by Patrick Neil McCormick A thesis submitted in conformity ... · Patrick Neil McCormick Doctor of Philosophy Institute of Medical Science University of Toronto 2010 Abstract The

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

by

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

© Copyright by Patrick Neil McCormick (2010)

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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

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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

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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.

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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.

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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

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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 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) 6 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

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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 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) 129 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 Figure 21. Binding potential (BPND) values for [11C]-(+)PHNO and [3H]-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 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) 151 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) 152

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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

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

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.

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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.

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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.

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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

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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.

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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

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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

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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

9

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-

innervated GABAergic neurons of the STR,54,55 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

AMPA67-69 and NMDA receptor channels,70-72 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

abundant than Gαi.80 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.


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).


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


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

In the human brain, highest [3H]-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.


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

Similar problems plague in vivo determinations of DAT Bmax.209,210 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:

FK

FBBD +

= max (1)

where B is the concentration of bound radioligand (mol·L-1), Bmax is the density of radioligand

specific binding sites (i.e. neurotransmitter receptor or transporter proteins, enzymes etc.)

(mol·L-1), F is the concentration of free radioligand (mol·L-1) 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

DKFBB max= (2)

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

DK

BFBBP max=≡ . (3)

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

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

off

on

kBk

FBBP max== (4)

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

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

governed by the rate constant k2 (min-1). 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

P11 CKJ = (5)

and the flux in the reverse direction by

122 CkJ = (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:

2

1

P

11 k

KCCV == (7)

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-

38

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.

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

2

1

P

NDND k

KCCV == (8)

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

ND33 CkJ = (9)

and, by expressing CND in terms of K1, k2 and CP, can be written as

P2

313 C

kkKJ ⋅= (10)

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

S44 CkJ = (11)

39

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

binding compartment is

P42

31

P

SS BP

kkkK

CCV ≡== (12)

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

analogue of the in vitro BP defined by equation (4).335 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:

SCB = (13)

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

FTCF = (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

PPCfF = (15)

40

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

parameters:

PP

S

CfCBP = (16)

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:

P

S

PF f

Vkkf

kKBP =42

31≡ (17)

Note that that BPF (cm3·mL-1) 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:

4

3

kk

CC

VVBP

NS

S

ND

SND === (18)

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

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

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

Binding potential

Specific binding relative to Volumes of distribution Kinetic rate constants

BPP Total plasma radiotracer (Cp) NDTS VVV -= 42

31

kkkK

BPF Free plasma radiotracer (fP·Cp) P

NDT

P

S

fV-V

fV

= 42

31

kkfkK

P

BPND Non-displaceable radiotracer in tissue (CND)

ND

NDT

ND

S

VV-V

VV

= 4

3

kk

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

0dt

)t(dCS = (19)

This condition defines so-called transient equilibrium. Equation (19) can be expressed in terms

of the rate constant for the specific binding compartment:

0*)(tCk*)(tCkdt

(t)dCS4ND3

S =−= (20)

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

NDND

NDT BPkk

*)t(C*)t(C*)t(C

==−

4

3 (21)

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:

*)t(C

*)t(C*)t(CBP

R

RTND

−= (22)

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

constants k3 and k4.339 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

below).333,334,340,341 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

the single time point concentrations.334,342 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

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

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

point method tends to overestimate BPND.344 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

subject groups, late time point methods provide a measure that is proportional to BPND.340,344 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

t)exp(-k21P1 KC)t(C = (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

21211 for TtT)]Tt(kexp[KCP)t(C 1 <≤−−= (24)

45

and after the second bolus by

212P11P1 Tt)]Tt(kexp[KC)]Tt(k[xpeKC(t)C ≥−−+−−= for 2212 (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:

... 3, 2, 1,ieKC(t)Ci

)Tt(kPi1

i ==∑ −− for 21 (26)

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

0

s)(tk1P1 dseK(s)C(t)C 2 (27)

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:

∫ −=⊗ dss)t(g)t(f)t(g)t(f (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

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

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:

ND

a BPk

kk

kk−

=+

=11

2

4

3

22 (29)

and the total volume of distribution given by

)BP(1V)BP(1kK

BP1kK

kKV NDNDND

2

1

ND

2

1

a2

1T +=+=

+

== (30)

47

Table 3. Common kinetic analysis methods.

