-593 or ethanol treatment594-596 is not associated with increased

D2 antagonist Bmax, and that unilateral 6-OHDA lesion results in increased Bmax in the ipsilateral striatum.597-599 Our results with [3H]-raclopride differ somewhat from in vivo reports showing

11 337 3 that the specific binding of [ C]-raclopride (Bmax) and [ H]-raclopride (striatum-to- cerebellum ratio)578 were decreased after withdrawal from chronic AMPH. The reason for this discrepancy is not clear, although it is not surprising considering that in vitro measures of antagonist specific binding have been seen to be increased or decreased in this animal model.594-

596 The increase in [3H]-raclopride SBR seen here for unilaterally 6-OHDA-lesioned rats is fully consistent with PET studies in this animal model.600,601

In AMPH-sensitized and ethanol-withdrawn rats, in which no increase in [3H]-raclopride

SBR was seen, the two-affinity-state model predicts that the increase in the proportion of high- affinity state receptors should be detected as an increase in [11C]-(+)-PHNO SBR. In unilaterally

6-OHDA-lesioned rats, an elevated proportion of high-affinity state receptors should be seen as an increase in [11C]-(+)-PHNO SBR over and above the change in [3H]-raclopride SBR resulting from increased D2 receptor expression. Contrary to these predictions, we observed no increase

116 in [11C]-(+)-PHNO in AMPH-sensitized or ethanol withdrawn rats, whereas in unilaterally 6-

OHDA-lesioned rats, the increase in [11C]-(+)-PHNO SBR was no different than that for [3H]- raclopride.

A second major finding of this study is that in response to AMPH pretreatment, the percent decrease in [11C]-(+)-PHNO SBR is the same as that seen for [3H]-raclopride, both in control and AMPH-sensitized rats. The two-affinity-state model predicts that the SBR of an agonist radiotracer should be inhibited to a greater extent by AMPH pretreatment than that of an antagonist radiotracer. This is because bound agonist (endogenous dopamine or exogenous agonist) should selectively preclude the binding of radiotracer to the high-affinity state, which at tracer dose represents the entire population of receptor sites to which an agonist radiotracer can bind, but only a fraction of the population to which an antagonist radiotracer can bind (sum of high- and low-affinity states). The lack of difference in AMPH effect on [11C]-(+)-PHNO and

[3H]-raclopride SBRs shown here replicates our previous results,513 and extends this finding to an animal model (AMPH-sensitization) shown to possess an in vitro increase in the proportion of high-affinity state receptors. This finding, however, is in disagreement with a report examining the effect of AMPH on the BPs of [3H]-(-)-NPA and [11C]-raclopride in mouse.559,602

This study, in contrast to the current study, used reserpine-induced dopamine depletion as the baseline for measuring the AMPH-sensitivity of the two radiotracers. Inspection of the data suggests that, were saline treated animals used as the control group, the agonist radiotracer would not show a stronger response to amphetamine challenge than would the antagonist radiotracer.

The findings of our current and previous study513 illustrate a major discrepancy between in vitro and in vivo measures of D2 receptor binding. There are two possible explanations for this discrepancy. First, methodological limitations in the current study could have obscured the measurement of the D2 high-affinity state. The basis for measuring the high-affinity state in this

117 study relies on the comparison of pharmacologically-induced changes in [11C]-(+)-PHNO and

[3H]-raclopride SBRs. From a theoretical perspective, this technique requires that three major methodological conditions be met. Firstly, the radiotracers used, [11C]-(+)-PHNO and [3H]- raclopride, must be full agonist and antagonist ligands, respectively. Secondly, [11C]-(+)-PHNO and [3H]-raclopride must label the same receptor type (D2) in vivo in rat striatum. Thirdly,

[11C]-(+)-PHNO must be administered at tracer dose such that it occupies only receptors in high-affinity state.

Based on the results of in vitro and in vivo experiments, there is little doubt that (+)-

PHNO is a potent full agonist526-528,531,567 and that raclopride is an antagonist603-606 – thus the first condition is clearly met. We have previously demonstrated that [11C]-(+)-PHNO and [3H]- raclopride SBR and BP can be blocked by D2, but not D3 receptor-selective drugs in both rat4,513 and cat striatum,343 indicating that these radiotracers label the D2 receptor type in this brain region. That [11C]-(+)-PHNO and [3H]-raclopride bind to the same receptor sites in striatum is further supported by data demonstrating that the specific binding of [11C]-raclopride can be fully blocked by cold (+)-PHNO, and that of [11C]-(+)-PHNO can be fully blocked by cold raclopride493 – thus one can be reasonably sure about the second condition. Finally, varying the injected dose of (+)-PHNO between ~0.1 µg/kg (8 µCi of [3H]-(+)-PHNO, 50 Ci/mmol) and the current injected dose of ~1 µg/kg has no impact on the measured SBR (data not shown). The insensitivity of the SBR to changes in injected radiotracer mass indicates that we are well within the tracer dose range for [11C]-(+)-PHNO. Under these conditions, [11C]-(+)-PHNO would not be expected to occupy receptors configured in the low-affinity state. For [3H]-raclopride, our injected dose of 0.1 µg/kg would be expected to result in occupancy under 1%, assuming an

EC50 of ~50 mg/kg (unpublished data and reference 444). Thus we feel that we have satisfied the necessary requirements for evaluation of the two-affinity-state model with these radiotracers in vivo.

118 The second possible explanation for the discrepancy between in vitro measurements of the high-affinity state and our current and previous ex vivo results513 is that the in vitro two- affinity-state model is not a valid description of D2 agonist binding in vivo. We show here that not only are the in vitro increases in the proportion of high-affinity states not reflected by in vivo changes in [11C]-(+)-PHNO SBR, but that changes in D2 expression (6-OHDA-lesion model) are associated with identical changes in [3H]-raclopride and [11C]-(+)-PHNO SBR. An alternative model consistent with these results is that in vivo, the D2 receptor exists in only one affinity state, labeled equally by [11C]-(+)-PHNO and [3H]-raclopride. Previous ex vivo [11C]-(+)-PHNO data from our group in non-anaesthetized rats513 and recent in vivo [11C]-(-)-MNPA PET data examining apomorphine pretreatment in monkey607 are also fully consistent with this model.

Although the present data do not fully resolve the discrepancy between one- and two- affinity state descriptions of in vivo agonist specific binding, what is clear is that two-affinity- state model of the D2 receptor does not sufficiently explain the available data with regard to the in vivo binding properties of agonist versus antagonist radiotracers. Furthermore, it is not necessary to adopt the more complex two-affinity-state model to describe the differential effect of anaesthesia on inhibition of agonist versus antagonist specific binding measures. For example it can be proposed that anaesthesia differentially affects the binding of agonist and antagonist radiotracers to a single affinity state. Thus we propose that a one-affinity-state model is a more useful description of the in vivo specific binding of D2 agonist radiotracers [11C]-(+)-PHNO,

[11C]-(-)-NPA and [11C]-(-)-MNPA. Further experiments are needed to investigate the differential effects of anaesthesia on agonist versus [11C]-raclopride specific binding in light of this one-affinity-state model.

A major limitation of the current study is that we examine radiotracer binding at only one time point (60 min) after radiotracer injection and thus do not benefit from a full kinetic analysis of time-activity curves. Our group has previously demonstrated an excellent correlation

119 between [11C]-raclopride ex vivo SBR at 60 min post radiotracer injection, and [11C]-raclopride

BP determined by a kinetic analysis (simplified reference tissue model) of time-activity curves.340 Although this study only examined the relationship between SBR and BP for [11]- raclopride, we believe based on several pieces of evidence that the same result is true for [11C]-

(+)-PHNO. First, we have demonstrated that the plasma-input curve for [11C]-(+)-PHNO is not altered by 4 mg/kg AMPH pretreatment (section 5, Figure 22). Second, the cerebellum time- activity curve for [11C]-(+)-PHNO is unaltered by AMPH pretreatment.493 Considering these data, and the fact that in the current study our 60 cerebellum %ID/g is not changed by 4 mg/kg

AMPH pretreatment, we feel that our AMPH pretreatment does not significantly affect plasma- to-brain or brain-to-plasma transfer kinetics of [11C]-(+)-PHNO. The other experimental conditions used in the current study, which involve rats drug-free for 1-4 weeks, are certainly less severe in terms of haemodynamic changes than acute AMPH pretreatment.608 We therefore feel that the SBR is, under the conditions of the current study, an accurate surrogate for more rigourous kinetically determined measures of specific binding, such as the BP. A second limitation of the current study is that, unlike the AMPH-sensitized and unilaterally 6-OHDA- lesioned rats, we provide no behavioural evaluation of the dopaminergic supersensitivity resulting from withdrawal from chronic ethanol treatment. However, the methods used in the current study were identical to those that were reported to result in increased in vitro D2 high- affinity state density.2 Finally, it would have been ideal to measure the number of receptors in high-affinity state using in-vitro techniques. However, the study required the dissolution of the extracted radioactivity-rich tissue in scintillation fluid precluding the use of that tissue for simultaneous in vitro experiments. However, the large (360%) in vitro increase in the high- affinity state has been documented several times in these animal models and it is thus very unlikely that our animals did not have the usual receptor changes.

120 5.6. Conclusions

In conclusion, despite the fact that an increased proportion of high-affinity state receptors is seen in vitro in animal models displaying dopaminergic supersensitivity, the SBR of

[11C]-(+)-PHNO, a full agonist D2 radiotracer, does not provide evidence for the existence of this increase, nor for the existence of two affinity states in vivo. Our results show that the SBRs binding of [11C]-(+)-PHNO and [3H]-raclopride are altered in an indistinguishable fashion in response to pretreatment with AMPH or after treatments that increase D2 receptor expression

(6-OHDA lesion model). These data, in combination with our previous results,513 do not support the in vivo validity of the two-affinity state model of the D2 agonist binding. Parsimoniously, a one-affinity-state model provides a better description of available data describing the binding characteristics of [11C]-(+)-PHNO, and perhaps those of the D2 agonist radiotracers in general.

121 6. Isoflurane anaesthesia differentially affects the amphetamine-sensitivity of agonist and antagonist D2/D3 positron emission tomography radiotracers: implications for in vivo imaging of dopamine release

Patrick N. McCormick1,2, Nathalie Ginovart2,4, Alan A. Wilson1,2,3

1 Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada M5S 1A8

2 PET Centre, Centre for Addiction and Mental Health, 250 College Street, Toronto, Ontario,

Canada M5T 1R8

3 Department of Psychiatry, University of Toronto, Toronto, Ontario, Canada M5S 1A8

4 University Department of Psychiatry, Neuroimaging Unit, University of Geneva, Geneva,

Switzerland

122 6.1. Abstract

Purpose: Using positron emission tomography in isoflurane-anaesthetized cat, we recently demonstrated that the effect of amphetamine was greater on the binding potential (BPND) of the agonist D2/D3 radiotracer [11C]-(+)-PHNO than on that of the antagonist [11C]-raclopride, a finding that we were unable to replicate in conscious rat. Herein we tested whether isoflurane differentially affects the AMPH-sensitivity of [11C]-(+)-PHNO and [11C]-raclopride.

Procedures: Conscious or isoflurane-anaesthetized rats pretreated i.v. with saline or 4 mg/kg

AMPH were co-injected i.v. with [11C]-(+)-PHNO/[3H]-raclopride or [3H]-(+)-PHNO/[11C]-(-)-

NPA. and sacrificed 2, 10, 20, 30, 40 or 60 min following [11C]-(+)-PHNO/[3H]-raclopride or 60 min following [11C]-(-)-NPA/[3H]-(+)-PHNO. Striatal binding at 60 min was estimated by the specific binding ratio (SBR) and the BPND for pseudodynamic data was calculated using the simplified reference tissue model.

Results: Isoflurane increased [11C]-(+)-PHNO, [3H]-(+)-PHNO and [11C]-(-)-NPA SBR (mean ±

SD) by 80±30%, 170±50% and 120±40%, and doubled the effect of amphetamine on the SBR of these radiotracers to -61±9%, -69±12% and -60±12% respectively. Neither effect was seen for

[3H]-raclopride SBR. Similar results were observed for [11C]-(+)-PHNO and [3H]-raclopride

BPND.

Conclusions: Isoflurane differentially increases the binding and AMPH-sensitivity of [11C]-(+)-

PHNO and [11C]-(-)-NPA relative to [11C]-raclopride, suggesting that agonist radiotracers will prove no more effective for imaging dopaminergic activity in human than antagonist radiotracers.

123 6.2. Introduction

Animal studies play an important role in brain positron emission tomography (PET) for evaluation of new radiotracers, validation of kinetic models and testing of biological and pharmacological hypotheses. By necessity, animal PET experiments typically use anaesthetics to render the subject motionless for the duration of the PET scan. Thus, for most animal PET experiments, the anaesthetized state serves as the pharmacological baseline against which PET outcome measures, such as the in vivo binding potential (BP), and changes in these parameters are quantified. Insofar as animal PET experiments are meant to inform the design and interpretation of human PET studies - which are generally performed without anaesthetics - the effects of anaesthesia on animal PET measurements must be considered.

With respect to the dopaminergic system, several anaesthetic effects have been measured using PET. Compared to the non-anaesthetized condition the inhaled anaesthetic isoflurane

11 increases the inhibitory effect of methamphetamine and nicotine on the BPND of [ C]-

236 11 raclopride. Halothane, another volatile anaesthetic, increases both the BPND of [ C]- raclopride and cerebral blood flow.609 The injected anaesthetic ketamine increases the striatal

11 BPF of the D1 radiotracer [ C]-SCH23390 and the dopamine transporter (DAT) radiotracers

[11C]-β-CFT and [11C]-βCIT-FE, increases the striatal accumulation of [11C]-L-DOPA, and

11 388,610,611 decreases the BPF of [ C]-raclopride. These examples are sufficient to illustrate that when interpreting the results of animal PET experiments, care must be taken to include consideration of anaesthetic effects and their confounds.

A recent PET study by our group reported that in isoflurane-anaesthetized cat, the BPND of the dopamine D2/D3 agonist radiotracer, [11C]-(+)-PHNO, was decreased by AMPH pretreatment to a much greater extent than that of the antagonist radiotracer [11C]-raclopride.343

This result was replicated in isoflurane-anaesthetized baboon.517 However, using ex vivo dual- radiotracer experiments in conscious rat, we were unable to demonstrate a greater vulnerability

124 of [11C]-(+)-PHNO versus [3H]-raclopride specific binding ratio (SBR) to AMPH

513 11 pretreatment. Furthermore, the AMPH-induced decrease in BPND for both [ C]-(+)-PHNO and [11C]-raclopride (83 ± 5% and 56 ± 8%, respectively) measured in anaesthetized cat,343 was substantially greater than seen for the SBR (40 ± 11% and 38 ± 7%, respectively) in conscious rat,513 despite the fact that the AMPH dose in the cat PET study (2 mg/kg i.v.) was only half that used in the ex vivo rat study (4 mg/kg i.v.). We considered it unlikely that species differences could account for such discrepancies between our two studies. Thus, in the current study, we test the hypothesis that isoflurane anaesthesia was responsible for the increased AMPH sensitivity of

11 11 [ C]-(+)-PHNO BPND over that of [ C]-raclopride.

Using ex vivo dual radiotracer experiments in rat, we demonstrate here that isoflurane

11 anaesthesia accounts for the increased AMPH sensitivity of [ C]-(+)-PHNO BPND and SBR

3 11 over those of [ H]-raclopride, and that in the absence of anaesthesia, the BPND and SBR of [ C]-

(+)-PHNO behave in a manner very similar to those of [3H]-raclopride in response to AMPH pretreatment. Implications of this finding for animal PET experiments, PET imaging of endogenous dopamine and the two-affinity state model of the D2 receptor are discussed.

6.3. Materials and Methods

All animal experiments were conducted with approval of the Animal Ethics Committee at the Centre for Addiction and Mental Health and in accordance with the Canadian Council on

Animal Care.

6.3.1. Anaesthesia and drug pretreatment

Male Sprague-Dawley rats weighing 300 ± 30 g were assigned to one of four treatment groups. The first two groups remained conscious (CON) and were pretreated, 50 min before radiotracer injection, with either i.v. saline vehicle (1 mL/kg) (CON + SAL) or i.v. AMPH (4

125 mg/kg) (CON + AMPH) in saline, respectively. The second two groups were anaesthetized with

2.5 % isoflurane (ISO) in oxygen gas (2.5 L/min, delivered by an isoflurane vaporizer, SurgiVet,

USA) ~30 min prior to i.v. pretreatment (i.e. 50 min prior to radiotracer injection) with either saline vehicle (ISO + SAL) or 4 mg/kg AMPH (ISO + AMPH), respectively, and remained anesthetized until sacrifice. Anaesthesia delivery was accomplished using two Plexiglas anaesthesia chambers, with six rats per chamber, connected in parallel to the output of the isoflurane vaporizer. Each rat was placed individually in its anaesthesia chamber and allowed to become completely immobilized before the next rat was introduced into the chamber. For drug and radiotracer injections, each rat was individually and briefly removed from its anaesthesia chamber. Anaesthesia was maintained during these periods using a nose cone to which isolfurane could be directed, briefly bypassing the anaesthesia chambers. Rats were placed alternatively in the two chambers, such that no two consecutive rats were removed from the same anaesthesia chamber, reducing the disturbance of isoflurane concentration in the chambers.

6.3.2. Ex vivo dual-radiotracer biodistribution studies

Fifty minutes after pretreatment, all rats received an i.v. co-injection of high specific activity [11C]-(+)-PHNO and [3H]-raclopride (n = 9 per treatment group) or [3H]-(+)-PHNO and

[11C]-(-)-NPA (n = 6 per treatment group). The injected masses and specific activities for the radiotracers were: [11C]-(+)-PHNO, 0.8 ± 0.2 nmol, 1500 ± 300 mCi/µmol; [3H]-raclopride,

~0.1 nmol, 62 mCi/µmol; [11C]-(-)-NPA, 1.19 ± 0.08 nmol, 996 mCi/µmol (single synthesis);

[3H]-(+)-PHNO, ~0.1 nmol, 78 mCi/µmol. Regional radiotracer brain biodistribution was determined as previously described (see section 3.3.2.1 for full details).4,390,612 Briefly, 60 minutes after radiotracer injection, rats were sacrificed by decapitation, a blood sample was taken from the trunk of the animal, and the brain was extracted from the skull and placed on ice until dissection. Blood samples from each animal were centrifuged to obtain plasma. Striatum

126 and cerebellum were excised and placed, as were blood plasma samples, in pre-weighed plastic sample tubes. Radioactivity due to 11C in the tissue and plasma samples was determined using a gamma counter and back corrected to the time of first radiotracer injection, using aliquots of the original injected dose as standard. For determination of radioactivity due to 3H, the samples were treated (after determination of 11C radioactivity) with 3 mL of 0.6 N NaOH, shaken for 24h, and 6 mL of AquassureTM scintillation fluid was then added. After a further 24 hours of mixing, the samples were counted in a liquid scintillation counter. Regional radioactivity was expressed as a percentage of the injected dose per gram of wet tissue weight (%ID/g), and as the standard uptake value (SUV), equal to the fraction of the injected dose per gram of tissue multiplied by the body weight in grams. Specific binding was estimated by the specific binding ratio (SBR), defined as:

%ID/g − %ID/g SBR = STR CER %ID/gCER

Between-group comparisons of average SUV and SBR values were done by ANOVA followed by Bonferroni’s multiple comparison test, with significance indicated by p < 0.05.

The SBR measures specific binding in a region of interest at only one time point after radiotracer injection. Therefore, we decided also to generate ex vivo data that would be amenable to kinetic analysis using the simplified reference tissue model (SRTM)339 in order to assess whether any changes seen in the SBR were also reflected by similar changes in the more kinetically robust binding potential (BPND). To generate time activity curves, rats were assigned to the same anaesthetic and drug pretreatment groups as above (n = 30 per group), but were sacrificed at various times (2, 10, 20, 30, or 40 min, n = 6 per time point) after radiotracer injection (data from the previous experiment were used for the 60 min time point). The average

BPND ± SD was estimated by a form of bootstrap analysis, as follows. Single striatum SUV values (each representing a measurement from one animal) were randomly selected, with

127 replacement, from each time point. Time-activity curves were assembled using one randomly- sampled SUV value from each time point. This sampling procedure was repeated thirty-six times, generating thirty six striatum time-activity curves for each treatment group. These thirty-six time activity curves were fit individually to generate radioligand BPND using the SRTM and the average cerebellum time-activity curve as the reference curve. The resulting thirty-six BPND values for each treatment group were averaged and the mean ± SD were used for statistical comparisons of BPND between treatment groups. Since average BPND was stably estimated (less than 6% change between 4 and 40 iterations), a practically-appropriate number of iterations was chosen by assessing the effect of iteration number on the stability of the SD. The number of iterations was increased by 2 until three subsequent increases (32, 34 and 36 iterations) resulted in <10% change in the SD for all treatment groups. Between-group comparisons of average

BPND values were done by ANOVA followed by Bonferroni’s multiple comparison test, with significance indicated by p < 0.05.

6.4. Results

The 60 min SUV values for all radiotracers in striatum and cerebellum are shown in

Table 6. Isoflurane anaesthesia increased the 60 min striatal SUV of [11C]-(+)-PHNO, [3H]-(+)-

PHNO, [11C]-(-)-NPA and [3H]-raclopride by 20 ± 16% (p < 0.05), 48 ± 19% (p < 0.001), 46 ±

11% (p < 0.001) and 38 ± 13% (p < 0.01) respectively. In conscious rats, pretreatment with 4 mg/kg AMPH resulted in a significant decrease in the striatal SUV of [11C]-(+)-PHNO, whereas for the other radiotracers this decrease did not reach statistical significance. In isoflurane- anaesthetized rats the AMPH-induced decrease in striatal SUV for each radiotracer was statistically significant (p < 0.001) and was greater than in the conscious condition (p < 0.01):

[11C]-(+)-PHNO, -64 ± 7% vs. -28 ± 10%; [3H]-(+)-PHNO, -64 ± 10% vs. -26 ± 10%; [11C]-(-)-

NPA, -44 ± 11% vs. -20 ± 7%; and [3H]-raclopride -58 ± 8% vs. -17 ± 17%. For

Table 7. Striatum (STR) and cerebellum (CER) standard uptake values (SUV) for conscious (CON) and isoflurane-anaesthetized (ISO) rats after pretreatment with saline (SAL) or 4 mg/kg AMPH.