Compartments Fitted parameters Method

Ref. region ROI Ref. region ROI Assumptions1

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

2-TC direct common VND --- 2-TC --- K1, k2, k3, k4

(constrained K1/k2) All regions same VND

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

1-TC/2-TC direct fixed VND 1-TC 2-TC K1, k2 k3, k4

1-TC ref. region VND equals ref. region VT

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

1-TC 1-TC 1-TC K1, k2 K1,

k2a = k2/(1+BPND)

1-TC ref. region 1-TC ROI VND equals ref. region VT

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

SRTM 1-TC 1-TC K1, k2 R1, k2, BPND 1-TC reference region 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

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

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

k2’, then fixes this value for all voxels in a subsequent SRTM procedure,357 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

BPND.355 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

(t)Ck(t)CKdt

)t(dC12p1

1 −= (31)

Integration of equation (30) gives

∫ ∫−= dt(t)Ckdt(t)CK(t)C 12p11 (32)

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

method:

50

21

p

2

1

1

1

k1

(t)C

dt(t)C

kK

(t)C

dt(t)C−= ∫∫ (33)

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

⎟⎟⎠

⎞⎜⎜⎝

⎛+−⎟⎟

⎠

⎞⎜⎜⎝

⎛+= ∫∫

4

3

2T

P

4

3

2

1

T

T

kk1

k1

(t)C

(t)C

kk1

kK

(t)C

dt(t)C (34)

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

b)t(C

)t(CV

)t(C

dt)t(C

T

PT

T

T+= ∫∫ (35)

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))

variables.347,358 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:

51

∫ ∫ ⎟⎟⎠

⎞⎜⎜⎝

⎛+=

'k(t)C

dt(t)C'K'k

dt(t)C2

RR

1

2P (36)

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

b(t)C

'k(t)Cdt(t)C

kk1

kK

'K'k

(t)C

dt(t)C

T

2

RR

4

3

2

1

1

2

T

T+

+

⎟⎟⎠

⎞⎜⎜⎝

⎛+=

∫∫ (37)

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:

b(t)C

'k(t)Cdt(t)C

kk1

(t)Cdt(t)C

T

2

RR

4

3

T

T−

+

⎟⎟⎠

⎞⎜⎜⎝

⎛+=

∫∫ (38)

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

parameter.348 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

the determination of VT.347 The operational equation for the Ichise multilinear analysis (MA)

method is obtained by rearrangement of equation (34):

∫ ∫+−= (t)dtCb

dt)t(Cb

V)t(C TPT

T1 (39)

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

2

4

3

22

1

4

3

2

1

2

1

4

3

2

1 111

kkk

'k'k'K

kk

kK

)t(Cdt)t(C

'k'K

kk

kK

)t(Cdt)t(C

T

R

T

T⎟⎟⎠

⎞⎜⎜⎝

⎛+

+⎟⎟⎠

⎞⎜⎜⎝

⎛+

−⎟⎟⎠

⎞⎜⎜⎝

⎛+

= ∫∫ (40)

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

22

111kBP

)t(C)t(C

'kBP

)t(C

dt)t(C)BP(

)t(C

dt)t(CND

T

RND

T

RND

T

T ++

+−+= ∫∫ (41)

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):

)t(C'k

kdt)t(Ckdt)t(CBPk)t(C RRT

NDT

2

22

2

1+−

+= ∫∫ (42)

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

specific binding (striatal BPND = 0.4-0.8).370,371 A slight improvement over [11C]-cocaine was

achieved with [11C]-methylphenidate ([11C]-MP), which had higher, but still relatively low

specific binding (BPND = ~1.5 in STR).372 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

binds to both DAT and SERT,395,398 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)

using the occipital cortex as a reference region.414 In the STR, [11C]-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

receptor (Ki = 0.14 nM)419 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

relatively low binding signal of this radiotracer (STR BPND ~ 0.8-1.0)420 and its activity at

serotonin 5-HT2A receptors421-423 inspired the search for other D1 radiotracers. Three other

high-affinity D1 radiotracers, [11C]-SCH-39166 (Ki = 3.4 nM),424 [11C]-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

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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

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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

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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

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

(BPND ~2.1 and 0.9 thalamus and temporal cortex).457 Like [11C]-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

D2/D3 receptors (Ki ~ 24 pM)464 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

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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

apparent KD of [11C]-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

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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

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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 absence of GTP. B) Two-affinity state model used to assign dissociation constants to the high- (KD

High) and low-affinity (KD

Low) 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 uncoupled form (R) (i.e. KD

High < KDLow). 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

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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.