CON + SAL CON + AMPH ISO + SAL ISO + AMPH STR CER STR CER STR CER STR CER [11C]-(+)-PHNO 1.4 ± 0.2 0.28 ± 0.08 1.0 ± 0.1 †† 0.26 ± 0.03 1.9 ± 0.2 † 0.22 ± 0.02 † 0.7 ± 0.1 ‡‡‡ 0.17 ± 0.03 [3H]-(+)-PHNO 1.7 ± 0.3 0.38 ± 0.07 1.2 ± 0.2 0.38 ± 0.09 2.5 ± 0.3 ††† 0.24 ± 0.04 † 0.9 ± 0.2 ‡‡‡ 0.24 ± 0.07 11 ††† ‡‡‡ [ C]-(-)-NPA 3.1 ± 0.6 0.8 ± 0.2 2.4 ± 0.2 0.8 ± 0.1 4.5 ± 0.3 0.67 ± 0.16 2.5 ± 0.5 0.7 ± 0.2 [3H]-Raclopride 1.6 ± 0.3 0.15 ± 0.08 1.3 ± 0.3 0.15 ± 0.04 2.2 ± 0.2 †† 0.21 ± 0.04 † 0.9 ± 0.2 ‡‡‡ 0.14 ± 0.04 † Significantly different from CON + SAL treatment group (ANOVA, Bonferroni’s multiple comparison test, p < 0.05) †† Significantly different from CON + SAL treatment group (ANOVA, Bonferroni’s multiple comparison test, p < 0.01) ††† Significantly different from CON + SAL treatment group (ANOVA, Bonferroni’s multiple comparison test, p < 0.001) ‡‡‡ Significantly different from ISO + SAL treatment group (ANOVA, Bonferroni’s multiple comparison test, p < 0.001)

128

129

Figure 18. Specific binding ratio (SBR) of [11C]-(+)-PHNO, [3H]-(+)-PHNO, [11C]-(-)-NPA and [3H]-raclopride in conscious (CON) and isoflurane-anaesthetized rats (ISO) after i.v. pretreatment with saline (SAL) or 4mg/kg amphetamine (AMPH). †† Significantly different from CON + SAL group (ANOVA, Bonferroni’s multiple comparison test, p > 0.01). ††† Significantly different from CON + SAL group (ANOVA, Bonferroni’s multiple comparison test, p > 0.001). ‡‡‡ Significantly different from ISO + SAL group (ANOVA, Bonferroni’s multiple comparison test, p > 001).

[11C]-(+)-PHNO and [3H]-(+)-PHNO the cerebellum SUV values were decreased in ISO + SAL treatment group relative to the CON + SAL treatment group (p < 0.5), whereas for [3H]- raclopride the cerebellum SUV values in this group were increased (p < 0.05).

The SBR values for all four radiotracers are shown in Figure 18. The SBR values for

[11C]-(+)-PHNO, [3H]-(+)-PHNO and [11C]-(-)-NPA were increased (p < 0.001) in the ISO +

130

SAL treatment group by 80 ± 30%, 170 ± 50% and 120 ± 40%, respectively, as compared to the conscious condition. Isoflurane anaesthesia had no significant effect on the SBR of [3H]- raclopride. In the conscious condition, pretreatment with 4 mg/kg AMPH significantly (p < 0.01) decreased the SBR of [11C]-(+)-PHNO (-29 ± 10%) and [3H]-raclopride (-24 ± 7%), whereas for

[3H]-(+)-PHNO and [11C]-(-)-NPA this decrease (-32 ± 18% and -29 ± 10%, respectively) was not statistically significant. In the conscious condition there was no significant difference between radiotracers in the effect of AMPH pretreatment on the SBR (Figure 19). The effect of

AMPH was significantly greater on the SBRs of the agonist radiotracers in the isoflurane- anaesthetized condition (Figure 19) than in the conscious condition (p < 0.001): [11C]-(+)-

PHNO, -61 ± 9%; [3H]-(+)-PHNO, -69 ± 12%; and [11C]-(-)-NPA, -60 ± 12%. For [3H]- raclopride the effect of AMPH on the SBR in the isoflurane-anaesthetized condition was -37 ±

16% and was not significantly larger than in the conscious condition.

131

) and ) and ND sacrifice at various times after radiotracer radiotracer after times various at sacrifice activity curves. Binding potentials (BP H]-raclopride time-activityH]-raclopride generated curves by 3 d closed circles represent cerebellum time- cerebellum represent closed circles d

C]-(+)-PHNO and[ 11 [ deviations are shown. deviations Ex vivo Ex

associated standard Figure 20. injection. Openrepresent circles an STR

132

11 3 Figure 21. Binding potential (BPND) values for [ C]-(+)PHNO and [ H]-raclopride. ††† Significantly different from average BPND in the CON + SAL treatment group (ANOVA, Bonferroni’s multiple comparison test, p < 0.001); ‡‡‡ Significantly different from average BPND in the ISO + SAL treatment group (ANOVA, Bonferroni’s multiple comparison test, p < 0.001).

Radiotracer time-activity curves and calculated BPND values are shown in Figure 20.

11 3 Data analysis resulted in BPND values for [ C]-(+)-PHNO and [ H]-raclopride of 3.2 ± 0.2 and

5.2 ± 0.5, respectively. Estimates of the SD were stable (less than 10% variation) when the number of time-activity curves included in the analysis reached 24 (data not shown). Isoflurane

11 anaesthesia and/or AMPH pretreatment resulted in a pattern of BPND changes for [ C]-(+)-

PHNO and [3H]-raclopride that is similar to that seen for the 60 min SBR (Figure 21). In the conscious condition AMPH caused 26 ± 6% and 21 ± 10% decreases (p < 0.001) in the BPND values of [11C]-(+)-PHNO and [3H]-raclopride, respectively. Isoflurane anaesthesia resulted in a

11 40 ± 20% increase in the BPND of [ C]-(+)-PHNO (p < 0.001) but had no effect on the BPND of

3 [ H]-raclopride. Under isoflurane anaesthesia, the effect of AMPH pretreatment on the BPND was significantly increased (p < 0.001) for both radiotracers (-51 ± 9% and -34 ± 8% for [11C]-

(+)-PHNO and [3H]-raclopride, respectively).

133 6.5. Discussion

Recent PET studies have shown that in anaesthetized animals, AMPH pretreatment

11 343 11 470 reduces the BPND of the D2/D3 agonist radiotracers [ C]-(+)-PHNO, [ C]-(-)-NPA and

[11C]-MNPA447 to a greater extent than that of the antagonist [11C]-raclopride. In conscious rat, however, we were unable to demonstrate a difference in AMPH-sensitivity between [11C]-(+)-

PHNO and [3H]-raclopride binding,513 a phenomenon we had previously observed in PET experiments in isoflurane-anaesthetized cat.343 Of several methodological differences between our ex vivo rat and in vivo cat studies – species, outcome measure (SBR versus BPND), conscious versus anaesthetized animals – we considered the use of anaesthesia to be the most likely explanation for this discrepancy. The goal of the current study was to test whether isoflurane anaesthesia was responsible for the increased AMPH-sensitivity of [11C]-(+)-PHNO versus

[11C]-raclopride binding. Since isoflurane anaesthesia has been used in several [11C]-(-)-NPA

PET studies,470,476,510,515 we decided to also investigate whether isoflurane was responsible for the increased AMPH-sensitivity of [11C]-(-)-NPA binding.

We show here that isoflurane anaesthesia has two important effects on the ex vivo binding of the agonist radiotracers [11C]/[3H]-(+)-PHNO and [11C]-(-)-NPA. First, isoflurane

11 3 11 anaesthesia increases the SBR and BPND of [ C]/[ H]-(+)-PHNO and the SBR of [ C]-(-)-NPA, but has no significant effect on either parameter for [3H]-raclopride. Second, isoflurane anaesthesia greatly increases the effect of AMPH pretreatment on the SBR and BPND of

[11C]/[3H]-(+)-PHNO and the SBR of [11C]-(-)-NPA, whereas for [11C]-raclopride this effect is much less pronounced. These results demonstrate that the use of isoflurane anaesthesia accounts for the difference in AMPH sensitivity between [11C]-(+)-PHNO or [11C]-(-)-NPA and [11C]-

343,470 raclopride BPND reported in recent PET studies, as well as for the discrepancy between these studies and our ex vivo comparison of [11C]-(+)-PHNO and [3H]-raclopride SBR in non- anaesthetized rats.513

134 6.5.1 Implications

The current findings have important implications both for the preclinical development of new agonist D2 radiotracers and for the interpretation of [11C]-(+)-PHNO and [11C]-(-)-NPA

PET data in humans. In terms of radiotracer development, preclinical studies conducted under anaesthesia may give an artificially high estimate of the BP that can be expected in later PET experiments using non-anaesthetized animals or ultimately, human subjects. A recent example is

11 given by PET experiments in which ketamine anaesthesia increased the BPND of [ C]-MNPA from 0.88 to 1.35 in monkey.613 There is no reliable way to predict how the BP of a candidate radiotracer will differ between preclinical animal and human PET studies, as species differences in radiotracer metabolism, kinetics or binding site density could conceivably result in either an increase or decrease in BP. For a radiotracer with modest preclinical binding, a reduction in BP due to species differences, combined with a ~50% decrease due to lack of anaesthesia (as we demonstrate here) could severely limit its utility in human studies.

A major consideration in development of D2 radiotracers is the susceptibility of their

BPs to changes in extracellular dopamine concentration, which provides an in vivo index of dopaminergic activity (for review see 475). The D2 agonist radiotracers [11C]-(+)-PHNO, [11C]-

11 (-)-NPA and [ C]-MNPA are considered to be more useful in this regard because their BPNDs in isoflurane-anaesthetized animals (or ketamine-anaesthetized for both [11C]-(+)-PHNO493 and

[11C]-MNPA447,522) are decreased by AMPH pretreatment to a greater extent than that of the benchmark antagonist radiotracer [11C]-raclopride.343,447,470 The current data show, however, that without anaesthesia the inhibition of [11C]/[3H]-(+)-PHNO and [11C]-(-)-NPA binding is the same as that of [3H]-raclopride. It has also recently been shown that the effect MAP

11 pretreatment on [ C]-MNPA BPND is 3- to 4-fold less in conscious than in ketamine- anaesthetized monkey. Our data suggest that the agonist radiotracers may prove no more useful

135 than the antagonist radiotracers for imaging of extracellular dopamine levels in conscious human subjects.

6.5.2. Mechanistic considerations

6.5.2.1. Consideration of cerebral blood flow and radiotracer metabolism

It is likely that the isoflurane-induced increases in [11C]/[3H]-(+)-PHNO and [11C]-(-)-

NPA binding are due to increased D2-receptor binding, as opposed to altered radiotracer delivery (i.e. changes in blood flow or radiotracer metabolism). The metabolite-corrected plasma curves for [11C]-(+)-PHNO (Figure. 22) show no evidence for changes in radiotracer

11 metabolism that could account for the observed changes in [ C]-(+)-PHNO SBR or BPND.

Furthermore, if the increase in [11C]/[3H]-(+)-PHNO or [11C]-(-)-NPA binding were due to altered radiotracer metabolism, one would not expect opposing changes in striatum and cerebellum SUV values, as is seen here (Table 6). The increased agonist radiotracer binding under isoflurane-anaesthesia is also difficult to explain in terms of changes in cerebral blood flow, which would be expected to similarly affect the regional SUVs of [11C]/[3H]-(+)-PHNO,

11 3 [ C]-(-)-NPA and [ H]-raclopride (Table 6).

6.5.2.2. Consideration of extracellular dopamine

In vivo microdialysis studies have shown that isoflurane causes a reproducible, though relatively small increase in baseline extracellular dopamine concentration (~20 and 50% in two studies614,615) and would therefore be expected to inhibit [11C]/[3H]-(+)-PHNO, [11C]-(-)-NPA and [3H]-raclopride binding. We observe, however, that under isoflurane anaesthesia, the binding of the agonist radiotracers is elevated relative to the conscious condition (Figures 18, 20 and 21). It is therefore also unlikely that the increase in [11C]/[3H]-(+)-PHNO and [11C]-(-)-NPA binding observed under isoflurane anesthesia is due to changes in dopamine levels.

136

Figure 22. Average metabolite-corrected plasma input curves for [11C]-(+)-PHNO in all treatment groups.

In conscious rats, we show that the binding of [11C]/[3H]-(+)-PHNO, [11C]-(-)-NPA, and

[3H]-raclopride are equally sensitive to the effects of AMPH-induced dopamine release (Figure

19). Thus, the increased effect of AMPH on agonist binding in the anaesthetized state (Figures

19 and 21) cannot be explained by a potentiation of AMPH-induced dopamine release as one would expect the reduction in binding to be elevated for all three radiotracers.

The effect of AMPH on D2/D3 radiotracer binding has also been described by the internalization model which proposes that D2 radiotracers susceptible to the AMPH treatment have different affinities for internalized versus cell-surface receptors, and that the change in BP after AMPH treatment is due to a dopamine-induced receptor internalization.475,483,616 It is similarly difficult with this model to simultaneously explain the similarity in AMPH sensitivity of [11C]-(+)-PHNO, [11C]-(-)-NPA and [3H]-(+)-raclopride in conscious rats, and the selective increase in the AMPH-sensitivity of the agonist radiotracers in anaesthetized rats.

6.5.2.3. Consideration of Bmax and/or KD changes

The BP of receptor radiotracers is proportional to the density of available binding sites

(Bmax) and the radiotracer affinity for these binding sites (1/KD). Changes in either of these

137 parameters would be expected to alter the measured BP or SBR. As discussed above, a change in KD due to an isoflurane-induced change in baseline extracellular dopamine concentration seems unlikely. Changes in the concentration of the receptor protein could result in increased or decreased Bmax. However, this mechanism is not relevant to the current study as the time scales involved are insufficient to permit significant changes in protein synthesis.481,617 Furthermore, an isoflurane-induced change in D2 receptor Bmax would be expected to affect the binding of

[11C]/[3H]-(+)-PHNO, [11C]-(-)-NPA and [3H]-raclopride in a similar fashion.

For agonist radiotracers, the BP is thought to be proportional to the Bmax of the high- affinity subset of the receptor population, rather than to the total receptor Bmax, as these radiotracers bind selectively to the high-affinity state in vitro.447,470,515 Thus, a model that could potentially explain our results is one in which the effect of isoflurane is to increase the proportion of D2 receptors configured in the high-affinity state. This model predicts an isoflurane-induced increase in the binding of [11C]/[3H]-(+)-PHNO and [11C]-(-)-NPA, but no such increase in that of [3H]-raclopride, and is thus in agreement with the current data from isoflurane-anaesthetized animals. However, this model also predicts that the agonist radiotracers should be more sensitive than [3H]-raclopride to AMPH-induced dopamine release, a prediction not borne out in the conscious condition. It should be pointed out also that in other situations a two-affinity-state model fails to predict the results of comparisons between [11C]-(+)-PHNO and

[3H]-raclopride. Recent work by our group has demonstrated that [11C]-(+)-PHNO and [3H]- raclopride are indistinguishably inhibited by pretreatment with both the exogenous agonist (-)-

NPA, and the partial agonist aripiprazole over a large range of doses.513 Also, in AMPH- sensitized and ethanol-withdrawn rats, which have been shown to display greatly increased high-affinity state in vitro,2,3 [11C]-(+)-PHNO (and [3H]-raclopride) SBR, are unchanged relative to control animals.618

138 6.5.3. Methodological considerations

The ex vivo dual-radiotracer methodology used here allows the examination of the SBR of two radiotracers simultaneously in the same animal, greatly reducing both the number of animals required and the variability associated with between radiotracer comparisons.559,619 We have previously shown (using pretreatment with the ~100-fold D3-selective drug SB277011), that [11C]-(+)-PHNO and [3H]-raclopride SBR in striatum is due solely to D2 receptor binding.513 The lack of SB277011 effect on in vivo striatal [11C]-(+)-PHNO binding has also

343 been found in cat PET experiments (using the BPND as the outcome measure), and is consistent with several reports on the D3 distribution in rat brain122,164,167,168,589 and with a recent report demonstrating that striatal [11C]-(+)-PHNO binding is abolished in D2 knockout mice but unchanged in D3 knockout mice.620 Thus, the pure D2 character of our striatal SBR obviates the need for consideration of the D3 receptor in the current study.

Another important methodological consideration is whether we have achieved tracer dose conditions. [3H]-(+)-PHNO and [3H]-raclopride were injected at mass doses (~0.3 nmol/kg) approximately an order of magnitude lower than the 11C-labeled radiotracers, [11C]-(+)-PHNO and [11C]-(-)-NPA (~3 nmol/kg). For the tritium-labeled radiotracers, [3H]-(+)-PHNO and [3H]- raclopride, the small injected masses (~0.3 nmol/kg), are undoubtedly within the tracer dose range for these radiotracers (vide infra). For [11C]-(+)-PHNO, the SBR seen here (4.1 ± 0.8) is similar to that in our previous reports (~4.5) which utilized similar injected mass,513,619 but also to more recent experiments (4.4 ± 1.0) which used ~10-fold higher injected mass (unpublished data), indicating no significant mass effects even at this relatively high dose. Moreover, the

SBRs obtained here with 3 nmol/kg [11C]-(+)-PHNO and with 0.3 nmol/kg [3H]-(+)-PHNO are similar (i.e. 4.1 ± 0.8 and 3.5 ± 1.1, respectively), indicating no significant mass effect in our

[11C]-(+)-PHNO data. Similarly, the SBR of [3H]-raclopride seen here (10.3) is similar to that in a previous report (11.6) using a higher mass dose (~2.5 nmol/kg) of [11C]-raclopride.340 Our

139 [11C]-(-)-NPA SBR of 2.7 is also comparable to that seen in a previous report (3.4) using similar mass doses (~0.3-3 nmol/kg).510 In addition, pretreatment experiments show that cold (-)-NPA

11 3 513 inhibits ex vivo [ C]-(+)-PHNO and [ H]-raclopride binding with an ED50 of ~81 nmol/kg, suggesting that at the mass dose of [11C]-(-)-NPA administered in the current study (~3 nmol/kg) the D2 receptor occupancy would be very low.

6.6. Conclusions

Although the mechanism for these effects is uncertain, the current results clearly demonstrate that isoflurane-anaesthesia differentially affects the BPND and SBR of agonist versus antagonist D2 radiotracers, as well as the influence of AMPH on these measures. In the interpretation of pre-clinical ex vivo biodistribution and in vivo PET experiments, and in the advancement of agonist D2 radiotracers to the clinical realm, care must be taken to consider the possibly confounding effects of anaesthesia. Most importantly, the current data demonstrate that

11 the commonly-reported greater effect of AMPH on D2 agonist versus [ C]-raclopride BPND is due to the effect of anaesthesia, not to any inherently greater sensitivity of agonist radiotracers to the effects of extracellular dopamine. These data suggest that the D2 agonist radiotracers, as a class, may prove to be no more effective than [11C]-raclopride, or other benzamide radiotracers, for monitoring in vivo dopamine release in non-anaesthetized human subjects.

140 7. The antipsychotics olanzapine, risperidone, clozapine and haloperidol are D2-selective ex vivo but not in vitro

Patrick N. McCormick1,2, Shitij Kapur2,3, Ariel Graff-Guerrero2,3, Roger Raymond3, José N.

Nobrega3,4, Alan A. Wilson1,2,3

1 Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada M5S 1A8

2 PET Centre, Centre for Addiction and Mental Health, 250 College Street, Toronto, ON,

Canada M5T 1R8

3 Department of Psychiatry, University of Toronto, Toronto, ON, Canada M5S 1A8

4 Neuroimaging Research Section, Centre for Addiction and Mental Health, 250 College Street,

Toronto, ON, M5T 1R8, Canada

5 Department of Pharmacology, University of Toronto, Toronto, ON, Canada M5S 1A8

141 7.1. Abstract

In a recent human [11C]-(+)-PHNO positron emission tomography (PET) study olanzapine, clozapine and risperidone occupied D2 receptors in striatum but, despite their similar in vitro D2 and D3 affinities, failed to occupy D3 receptors in globus pallidus. The current study had two goals: 1) to characterize the regional D2/D3 pharmacology of in vitro and ex vivo [3H]-(+)-PHNO binding sites in rat brain; 2) to compare, using [3H]-(+)-PHNO autoradiography, the ex vivo and in vitro pharmacology of olanzapine, clozapine, risperidone and haloperidol. Using the D3-selective drug SB277011 we found that ex vivo and in vitro [3H]-

(+)-PHNO binding in striatum is due exclusively to D2 whereas that in cerebellar lobes 9 and 10 is due exclusively to D3. Surprisingly, the D3 contribution to [3H]-(+)-PHNO binding in the islands of Calleja, ventral pallidum, substantia nigra and nucleus accumbens was greater ex vivo than in vitro. Ex vivo, systemically administered olanzapine, risperidone and haloperidol, at doses occupying ~80% D2, did not occupy D3 receptors. Clozapine, which also occupied ~80% of D2 receptors ex vivo, occupied a smaller percentage of D3 receptors than predicted by its in vitro pharmacology. Across brain regions, ex vivo occupancy by antipsychotics was inversely related to the D3 contribution to [3H]-(+)-PHNO binding. In contrast, in vitro occupancy was similar across brain regions, independent of the regional D3 contribution. These data indicate that at clinically relevant doses, olanzapine, clozapine, risperidone and haloperidol are D2- selective ex vivo. This unforeseen finding suggests that their clinical effects cannot be attributed to D3 receptor blockade.

142 7.2. Introduction

In agreement with its high in vitro affinity for both D2 and D3 receptors,506,532 the agonist positron emission tomography (PET) radiotracer [11C]-(+)-PHNO is thought to label both receptor subtypes in vivo. In both human and baboon the regional pattern of in vivo [11C]-

(+)-PHNO binding is unique among D2/D3 radiotracers, with highest binding in the globus pallidus (GP) followed by ventral striatum (VS), caudate (CAU) and putamen (PUT) and substantia nigra (SN).5,517,548 In general agreement with the distribution of D2 and D3 receptors,

[11C]-(+)-PHNO binding sites in CAU and PUT are thought to represent primarily D2 receptors whereas those in GP and SN are thought to be primarily of the D3 receptor type.5,517,548 The pharmacological dissimilarity between [11C]-(+)-PHNO binding sites in CAU/PUT and GP was first suggested by their anatomical distribution and the slower washout of the radiotracer from

GP than from CAU and PUT,5,548 but has since been supported by pharmacological evidence

11 showing that [ C]-(+)-PHNO BPND (binding potential with respect to non-displaceable binding) in the GP can be blocked in a regionally-selective fashion by the D3-selective drugs BP897 and

SB277011 in baboon7,517 and pramipexole550 and ABT-925549 in human. Further support for the binding of [3H]-(+)-PHNO to both receptor subtypes is provided by mouse experiments demonstrating that D2 receptor knockout abolishes [3H]-(+)-PHNO binding in the striatum while leaving SB277011-sensitive binding in midbrain and cerebellum lobes 9 and 10 largely intact.7

The observation that [11C]-(+)-PHNO labels both D2 and D3 receptors in vivo, coupled with the anatomical separation between regions with primarily D2 (CAU/PUT) or D3 (GP) binding sites, makes [11C]-(+)-PHNO a potentially-useful radiotracer for measuring occupancy by drugs that bind to either or both receptor types. Antipsychotic drugs, which have similar affinity for D2 and D3 receptors in vitro (Figure 23), are expected to occupy comparable proportions of brain D2 and D3 receptor populations. Although it is well established that D2

143

Figure 23. Affinity (pKi) of various antipsychotic drugs for cloned dopamine D2 and D3 receptors (human and rat). Data are from the National Institutes of Mental Health (NIMH) Psychoactive Drug Screening Program (PDSP) Ki database located at http://pdsp.cwru.edu/pdsp.asp and references therein. The number of individual values included in the average D2 and D3 affinities, respectively are: quetiapine 17, 10; remoxipride 8, 7; clozapine 33, 20; olanzapine 19, 12; loxapine 13, 4; ziprazidone 7, 5; risperidone 24, 13; chlorpromazine 19, 10 for; haloperidol 39, 3 3 125 22. Radioligands used in determination of Ki values were [ H]-raclopride, [ H]-nemonapride, [ I]-iodosulpiride, [3H]-spiperone, or [3H]-N-methylspiperone.

receptor blockade is required for the therapeutic efficacy of these drugs,310,313,316,621 it has been suggested that the D3 receptor may be at least partially responsible for their clinical effect.135,622

However, there has traditionally been no way to test the D2 versus D3 binding properties of these drugs in the living brain.