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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-

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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-

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

striatum ranged from 1.29 with full kinetic analysis to 1.41 with the SRTM.514 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

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

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

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amphetamine challenge.470 The lower BPND of [11C]-(-)-NPA versus [11C]-raclopride is largely

the result of relatively higher volume of distribution in the non-displaceable binding

compartment [VND, estimated by the total volume of distribution (VT) in cerebellum].470,515 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

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

[11C]-raclopride in the same subjects (BPND = 2.6),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

experiments is the prominent binding of [11C]-(-)-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

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

(human and baboon) dorsal STR (BPND = ~2.6) than in the globus pallidus (BPND = ~1.5),6,517

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

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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

determined.523 As with [11C]-(-)-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

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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

for the CER.525 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

methods are appropriate for measurement of [11C]-(-)-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

against [3H]-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

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

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

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

(IC50 = 77-85 nM),526,528 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

membranes (Ki = 10.3 µM)531 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

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[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

dopaminergic activity.526,527,536 Specifically, (+)-PHNO induced hypothermia in mice (ED50 = 3

and 13 µg/kg in two studies),527,536 postural asymmetry in unilaterally caudectomized mice

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

lesioned rats (ED50 = 10 and 5 µg/kg, respectively)527,537 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-

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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

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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-

2.1).5,302,545-548 The BPND of [11C]-(+)-PHNO could be reduced by AMPH547 or haloperidol548

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

in both regions).5 PET studies in baboon confirmed that [11C]-(+)-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

reduction seen in CAU and PUT.7,517 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

77

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.

SV-156.7 In human as well, [11C]-(+)-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

the portion of [11C]-(+)-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

D2 binding. Comparing the regional rank order of BPND for [11C]-(+)-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

regional total distribution volumes, k3/k4 ratios (BPND) were poorly identified.5 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

obtained in cerebellum (the reference region).5 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

scanning time appropriate for a 11C-labeled radiotracer (80 min).5 Thus, despite a small BPND

underestimate, the SRTM has become the method of choice for determination of [11C]-(+)-

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

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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.

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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.

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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

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

NPA ED50 0.027 and 0.023 mg/kg for [11C]-(+)-PHNO and [3H]-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.

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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

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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.

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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:

bellum%ID/g Cerelum/g Cerebelatum - %ID%ID/g Stri SBR = .

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

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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

fitted (R2 > 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]-

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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).

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). Error bars represent the SD of the means.

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

Drug Drug type ED50 (mg/kg)

95% Confidence interval

Hill slope


ED50 (mg/kg)


Hill slope


(-)-NPA D2 full agonist 0.027 0.015, 0.050 -0.92 -1.40, -0.45 0.023 0.019, 0.028 -1.04 -1.25, -0.85

Aripiprazole D2 partial agonist 0.33 0.13, 0.88 -0.79 -1.2, -0.35 0.15 0.069, 0.34 -0.89 -1.48, -0.33

Haloperidol D2 antagonist 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

Clozapine D2 antagonist 4.6 3.5, 6.2 -1.4 -2.02, -0.74 6.2 4.4, 8.6 -1.7 -2.64, -0.79

a Significantly different from Hill slope of (-)-NPA vs. [11C]-(+)-PHNO; Bonferroni’s multiple comparison (p<0.05). b Significantly different from Hill slope of aripiprazole vs. [11C]-(+)-PHNO; Bonferroni’s multiple comparison (p<0.01).

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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.

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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

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

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.

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

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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

specific binding (V3”) is more potently inhibited by AMPH treatment than that of [11C]-

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

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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

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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

pmol/mL, respectively).343 Similar striatal Bmax values for the agonist radiotracer [3H]-(-)-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]-(+)-

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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

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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

BPratio-ex vivo, in their nomenclature) correlates well with the kinetically modeled BP.340 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.

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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*

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



Canada M5T 1R8


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

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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.

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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,

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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

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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.

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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-

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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

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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:

CER

CERSTR

%ID/g%ID/g%ID/g

SBR−

=

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).

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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).

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.

[11C]-(+)-PHNO [3H]-Raclopride Sensitization regime

Chronic treatment

Acute pretreatment STR %ID/g CER %ID/g SBR STR %ID/g CER %ID/g SBR

Chronic AMPH 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

4 mg/kg AMPH 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 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 AMPH 0.27 ± 0.04a 0.074 ± 0.009 2.6 ± 0.5d 0.27 ± 0.03c 0.053 ± 0.005 4.2 ± 0.5d

Ethanol withdrawal Saline none 0.4 ± 0.1 0.08 ± 0.02 4.3 ± 0.5 0.32 ± 0.07 0.030 ± 0.008 10 ± 1

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)

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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

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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).

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).