A recent [11C]-(+)-PHNO PET study in schizophrenic patients conducted at our centre revealed that chronic treatment with the antipsychotic drugs olanzapine, clozapine or risperidone, produced high levels of receptor occupancy in D2-rich CAU, PUT and VS, but no receptor

144 occupancy in D3-rich GP.550 The purpose of the current study was to clarify this discrepancy by comparing ex vivo and in vitro D2 and D3 occupancy by olanzapine, clozapine, and risperidone, as well as the typical antipsychotic haloperidol, using [3H]-(+)-PHNO autoradiography in rat brain. We first describe the ex vivo and in vitro distribution of [3H]-(+)-PHNO binding in rat brain, and use the D3-selective drug SB277011 to estimate the D3 receptor contribution to the binding signal in each of the major dopaminergic regions of interest (ROIs). We then use ex vivo and in vitro [3H]-(+)-PHNO autoradiography to examine the regional receptor occupancy produced by each of the above antipsychotic drugs in order to clarify their D2 versus D3 binding properties.

7.3. Materials and methods

7.3.1. General

Male Sprague-Dawley rats weighing 250-275 g at the beginning of the study were housed two per cage under a 12 h light 12 h dark photocycle and were allowed unlimited access to food and water. All rats were housed in the animal facility at the Centre for Addiction and

Mental Health for one week prior to use in experiments. [3H]-(+)-PHNO (two batches, 50 and

78 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA, USA). Olanzapine, risperidone, haloperidol and clozapine were purchased from Bosche Scientific (New Brunswick,

NJ, USA) and SB277011 was a gift from Dr. Bernard Le Foll at the Centre for Addiction and

Mental Health. All animal experiments were conducted with approval of the Animal Ethics

Committee at the Centre for Addiction and Mental Health and in accordance with the Canadian

Council on Animal Care.

145 7.3.2. Drug treatments

Antipsychotic drugs were administered chronically so as to mimic the conditions of the human PET experiment from which this study arose. High dose chronic olanzapine (7.5 mg/kg/d;

3, 7, 14, or 21 days; n = 4 per group), risperidone (4.2 mg/kg/days; 3 or 21 d; n = 5 per group), haloperidol (0.3 mg/kg/d; 3 or 21 days; n = 5 per group) or chronic vehicle (1% acetic acid in saline; 21 days; n = 18) were administered via implanted osmotic minipumps. Antipsychotic doses were chosen to target 80% striatal D2/D3 receptor occupancy based on the results of previous reports.330,513 The concentration of antipsychotic drugs required to deliver the above doses was calculated using the predicted rat weight at the midpoint of the treatment period

(assuming 7 g daily weight gain) and the known delivery rate of the osmotic minipumps (2.5 and 5 µL/hr for 2ML4 and 2ML2 models, respectively). Osmotic minipumps were filled (2 mL total volume) with either vehicle (1% acetic acid in saline) or antipsychotic drug solution and implanted subcutaneously under isoflurane anaesthesia. Anaesthesia was induced with 5% and maintained with 2.5% isoflurane (in oxygen). A small area on the upper back of the animal was shaved, disinfected with 95% ethanol, and a ~2 cm lateral incision was made. The osmotic minipump was disinfected with 95% ethanol and inserted into the subcutaneous space with the minipump outlet port facing posteriorly. The wound was closed with four 14 x 3 mm wound clips, wiped with 95% ethanol and gently dried with gauze. The animal was then allowed to recover from anaesthesia in a recovery cage for ~30 min before being placed back into its home cage. All animals were weighed and monitored for signs of stress daily for 7 days following surgery. The procedure was well tolerated by all animals; weight gain was similar to that in saline-treated rats from previous non-surgical experiments (data not shown) and there were no signs of unacceptable stress.

The combination of low potency and low solubility of clozapine relative to olanzapine, risperidone and haloperidol limits the maximum dose that can be delivered via osmotic

146 minipump.330 Therefore, in order to achieve striatal D2/D3 receptor occupancy comparable to that of the other antipsychotics, we chose instead to administer clozapine acutely at a dose of 60 mg/kg, s.c. (n = 12). Acute vehicle-treated rats (2% acetic acid in saline, n = 6) were used as a control group.

To estimate the relative contribution of D2 versus D3 receptors to regional [3H]-(+)-

PHNO binding, rats were treated acutely with the D3-selective drug SB277011 at a D3-selective dose513,623 of 10 mg/kg i.p. or with vehicle (30% β-cyclodextrin in saline), during day 7 of chronic olanzapine or chronic vehicle treatment (1% acetic acid in saline, n = 5 per group).

7.3.3. Ex vivo [3H]-(+)-PHNO autoradiography

Rats were injected i.v. with ~2 nmol of [3H]-(+)-PHNO via the tail vein either during chronic olanzapine, risperidone or haloperidol treatment, or 30 or 60 min after acute treatment with clozapine or SB277011, respectively, and sacrificed 60 min later by decapitation. A sacrifice time of 60 min post radiotracer injection was chosen because it allows for substantial clearance of non-displaceable radiotracer binding, resulting in good signal contrast between

ROIs and the CER reference region. A blood sample was collected from the trunk of each rat for analysis of plasma antipsychotic concentrations. Whole brains were excised, rinsed in saline and frozen at -80 ºC until further use. Plasma samples were sent to the clinical laboratory at the

Centre for Addiction and Mental Health for analysis of antipsychotic concentrations.

20 µm brain sections were cut at -10 ºC on a Hacker Bright cryostat and thaw-mounted onto microscope slides. For each brain, duplicate tissue sections were collected at the following anterior-posterior coordinates (anterior to bregma)624 and included the following regions of interest: 1) 1.6 mm, cerebral cortex (CRT), striatum (STR), nucleus accumbens (NACC),

Islands of Calleja (ICj); 2) -0.3 mm, STR, ventral pallidum (VP); 3) -5.2 mm, substantia nigra

(SN); 4) -12.7 mm, cerebellar cortex (CER), cerebellum lobe 10 (LOB). The slide-mounted

147 brain sections were dried in a dessicator containing Drierite for at least 24 h at 4 ºC, and were then exposed to Fujifilm tritium-sensitive imaging plates (model BAS TR2025) for 4 weeks.

Regional tissue radioactivity was determined using a BAS 5000 Fujifilm image plate reader. Ex vivo [3H]-(+)-PHNO binding was quantified using radioactivity in the ROI and CER (as an estimate of non-displaceable binding) as the specific binding ratio, SBR = (ROI-CER)/CER. For each ROI, occupancy was calculated as:

⎛ SBR − SBR ⎞ ⎜ Drug Control ⎟ Percent occupancy = 100 × ⎜ ⎟ ⎝ SBRControl ⎠ where SBRDrug is the SBR in an individual drug-treated animal and SBRControl is the average SBR in the control vehicle-treated group.

7.3.4. In vitro [3H]-(+)-PHNO autoradiography

Five rats were sacrificed by decapitation and their brains removed, frozen on dry ice, and stored at -80 ºC until further use. For each anterior-posterior brain coordinate given above, 16 adjacent 10 µm brain sections were collected. The tissue sections were thaw-mounted such that slide #1 contained the most anterior tissue section cut at each brain coordinate, slide #2 contained the next most posterior section from each coordinate, slide #3 the third most posterior, etc. For each brain, all of the 16 resulting sequential slides therefore contained all ROIs but at progressively more posterior positions. In total 80 slides were produced (5 brains, 16 slides per brain). Each of these 80 slides was then randomly assigned to one of 16 treatment groups. The treatment groups were as follows: control; 47, 180 or 380 nM olanzapine; 6, 23 or 48 nM risperidone; 4, 14 or 30 nM haloperidol; 330, 1270 or 2680 nM clozapine; 26, 100 or 210 nM

SB277011. The concentrations of each drug were chosen to produce approximately 70, 90 and

95% occupancy at D2 receptors (for antipsychotic drugs) or D3 receptors (for SB277011) based on a pKi value for each drug obtained by averaging all of the appropriate entries listed in the

148

NIMH Psychoactive Drug Screening Program (PDSP) Ki database and extrapolating using a sigmoidal dose-response relationship with Hill slope = 1.

Slides were incubated for 2 h at room temperature in buffer (50 mM Tris·HCl, 1 mM

3 EDTA, 1.5 mM CaCl2, 4 mM MgCl2 and NaCl either 0.6 nM) containing either 0.6 nM [ H]-

(+)-PHNO alone (control) or one of the above drug concentrations. After incubation, the slides were rinsed in ice-cold buffer (3 x 5 min), dipped for 10 s in ice-cold deionized water and dried under a stream of room temperature air. The slides were left to dry further in a fume hood overnight then exposed to tritium-sensitive image plates for 3 weeks in the presence of calibrated methacrylate radioactivity standards. The image plates were then scanned and regional [3H]-(+)-PHNO specific binding (SB) was calculated as the total binding in the ROI minus the average total binding in CER (as an estimate of non-displaceable binding). Effort was made to draw ROIs of the same shape, size and location as in the ex vivo autoradiography experiments. However, in vitro [3H]-(+)-PHNO binding in the ICJ could not be reliably distinguished from the binding seen in the olfactory tubercle. Therefore, in the in vitro condition, no ROIs were drawn for ICJ. For each ROI, drug occupancy was calculated in the same way as for the ex vivo experiments (i.e. with the substitution of SB for SBR)

7.3.5. Statistical analysis

SBR, SB and percent occupancy are expressed as mean ± SD. For statistical comparisons, means were considered significantly different when p < 0.05. Comparison of ex vivo [3H]-(+)-

PHNO SBR between individual vehicle-treated groups or between treatment durations (e.g. 3 days versus 7 days olanzapine treatment) was done by ANOVA followed by post-hoc

Bonferroni’s multiple comparison test. Comparison of average [3H]-(+)-PHNO binding (SBR for ex vivo and SB for in vitro experiments) between drug-treated and control groups was done by

ANOVA followed by Dunnett’s multiple comparison test. Occupancy was considered

149 significant when the SBR or SB in the drug-treated group was significantly different from that in the respective control group. Evaluation of occupancy differences between ex vivo and in vitro conditions was done by ANOVA followed by Bonferroni’s multiple comparison test.

7.4. Results

7.4.1. Ex vivo [3H]-(+)-PHNO autoradiography

Typical ex vivo and in vitro [3H]-(+)-PHNO autoradiographs are shown in Figure 24 (left side). [3H]-(+)-PHNO SBRs were very similar between individual vehicle-treated groups and the data were therefore pooled to produce a single control group. Regional ex vivo [3H]-(+)-

PHNO SBR in the resulting pooled vehicle-treated group is shown in Figure 25 (top panel). The highest SBR was seen in the ICJ (8.0 ± 2.0) followed by STR (4.4 ± 1.0), NACC (2.5 ± 0.7),

LOB (2.0 ± 0.5), VP (1.6 ± 0.4) and SN (0.5 ± 0.2). The lowest SBR was seen in CRT (0.1 ±

0.1).

For rats treated with chronic risperidone or haloperidol, regional occupancy and plasma drug concentrations (Table 7) were similar for all treatment durations groups (p > 0.05). We therefore chose to pool the data to produce a single group for each drug in order to increase statistical power. During chronic olanzapine treatment, plasma olanzapine concentration decreased over time as has been previously reported625 (Table 7), resulting in slightly reduced

STR occupancy in the 14 and 21 day treatment groups (p < 0.01, 74 ± 7% and 71 ± 8 %, respectively, versus 88 ± 7% in the 3 day treatment group). No other differences in regional occupancy were seen between olanzapine-treated groups. Consequently, we chose to also pool the chronic olanzapine data were, with the understanding that this pooled group underestimates the occupancy in STR by ~7% relative to the 3 day treatment group. Figure 26 shows ex vivo occupancy (left panels) in comparison to that measured in vitro (right panels). Treatment with chronic 7.5 mg/kg/d olanzapine plus 10 mg/kg acute SB277011 resulted in extensive

150

Figure 24. Typical control [3H]-(+)-PHNO autoradiographs in rat brain measured ex vivo (left) and in vitro (right). The anterior-posterior coordinate (anterior to bregma) is shown to the right of each autoradiograph. Regions of interest at each coordinate are 1.60 mm, cerebral cortex (CRT), striatum (STR), nucleus accumbens (NACC), islands of Calleja (ICJ); -0.3 mm, STR, ventral pallidum (VP); -5.2 mm, substantia nigra (SN); -12.7 mm, cerebellar cortex (CER), cerebellar lobes 9 and 10 (LOB).

151

Figure 25. Regional [3H]-(+)-PHNO binding in striatum (STR), nucleus accumbens (NACC), cerebellar lobes 9 and 10 (LOB), substantia nigra (SN) and cerebral cortex (CRT), measured ex vivo in vehicle-treated rats (top) and in vitro in control brain sections (bottom). To facilitate direct visual comparison between ex vivo and in vitro [3H]- (+)-PHNO binding, only those brain regions examined in both the ex vivo and in vitro conditions are shown on the main horizontal axis. Ex vivo binding in islands of Calleja (ICJ) are shown in the inset of the top graph (note difference in scale). Note also that in vitro STR binding, as opposed to that in the other regions, was measured in two tissue sections per slide resulting in a total of ten separate measurements.

Table 8. Antipsychotic drug concentrations in blood plasma

Duration Olanzapine Haloperidol Risperidone Clozapine (d) (nM) (nM) (nM) (µM)

Acute ------5.6 ± 2.2 3 244 ± 52 7.6 ± 1.3 152 ± 47 --- 7 190 ± 27 ------14 153 ± 33** ------21 134 ± 26** 7.4 ± 1.7 117 ± 26 --- ** p < 0.01 versus 3 day treatment group. Dunnett’s multiple comparison test

152

Figure 26. Ex vivo (left) and in vitro (right) SB277011 and antipsychotic occupancy in cerebellar lobes 9 and 10 (LOB), ventral pallidum (VP), islands of Calleja (ICJ, ex vivo condition only), nucleus accumbens (NACC) and striatum (STR). Dashed lines indicate the 0 and 80% occupancy levels. Note the similarity in STR occupancy both between antipsychotic drugs and between the ex vivo and in vitro conditions. Note also the similarity in LOB occupancy for SB277011 ex vivo versus in vitro. * p < 0.05, ** p < 0.001 significant occupancy (i.e. significant reduction in [3H]-(+)-PHNO binding relative to control group); ## p < 0.01, ### p < 0.001 occupancy significantly different from ex vivo condition.

153 Table 9. Regional ex vivo occupancy by antipsychotic drugs or SB277011

STR NACC VP SN ICJ LOB SB277011a 5 ± 11 25 ± 15* 62 ± 8** 52 ± 17* 64 ± 8** 82 ± 6**

Olanzapine 80 ± 11** 54 ± 21** 18 ± 26** 21 ± 51** 31 ± 20** -21 ± 32

Olanzapine + SB277011 90 ± 3** 80 ± 5** 83 ± 5** 89 ± 10** 68 ± 14** 82 ± 6**

Risperidone 83 ± 8** 69 ± 13** 14 ± 25** 27 ± 41 32 ± 13** -6 ± 14

Haloperidol 79 ± 4** 58 ± 10** -1 ± 21 9 ± 40 13 ± 11 -10 ± 15

Clozapine 80 ± 7** 71 ± 9** 63 ± 10** 62 ± 22** 43 ± 14** 35 ± 21** * p < 0.05; ** p < 0.01 Significant reduction in SBR relative to vehicle-treated group, Bonferroni’s

multiple comparison test a drug doses: SB277011 10 mg/kg; olanzapine 7.5 mg/kg/d; risperidone 4.2 mg/kg/d; haloperidol 0.3

mg/kg/d; clozapine 60 mg/kg

receptor occupancy across all brain regions: STR, 90 ± 3%; NACC, 80 ± 5%; 83 ± 5%; SN, 89

± 10%; ICJ, 68 ± 14%; LOB, 82 ± 6%. Average ex vivo occupancy ± standard deviation can also be found in Table 8.

7.4.2. In vitro [3H]-(+)-PHNO autoradiography

Typical in vitro [3H]-(+)-PHNO autoradiographs are shown in Figure 24 (right panels) and regional in vitro SB is shown in Figure 25 (bottom panel). [3H]-(+)-PHNO SB was highest in STR (1164 ± 129 fmol/g), followed by NACC (836 ± 77 fmol/g), VP (118 ± 29 fmol/g), SN

(85 ± 20 fmol/g), LOB (65 ± 7 fmol/g) and CRT (23 ± 7 fmol/g). Total binding in CER (used as an estimate of non-displaceable binding) was 29 ± 7 fmol/g.

Figure 26 (right panels) shows the regional occupancy produced by the highest concentration SB277011 and each of the antipsychotic drugs in comparison to the occupancy observed ex vivo. The occupancy ± produced by the remaining two antipsychotic concentrations can be found in Table 9.

154

Table 10. Regional in vitro occupancy in antipsychotic- or SB277011-treated brain sections STR NACC VP SN LOB SB277011 (nM) 26 8 ± 14 5 ± 26 27 ± 5** -12 ± 12 26 ± 30 100 7 ± 16 -6 ± 17 32 ± 10** -9 ± 12 66 ± 7** 210 12 ± 13 1 ± 14 42 ± 10** 7 ± 27 73 ± 7** Olanzapine (nM) 47 45 ± 11** 32 ± 13** 46 ± 14** 42 ± 23* 12 ± 42 180 74 ± 4** 69 ± 7** 70 ± 2** 64 ± 10** 52 ± 12** 380 83 ± 2** 78 ± 4** 73 ± 2** 73 ± 10** 66 ± 10** Risperidone (nM) 6 40 ± 5** 27 ± 10** 50 ± 11** 50 ± 13** 18 ± 26 23 65 ± 2** 59 ± 11** 71 ± 8** 83 ± 10** 47 ± 21* 48 80 ± 4** 77 ± 4** 78 ± 5** 81 ± 7** 59 ± 13** Haloperidol (nM) 4 57 ± 6** 33 ± 17** 42 ± 7** 35 ± 28 7 ± 29 13 68 ± 4** 61 ± 7** 67 ± 4** 70 ± 21** 31 ± 29 30 81 ± 3** 72 ± 10** 77 ± 6** 86 ± 17** 46 ± 21* Clozapine (µM) 0.33 40 ± 9** 44 ± 13** 67 ±12** 64 ± 21** 32 ± 17 1.27 64 ± 5** 64 ± 6** 84 ±6** 88 ± 9** 73 ± 12** 2.68 79 ± 3** 84 ± 5** 96 ± 4** 93 ± 2** 80 ± 12** ** p < 0.01; * p < 0.05 Significant occupancy relative to control brain sections, Bonferroni’s multiple

comparison test

7.5. Discussion

Our previous ex vivo rat experiments with [11C]-(+)-PHNO using brain dissection4,513,619 allowed the examination of [11C]-(+)-PHNO binding in large brain regions such as STR, CRT and whole cerebellum, but not in smaller regions such as NACC, VP and LOB. The ex vivo

SBRs measured here in STR (4.4 ± 1.0) and CRT (0.1 ± 0.1) of vehicle-treated rats are in agreement with our previous ex vivo measurements.4,513,619 The distribution of [3H]-(+)-PHNO

SBR in both striatal and non-striatal regions (Figure 25, top panel) is consistent with the known distribution of D2/D3 receptors in rat brain.122,164 Our in vitro [3H]-(+)-PHNO measurements

(Figure 25, bottom panel) are also consistent with the D2/D3 receptor binding pattern described in the above-cited reports and with previous in vitro [3H]-(+)-PHNO autoradiographic data reported by Nobrega and Seeman (although the authors did not describe binding in LOB).535

155 However, the ex vivo and in vitro distribution patterns of [3H]-(+)-PHNO reported here are quantitatively very different from one another. Visual inspection of Figure 25 suggests that these differences are due to reduced binding in D3-rich areas. Although the SBR (ex vivo) and

SB (in vitro) cannot be directly compared, quantitative differences between ex vivo and in vitro

[3H]-(+)-PHNO binding can be illustrated by normalization of regional binding to that in STR, which, as discussed below, is due exclusively to D2 binding and should thus be unaffected by changes in D3 binding. Relative to STR, ex vivo binding in LOB (45% of SBR in STR), VP

(36% of STR) and SN (11% of STR) is significantly greater than seen in vitro (6, 10% and 7% of STR, respectively; t test, p < 0.001 for LOB and VP, p < 0.05 for SN), whereas no difference is seen in NACC (p > 0.05). The decrease in LOB, VP and SN binding relative to STR is also reflected in the distinctive change in regional rank order of [3H]-(+)-PHNO binding between the ex vivo (STR > NACC > LOB > VP > SN > CRT) and in vitro (STR > NACC > VP ~ SN ~

LOB > CRT) conditions, which would not be expected if the ex vivo versus in vitro differences in baseline binding were due to altered D2 receptor binding. Unfortunately, because ROIs could not be reliably drawn around ICJ in the in vitro condition, a direct comparison between ex vivo and in vitro binding in this region was not possible. However, the fact that the ICJ were more easily distinguishable ex vivo suggests an increased ex vivo versus in vitro signal in this region similar to that seen in LOB and VP.

Although the anatomical pattern of D2-like receptor binding is well established,167,168 the ratio of D2 to D3 receptor binding sites within individual brain region is not precisely known.

Pretreatment with the ~100-fold D3-selective drug SB277011626 resulted in >50% occupancy in

LOB, ICJ, VP and SN (Table 8), indicating that the D3 receptor is the major contributor to [3H]-

(+)-PHNO SBR in these regions. No occupancy was seen in STR demonstrating, as we have previously reported,513 that ex vivo striatal [3H]-(+)-PHNO SBR is due exclusively to D2 receptor binding in rat brain. Combined treatment with SB277011 and chronic olanzapine

156 producing similarly large levels of occupancy in STR, NACC, SN, VP and ICJ (~80%) but failed to increase occupancy in LOB above that seen after SB277011 treatment alone (Table 8).

Thus, LOB appears to be the only region in which [3H]-(+)-PHNO SBR is due exclusively to D3 receptor binding. Assuming that the SB277011-induced D3 receptor occupancy is similar across brain regions (~80%), the percentage D3 contribution to [3H]-(+)-PHNO can be calculated as the ratio of SB277011 occupancy in the ROI to that in LOB. Thus, the D3 contribution to ex vivo SBR is ~30% in NACC, ~63% in SN and ~75-80% in VP and ICJ. It is likely, however, that the D3 contribution to [3H]-(+)-PHNO binding in ICJ is larger than indicated here, as the

ROIs drawn around these small structures probably included some spillover from the D2-rich olfactory tubercle. The anatomical separation and relatively large size of the other regions is more amenable to accurate ROI delineation, and our reported D3 contribution in these regions is more certain.