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

Right-lesioned 0.58 ± 0.09 0.8 ± 0.1a 0.10 ± 0.01 0.54 ± 0.07 0.8 ± 0.1a 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.

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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

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

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

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

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.

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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

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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

that the specific binding of [11C]-raclopride (Bmax)337 and [3H]-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

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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

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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.

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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

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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.

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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.

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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



Canada M5T 1R8


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

Switzerland

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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.

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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

increases the inhibitory effect of methamphetamine and nicotine on the BPND of [11C]-

raclopride.236 Halothane, another volatile anaesthetic, increases both the BPND of [11C]-

raclopride and cerebral blood flow.609 The injected anaesthetic ketamine increases the striatal

BPF of the D1 radiotracer [11C]-SCH23390 and the dopamine transporter (DAT) radiotracers

[11C]-β-CFT and [11C]-βCIT-FE, increases the striatal accumulation of [11C]-L-DOPA, and

decreases the BPF of [11C]-raclopride.388,610,611 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

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of [11C]-(+)-PHNO versus [3H]-raclopride specific binding ratio (SBR) to AMPH

pretreatment.513 Furthermore, the AMPH-induced decrease in BPND for both [11C]-(+)-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

[11C]-(+)-PHNO BPND over that of [11C]-raclopride.

Using ex vivo dual radiotracer experiments in rat, we demonstrate here that isoflurane

anaesthesia accounts for the increased AMPH sensitivity of [11C]-(+)-PHNO BPND and SBR

over those of [3H]-raclopride, and that in the absence of anaesthesia, the BPND and SBR of [11C]-

(+)-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

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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

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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:

CER

CERSTR

%ID/g%ID/g%ID/g

SBR−

=

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

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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

[11C]-(-)-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)

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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 +

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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. ††† Significantly different from conscious condition (ANOVA, Bonferroni’s multiple comparison test, p < 0.001).

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

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

Data analysis resulted in BPND values for [11C]-(+)-PHNO and [3H]-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

anaesthesia and/or AMPH pretreatment resulted in a pattern of BPND changes for [11C]-(+)-

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

40 ± 20% increase in the BPND of [11C]-(+)-PHNO (p < 0.001) but had no effect on the BPND of

[3H]-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).

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6.5. Discussion

Recent PET studies have shown that in anaesthetized animals, AMPH pretreatment

reduces the BPND of the D2/D3 agonist radiotracers [11C]-(+)-PHNO,343 [11C]-(-)-NPA470 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

anaesthesia increases the SBR and BPND of [11C]/[3H]-(+)-PHNO and the SBR of [11C]-(-)-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]-

raclopride BPND reported in recent PET studies,343,470 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

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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

given by PET experiments in which ketamine anaesthesia increased the BPND of [11C]-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]-

(-)-NPA and [11C]-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

pretreatment on [11C]-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

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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

metabolism that could account for the observed changes in [11C]-(+)-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,

[11C]-(-)-NPA and [3H]-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.

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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

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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

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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

been found in cat PET experiments (using the BPND as the outcome measure),343 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

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[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

inhibits ex vivo [11C]-(+)-PHNO and [3H]-raclopride binding with an ED50 of ~81 nmol/kg,513

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

the commonly-reported greater effect of AMPH on D2 agonist versus [11C]-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.

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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


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

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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.

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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

showing that [11C]-(+)-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, 22. Radioligands used in determination of Ki values were [3H]-raclopride, [3H]-nemonapride, [125I]-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

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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.

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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

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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:

⎟⎟⎠

⎞⎜⎜⎝

⎛ −×=

Control

ControlDrug

SBRSBRSBR

cupancy Percent oc 100

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

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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

EDTA, 1.5 mM CaCl2, 4 mM MgCl2 and NaCl either 0.6 nM) containing either 0.6 nM [3H]-

(+)-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

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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

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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 (d)

Olanzapine (nM)

Haloperidol (nM)

Risperidone (nM)

Clozapine (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

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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.

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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.

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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

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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

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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,

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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

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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

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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-

called transient equilibrium) to estimate the non-displaceable binding potential (BPND).333,334,341

The late time point method, which is used in the current study, also provides an estimate of

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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

provides accurate estimates of the BPND,333,334,341 the late time point method can result in modest

overestimate of BPND,334,344 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

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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

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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.

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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.

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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

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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

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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

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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

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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

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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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by Patrick Neil McCormick A thesis submitted in conformity ... · Patrick Neil McCormick Doctor of Philosophy Institute of Medical Science University of Toronto 2010 Abstract The