In vitro, the same regional trend in SB277011 occupancy was seen (Figure 26). However, in vitro, SB277011 had a smaller effect on [3H]-(+)-PHNO binding in VP and SN than seen ex vivo. In addition, SB277011 had no measurable effect on in vitro [3H]-(+)-PHNO binding in the

NACC, in contrast to the significant 25% NACC occupancy by seen ex vivo. The estimated D3 contribution to in vitro [3H]-(+)-PHNO SB, derived from the regional effect of SB277011 treatment is ~10% in SN and ~57% in VP, whereas the D3 receptor does not appear to contribute to in vitro binding in NACC or STR. Thus, the D3 receptor appears to contribute more heavily to ex vivo than to in vitro [3H]-(+)-PHNO binding. This conclusion is also supported by the increased ex vivo versus in vitro [3H]-(+)-PHNO SBR in LOB, VP, and SN, regions that have the highest D3 contribution. Although the mechanism of this ex vivo versus in vitro difference is unclear, possible contributing factors could include differences in levels of endogenous dopamine or the proportion of receptors in the high-affinity state. Under basal conditions the D3 receptor has been reported to be extensively occupied by dopamine,

157 preventing in vitro D3 binding of [125I]-iodosulpiride627 and [3H]-7-OH-DPAT.628 However, endogenous dopamine does not seem to be relevant to the current data as the extensive dilution of dopamine into the in vitro assay volume (12 mL volume over 60 min) would be expected to increase, rather than decrease, [11C]-(+)-PHNO binding in the VP (relative to the ex vivo condition), and to have little or no effect on binding in LOB, which lacks dopaminergic innervation.629,630 It is possible that the D3 receptor high-affinity state is selectively disrupted in vitro, resulting in lower D3 receptor [3H]-(+)-PHNO binding. However, this explanation also seems unlikely given that the D3 receptor-G protein complex responsible for high-affinity agonist binding is thought to be more stable than that of the D2 receptor.135,631 Many other potential differences exist between our in vitro and ex vivo conditions – e.g. disruption of ionic gradients, membrane voltage and protein kinase/phosphatase cycles – and further investigation would be necessary to unravel their relevance to the current data. Fortunately, both ex vivo and in vitro, the pure D3 and D2 [3H]-(+)-PHNO binding signal in LOB and STR, respectively, make these regions ideally suited for the examination of drug occupancy at these receptor types.

However, in other regions, the D3 versus D2 contribution to [3H]-(+)-PHNO binding depends, at least under our experimental conditions, on whether the measurements are made in vitro or ex vivo and drug occupancy in these regions must be interpreted accordingly.

The antipsychotic drugs olanzapine (7.5 mg/kg/d), clozapine (60 mg/kg), risperidone

(4.2 mg/kg/d) and haloperidol (0.3 mg/kg/d) occupied a large proportion (~80%) of ex vivo

[3H]-(+)-PHNO binding sites in STR, in agreement with their well-known D2 antagonism.

However, despite their reportedly similar affinity for D2 and D3 receptors, olanzapine, risperidone and haloperidol did not measurably occupancy D3 receptors in LOB. As well, clozapine occupancy in LOB was lowest among the ROIs examined. Ex vivo antipsychotic occupancy across all regions followed a trend that was the inverse of that seen for the D3- selective drug SB277011 (Figure 26). Using in vitro [3H]-(+)-PHNO autoradiography we

158 confirmed that all of these drugs produce similar occupancy in STR, NACC, SN, VP and LOB.

These data provide strong pharmacological evidence that these drugs are in fact D2-selective ex vivo – a surprising finding given their in vitro pharmacological profile. Similar results have been reported by Schotte et al.628 Their autoradiographic experiments involved in vivo antipsychotic treatment followed by measurement of receptor occupancy using in vitro [3H]-7-OH-DPAT (D3 receptors in ICJ) or [125I]-iodosulpiride (nominally D2 receptors in NACC). They report that when administered in vivo, clozapine, olanzapine, risperidone and haloperidol had ratios of D2 to D3 potency 2-10 times higher than when determined using in vitro competitive binding experiments, and argued that this effect was due to the in vivo inhibitory influence of endogenous dopamine on antipsychotic D3 receptor binding. However, this explanation cannot account for our observation that the antipsychotic drugs have lower potency for D3 receptors in

LOB, which lacks dopaminergic innervation,629,630 than for D2 receptors in STR, nor can it explain the inverse regional trend between antipsychotic occupancy and SB277011 occupancy.

Our data are explained more completely by a model in which the in vivo D2/D3 affinity ratio for antipsychotics is greater than predicted by in vitro measurements.

Clinically, most antipsychotic drugs produce therapeutic effects at doses producing 65-

80% D2 receptor occupancy.310,313,316,621 The levels of D2 occupancy in the current study are on the high end of this occupancy range (~80%) suggesting that at clinically relevant doses, the therapeutic effects of olanzapine, risperidone, and haloperidol are not attributable to D3 receptor blockade. Clozapine was the only antipsychotic to produce significant occupancy in LOB, suggesting that its selectivity for D2 over D3 receptors in living brain is less than that of the other antipsychotics. This does not appear to be a consequence of the fact that clozapine was administered acutely rather than chronically, as the duration of treatment had little effect on regional occupancy produced by the other antipsychotics. Nevertheless, the clinical importance of the D3 receptor to clozapine’s clinical effect is likely small, as the D2 occupancy of

159 therapeutic clozapine (20-60%)310,314 is typically less than for the other antipsychotic, indicating that clozapine’s D3 receptor occupancy in the clinical setting is likely also much lower than the

35% occupancy seen here. Indeed the recent PET study by our group in schizophrenic patients found that chronic clozapine treatment, while producing ~50% occupancy in STR, did not reduce [11C]-(+)-PHNO binding in the GP.550

The current data point to the importance of in vivo or ex vivo experiments in elucidating the mechanism of action of therapeutic agents within the brain. As we show here, in vitro pharmacological measurement, although invaluable in drug and radiotracer development, can sometimes lead to erroneous assumptions regarding in vivo drug action. Ex vivo [3H]-(+)-PHNO autoradiography is a powerful technique for determining, with high anatomical resolution, the interaction of drugs with both D2 (STR) and D3 (LOB) receptors in the living brain. In particular, ex vivo autoradiography has two major strengths for pre-clinical drug evaluation in rat or other small research animals. First, unlike small animal PET experiments, ex vivo autoradiography does not require the use of anaesthesia, which can confound interpretation of radiotracer binding results.587,610 Second, the spatial resolution of autoradiography is very high in comparison to either ex vivo dissection experiments or in vivo small animal PET experiments.

The major limitation of this technique, however, is that, barring the use of prohibitively large numbers of animals, it is not easily amenable to generation of multiple time point data and analysis of radiotracer kinetics. The so-called ratio methods, such as that used to generate our ex vivo SBR (equivalent to STR/CER – 1), are common non-kinetic methods for analysis of in vivo or ex vivo radiotracer binding. The two most common ratio methods are the transient equilibrium and late time point methods. The transient equilibrium method uses the ratio of ROI to reference tissue radioactivities at a time point corresponding to peak specific binding (so-

333,334,341 called transient equilibrium) to estimate the non-displaceable binding potential (BPND).

The late time point method, which is used in the current study, also provides an estimate of

160

BPND, but instead uses the ratio of ROI to reference tissue radioactivities at some time after transient equilibrium has been reached (~30 min for [11C]-(+)-PHNO in rat).333,334,341,344 Relative to the transient equilibrium method, which for several reversible neuroreceptor radiotracers

333,334,341 provides accurate estimates of the BPND, the late time point method can result in modest

334,344 overestimate of BPND, the magnitude of this bias presumably related to the kinetics of the radiotracer used. This may have bearing on interpretation of the current results. In human, the kinetics of [11C]-(+)-PHNO binding in the D2-rich dorsal striatum are very different from those in the D3-rich globus pallidus.5 If they were to exist in rat, such kinetic differences could potentially result in greater overestimate of SBR in D3-rich versus D2-rich regions, possibly helping to explain some of the quantitative difference between our ex vivo and in vitro results.

However, inspection of published human [11C]-(+)-PHNO PET kinetic data5 suggests exactly the opposite; that a SBR calculated using a late time point (60 min.) overestimates BPND to a larger extent in D2-rich caudate-putamen than in the D3-rich globus pallidus. However, in the absence of detailed regional [3H]-(+)-PHNO kinetic data in rat it is impossible to make firm statements regarding the influence of regional kinetics on the current data.

Ratio methods can be expected to result in reliable quantification of inter-group differences in radiotracer binding and receptor occupancy provided that no large differences in radiotracer delivery exist between groups.334,341,345 Such delivery differences, typically attributable to inter-group differences in cerebral blood flow, can alter ROI-to-reference tissue ratios and thus confound interpretation of group differences in radiotracer binding. To the authors’ knowledge, antipsychotic drugs are not known to cause changes in cerebral blood flow large enough to explain our results.632 Further supporting the validity of the ratio method used here is the general agreement between the current findings and the results of our previous [11C]-

(+)-PHNO PET study of antipsychotic occupancy in human subjects550 which used the more

161 kinetically-robust simplified reference tissue model, which has been validated for [11C]-(+)-

PHNO.5

In addressing the limitations of our study, it is worthwhile also to consider whether our relatively large injected [3H]-(+)-PHNO dose (~5.7 nmol/kg vs. 0.3-3 nmol/kg in our previous studies) falls within the tracer dose range (commonly defined by receptor occupancy not exceeding 5%). Within this dose range, our ex vivo SBR (a saturable signal) should be relatively insensitive to changes in injected mass, as opposed for example to a dose range surrounding the

EC50 where small changes in injected dose would result in large reductions in the SBR. Our striatal SBR of 4.5 is identical to that in our previous reports which utilized 2- to 10-fold lower injected mass513,619 (and unpublished data), suggesting that the tracer dose range for the D2 receptor extends at least as high as the current injected dose of 5.7 nmol/kg. Secondly, exceeding the tracer dose range would result in an underestimate of antipsychotic D2 receptor occupancy. We find however, that our current striatal clozapine D2 occupancy (80 ± 7%) is very similar to that of our previous report (80 ± 8%) in which a 2-fold lower injected [3H]-(+)-PHNO dose was used.513 (+)-PHNO has been reported to have 30- to 45-fold higher in vitro affinity for the D3 receptor than the D2 receptor506,532 suggesting that a lower injected dose may be required to fulfill tracer dose conditions for D3 compared to D2. In the absence of reports comparing the in vivo affinity of [3H]-(+)-PHNO, it remains possible that the tracer dose range has been exceeded with respect to the D3 receptor, consequently presenting the possibility of underestimated drug D3 receptor occupancy. However, it seems unlikely that this alone could account for such a large discrepancy between our ex vivo D2 and D3 occupancies (80% vs. 0%), especially given the general agreement of our results with those of our recent PET study in human subjects using an injected [11C]-(+)-PHNO dose of about 0.1 nmol/kg, ~60-fold lower than in the current study.550 The greater in vitro affinity of (+)-PHNO for D3 versus D2 receptors implies that exceeding tracer concentration in our in vitro experiments would result in

162 a greater underestimate of drug occupancy at D3 versus D2 receptors. Thus, if we have exceeded tracer dose in vitro, the “true” antipsychotic D3 receptors occupancy will be numerically closer to, or even exceeding, that of D2 receptors. Thus we don’t feel that this would change the major conclusion of our paper i.e. that there is a major discrepancy between ex vivo and in vitro measures of antipsychotic occupancy.

7.6. Conclusions

In conclusion, the current study demonstrates that the antipsychotic drugs olanzapine, clozapine, risperidone and haloperidol do not occupy the D3 receptor as measured ex vivo. This is in contrast to our in vitro measurements demonstrating similar occupancy at both receptor types, and to the large body of literature indicating similar in vitro affinity of these drugs for D2 and D3 receptors. These findings corroborate and clarify the results of a recent human [11C]-(+)-

PHNO PET study conducted at our centre showing that, despite significant occupancy in STR, olanzapine, clozapine and risperidone did not reduce [11C]-(+)-PHNO D3 receptor binding in

GP. Furthermore, the data suggest that the therapeutic effects of olanzapine, clozapine, risperidone and haloperidol are not attributable to blockade of the D3 receptor.

163 8. Concluding remarks and future directions

On theoretical grounds, the dopamine D2 receptor agonist radiotracers [11C]-(+)-PHNO,

[11C]-(-)-NPA and [11C]-(-)-MNPA, have been proposed to have two major advantages over common antagonist radiotracers such as [11C]-raclopride. Firstly, the agonist D2 radiotracers should allow the direct in vivo measurement of the D2 high-affinity state, which based on in vitro evidence, is thought to represent a sub-population of the D2 receptor responsible for the neuromodulatory effects of dopamine,253,254 and to be involved in the pathophysiology of schizophrenia and substance abuse.1-3 Consequently, the agonist radiotracers should be more sensitive to competition with agonists than traditional antagonist radiotracers, giving them an advantage for in vivo imaging of extracellular dopamine levels. These predictions however, rely on the assumption that the two-affinity-state model, i.e. that the D2 receptor exists in two separate states with high and low agonist affinity, is a valid description of the D2 receptor in vivo. Although the existence of two affinity states of the D2 receptor (and other G protein- coupled receptors) in membrane preparations in vitro is beyond doubt, there has been little exploration of the validity of this model in the living brain. The main goal of this thesis (sections

3-5) was to clarify the nature of [11C]-(+)-PHNO binding sites in living brain in the context of the two affinity model of the D2 receptor.

Soon after its original characterization as a PET radiotracer, [11C]-(+)-PHNO was reported to bind both D2 and D3 receptors in vivo. Subsequently, a [11C]-(+)-PHNO PET study in human subjects produced the surprising finding that several antipsychotic drugs, which bind equally to D2 and D3 receptors in vitro, did not occupy [11C]-(+)-PHNO binding sites in globus pallidus, thought to represent primarily D3 receptor binding. Thus, our second goal was to dissect the regional contribution of D2 versus D3 receptors to ex vivo [11C]-(+)-PHNO binding, and to use this knowledge to clarify the D2 versus D3 pharmacology of antipsychotic drugs in the living brain.

164 With respect to our examination of the in vivo validity of the two-affinity-state model, our experiments produced several surprising findings. First, in conscious rats, ex vivo [11C]-(+)-

PHNO and [3H]-raclopride striatal D2 receptor binding were found to be equally sensitive to inhibition not only by antagonist drugs but also by exogenous partial and full direct agonists and by extracellular dopamine released in response to AMPH challenge. Second, neither [11C]-(+)-

PHNO nor [3H]-raclopride ex vivo binding were altered in rat models that display increased D2 high affinity state binding in vitro,1-3 whereas the binding of both radiotracers was increased in rats unilaterally lesioned with 6-OHDA, a treatment known to increase D2 receptor expression.601,633 These findings provide no experimental support for the in vivo validity of the two-affinity-state model of the D2 receptor, and instead support a model in which [11C]-(+)-

PHNO and [3H]-raclopride label a pharmacologically-similar form of the D2 receptor in the living brain. Third, the sensitivity of ex vivo [11C]-(+)-PHNO and [11C]-(-)-NPA binding (both full agonists) to extracellular dopamine was increased in isoflurane-anaesthetized rats compared to conscious rats, an effect not seen for the antagonist radiotracer [3H]-raclopride. Thus, our results also indicate that reports of increased sensitivity of [11C]-(+)-PHNO and [11C]-(-)-NPA to extracellular dopamine in animal models stem from the differential effects of isoflurane anaesthesia on agonist versus antagonist radiotracer binding, not to any inherently greater sensitivity of the agonist radiotracers to competition with dopamine. These results have major implications for PET imaging of the dopamine D2 receptor, suggesting that the agonist radiotracers may be no more effective than antagonist radiotracers for imaging of extracellular dopamine levels in conscious human subjects. To test this definitively, a direct comparison of the effect of AMPH on the binding of the agonist radiotracers and [11C]-raclopride in the same human subjects will be necessary using PET. This should now be possible as [11C]-(+)-PHNO,

[11C]-(-)-NPA and [11C]-(-)-MNPA have been characterized in human subjects.6,548,634

165 More fundamentally, these results present a major challenge for our understanding of the in vivo function of the dopamine D2 receptor and other G protein-coupled receptors. Why do agonist ligands distinguish between two affinity states of the D2 receptor in vitro but not in vivo?

The presence of high- and low-affinity receptor states in vitro is classically described by the ternary complex model which ascribes high-affinity agonist binding to the G protein-coupled form of the receptor and low-affinity binding to the G protein-uncoupled form. Within the context of this model, a potential explanation for the results of the current thesis is that in vivo the vast majority of D2 receptors exist in the high-affinity state, and that the proportion of receptors in the low-affinity state is too small to have a measurable impact on our ex vivo results.

This model, however, is difficult to reconcile with the undoubtedly higher concentration of GTP in intact neurons, which should inhibit high-affinity agonist binding compared to membrane preparations which are typically washed, and thus deficient in soluble intracellular factors. A second possibility is that in vivo D2 agonist binding sites represent predominantly the low- affinity G protein-uncoupled form, which would agree in general with higher GTP concentrations found in vivo, and with observations by Sibley et al. indicating that in intact cells, the D2 receptor exists in a single state whose affinity corresponds to the low-affinity state seen in membrane preparations.505,560 However, the low affinity of [11C]-(+)-PHNO, [11C]-(-)-NPA and [11C]-(-)-MNPA for this receptor state (>60, 180 and 300 fold lower than for the high- affinity state)509,529 would be expected to result in STR/CER ratios much lower than observed.

A third possibility is that the ternary complex model is a fundamentally inaccurate description of the D2 and other G protein-coupled receptors in the living brain. Explicit in the ternary complex model is the notion that the receptor and the G protein participate in an association-dissociation equilibrium, the position of which determines the proportion of receptors in high and low agonist affinity states. The model further implies that the receptor and effector enzyme are physically separated, and that G protein must diffuse between receptor and

166 effector for signal transduction to occur. However, in recent years substantial evidence has accumulated indicating that G protein-coupled receptors in living cells instead function constitutively as part of large heteromultimeric signaling complexes involving receptor, G protein, effector enzymes, other regulatory proteins and various scaffolding proteins that anchor the complex to the cytoskeleton (see references 635-637). In living cells, fluorescence and bioluminescence energy transfer (FRET and BRET) experiments, which use changes in bioluminescence of fluorescence to indicate the proximity of protein species to one another, indicate that complexes containing various G protein-coupled receptors (including adrenergic β2 and α2, and δ-opiod receptors) are very stable under in vivo conditions, and appear not to dissociate even during receptor activation by agonists.638-640 Such signaling complexes provide the basis for a fundamentally different view of G protein-coupled receptor function. A mechanistic description of the function of G protein-coupled receptors in membrane signaling complexes is far from complete. However, because the receptor appears constitutively and stably bound to its G protein, it is conceivable that such complexes exist entirely in an agonist high-affinity state. Furthermore, agonist binding, rather than causing receptor-G protein dissociation as described by the ternary complex model, may instead function by producing a concerted conformational change throughout the signaling complex, allowing both G protein and effector to simultaneously adopt active conformations, with the exchange of GDP for GTP and subsequent GTP hydrolysis acting as an enzymatic timing switch regulating the duration of signal transduction. In this context, the low affinity state need not represent a separate, relatively long-lived receptor state, but potentially only a short-lived transitional conformation in the catalytic cycle of the complex. Alternatively, the in vitro low-affinity state could be viewed as entirely artifactual, resulting from the disruption of the native signaling complex during membrane preparation. The participation of the D2 receptor in large signaling complexes can also offer insight into the apparent paradox whereby the increased in vitro D2 high-affinity state

167 in animal models is not reflected by increased ex vivo [11C]-(+)-PHNO binding (section 4). If the in vitro low-affinity state is assumed to represent receptors fully or partially dissociated from the functional signaling complex during membrane preparation (e.g. as the result of homogenization, disruption of ionic conditions, etc.), then an increase in the stability of the in vivo signaling complex would be expected to result in an apparent increase in the in vitro high- affinity state, as more functional (i.e. high-affinity) complexes would remain intact. In summary, the current thesis supports the idea that the D2 receptor exists in a single affinity state for agonists. Clarification of the exact biochemical nature of this binding site, however, awaits a more complete biochemical characterization of D2 receptor function in vivo.

Another unresolved issue arising from the current work is the mechanism by which isoflurane differentially affects the ex vivo binding of the agonist radiotracers [11C]-(+)-PHNO and [11C]-(-)-NPA, and the antagonist [3H]-raclopride. As discussed in section 5.5, it is difficult to rationalize the effects of isoflurane anaesthesia in terms of the two-affinity state model of the

D2 receptor. Furthermore, interpretation of isoflurane’s effects in the context of this model would be inconsistent with the results of sections 3 and 4 of this thesis, which support a one affinity state model. As antagonists bind with equal affinity to the in vitro high- and low-affinity states, which presumably represent different receptor conformations, it is not difficult to imagine that an isoflurane-induced D2 receptor conformational change could increase agonist affinity without having measurable impact of the affinity of an antagonist radiotracer. Thus, our ex vivo binding studies with isoflurane anaesthesia, while not providing direct support for a one affinity state model, are not inconsistent with such a model. Clarification of this issue could be accomplished by in vivo or ex vivo saturation studies in which the affinity (KD) and density (Bmax) of these radiotracers is assessed in conscious and isoflurane-anaesthetized animals. The differential effect of isoflurane on [11C]-(+)-PHNO and [11C]-(-)-NPA versus [3H]-raclopride implies a dependence on the chemical structure of the radiotracer (i.e. the structure of the

168 pharmacophore, which presumably imparts ligands with either agonist or antagonist properties).

Although the similarity in isoflurane’s effects on [11C]-(+)-PHNO and [11C]-(-)-NPA could be related to conservation in the agonist pharmacophore recognized by the D2 receptor, it might be useful to test whether isoflurane has differential effects on the in vivo binding of benzamide versus butyrophenone radiotracers, which have substantially different core structures.

The final study (section 6) of this thesis using [3H]-(+)-PHNO autoradiography indicates that the antipsychotic drugs olanzapine, risperidone, clozapine and haloperidol, despite producing high levels of D2 receptor occupancy, occupy a far smaller proportion of D3 receptors than would be predicted from their similar in vitro affinity for the two receptor subtypes. This has major relevance for the in vivo therapeutic action of these drugs, which has been proposed by some authors to be due at least in part to D3 receptor blockade. Important future work in this realm would be to determine, potentially using methods similar to those used here or using PET in human subjects, whether in vivo D2-selectivity is a property of antipsychotic drugs as a class, or whether this phenomenon is limited to the drugs examined in the current work. Importantly, this study, like the first three studies in this thesis, points to major discrepancies between the ex vivo and in vitro binding not only of antipsychotic drugs but also of [3H]-(+)-PHNO binding, which appears to label a higher proportion of D3 receptors ex vivo compared to in vitro. An interpretation of these discrepancies is a complicated matter, requiring an understanding of the many factors that can potentially result in differences between in vitro and in vivo ligand binding (for a detailed review see reference 641). These factors, which potentially apply to interpretation of all of the results of this thesis, could include disruption of ionic conditions surrounding the receptor, membrane voltage, protein kinase/phosphatase cycles, and levels of guanine nucleotide, all known to affect the activity and/or ligand binding of D2 and other G protein-coupled receptors.642-646

169 In conclusion, the results of this thesis provide no support for the existence of the D2 receptor in vivo in separate states of affinity for agonists, being instead supportive of a single affinity state model in which the [11C]-(+)-PHNO and the other D2/D3 agonist radiotracers label a receptor state pharmacologically similar to that labeled by [11C]-raclopride in conscious rats.

Consequently, it is proposed that the agonist PET radiotracers, [11C]-(+)-PHNO, [11C]-(-)-NPA and [11C]-(-)-MNPA, will prove no more effective than [11C]-raclopride or other benzamide radiotracers for measurement of extracellular dopamine levels in conscious human subjects.

This thesis also demonstrates the utility of [3H]-(+)-PHNO as a radiotracer for measurement of drug occupancy at D2 and D3 receptor subtypes, and contributes novel findings to our understanding of the dopaminergic pharmacology of several antipsychotic drugs. Importantly, the findings of this thesis illustrate major discrepancies between in vitro and in vivo or ex vivo radioligand binding and point to limitations in our understanding of the function of the D2 receptor in living brain.

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