In Vivo Vulnerability to Competitionby Endogenous Dopamine:

Comparison of the D2 Receptor AgonistRadiotracer (–)-N-[11C]propyl-

norapomorphine ([11C]NPA) With theD2 Receptor Antagonist Radiotracer

[11C]-RacloprideRAJESH NARENDRAN,1* DAH-REN HWANG,1,2 MARK SLIFSTEIN,1 PETER S. TALBOT,1

DAVID ERRITZOE,1 YIYUN HUANG,1,2 THOMAS B. COOPER,1 DIANA MARTINEZ,1LAWRENCE S. KEGELES,1 ANISSA ABI-DARGHAM,1,2 AND MARC LARUELLE1,2

1Department of Psychiatry, Columbia University College of Physicians and Surgeonsand the New York State Psychiatric Institute, New York, NY 10032

2Department of Radiology, Columbia University College of Physicians and Surgeonsand the New York State Psychiatric Institute, New York, NY 10032

KEY WORDS [11C]NPA; [11C]raclopride; PET; dopamine; amphetamine; agonist; an-tagonist

ABSTRACT (–)-N-Propyl-norapomorphine (NPA) is a full dopamine (DA) D2 receptoragonist and [11C]NPA is a suitable radiotracer to image D2 receptors configured in astate of high affinity for agonists with positron emission tomography (PET). In this studythe vulnerability of the in vivo binding of [11C]NPA to acute fluctuation in synaptic DAwas assessed with PET in baboons and compared to that of the reference D2 receptorantagonist radiotracer [11C]raclopride. Three male baboons were studied with [11C]raclo-pride and [11C]NPA under baseline conditions and following administration of the potentDA releaser amphetamine (0.3, 0.5, and 1.0 mg kg–1 i.v.). Kinetic modeling with an arterialinput function was used to derive the striatal specific-to-nonspecific equilibrium partitioncoefficient (V3�). [11C]Raclopride V3� was reduced by 24 � 10%, 32 � 6%, and 44 � 9%following amphetamine doses of 0.3, 0.5, and 1.0 mg kg–1, respectively. [11C]NPA V3� wasreduced by 32 � 2%, 45 � 3%, and 53 � 9% following amphetamine doses of 0.3, 0.5, and 1.0mg kg–1, respectively. Thus, endogenous DA was more effective at competing with [11C]NPAbinding compared to [11C]raclopride binding, a finding consistent with the pharmacology ofthese tracers (agonist vs. antagonist). These results also suggest that 71% of D2 receptorsare configured in a state of high affinity for agonists in vivo. In conclusion, [11C]NPA mightprovide a superior radiotracer to probe presynaptic DA function with PET in health anddisease. Synapse 52:188–208, 2004. © 2004 Wiley-Liss, Inc.

INTRODUCTION

Over the past decade, several groups have providedevidence that competition between neurotransmittersand radioligands for neuroreceptor binding allowsmeasurement of changes in synaptic neurotransmitterlevels with in vivo imaging techniques (for review, seeLaruelle, 2000). The reduction in D2 receptor in vivoavailability following administration of the potent do-pamine (DA) releaser amphetamine is one example ofsuch an interaction. The amphetamine-induced reduc-tion of [11C]raclopride or [123I]IBZM specific binding

has been well validated as a measure of change inendogenous DA in both nonhuman primates (Carson et

Contract grant sponsors: NARSAD, the NIMH; Contract grant numbers:1RO1MH62089, 1-K02-MH01603-01, MH59342-01; Contract grant sponsor: theLieber Center for Schizophrenia Research at Columbia.

*Correspondence to: R. Narendran, MD, New York State Psychiatric Institute,1051 Riverside Drive, Box #31, New York, NY 10032.E-mail: rn2012@columbia.edu

Received 15 July 2003; Accepted 20 January 2004

DOI 10.1002/syn.20013

SYNAPSE 52:188–208 (2004)

© 2004 WILEY-LISS, INC.

al., 1997; Dewey et al., 1993; Drevets et al., 1999;Endres et al., 1997; Innis et al., 1992; Laruelle et al.,1997; Tsukada et al., 1999) and humans (Breier et al.,1997; Drevets et al., 2001; Kegeles et al., 1999; Laruelleet al., 1995; Martinez et al., 2003). This technique hasbeen used as a probe of presynaptic DA function inhealth and disease (Abi-Dargham et al., 1998; Anand etal., 2000; Breier et al., 1997; Laruelle et al., 1996;Parsey et al., 2001; Singer et al., 2002).

A well-described limitation of this technique is itslow sensitivity, in that relatively large increases inextracellular DA are associated with relatively modesteffects on [123I]IBZM or [11C]raclopride binding (Breieret al., 1997; Laruelle et al., 1997). This low sensitivitymight be related to the fact that D2 receptors are con-figured in interconvertible states of high (D2high) or low(D2low) affinity for agonists (George et al., 1985a; Rich-field et al., 1989b; Seeman and Grigoriadis, 1987; Sib-ley et al., 1982; Zahniser and Molinoff, 1978). Antago-nists, such as [123I]IBZM or [11C]raclopride, bind withequal affinity to D2high and D2low sites. The agonist DAis not expected to compete efficiently with [123I]IBZMor [11C]raclopride at the D2low sites. These consider-ations imply that the ideal radiotracer for endogenouscompetition studies would be a D2 receptor agonist.

Another limitation of endogenous competition stud-ies with radiolabeled antagonists resides at the level ofthe interpretation of the results. In theory, changes inamphetamine-induced reduction in [11C]raclopride or[123I]IBZM binding in pathological states could be dueto presynaptic factors (differences in the magnitude ofchanges in synaptic DA) or to postsynaptic factors (dif-ferences in affinities of D2 receptors for agonists), or tosome combination of pre- and postsynaptic factors.Here again, a radiolabeled agonist would be advanta-geous, as it would provide information about the affin-ity of D2 receptors for agonists, allowing researchers tocontrol for this important parameter in clinical studies.

Hwang et al. (2000) recently reported a procedure toradiolabel the potent DA D2 agonist PET radiotracer(–)-N-propyl-norapomorphine (NPA) with C-11, as wellas initial imaging experiments in baboons using[11C]NPA. NPA is a potent and full agonist at D2 andD3 receptors (Gardner and Strange, 1998; Gulat-Mar-nay et al., 1985; Menon et al., 1978; Neumeyer et al.,1973; Rinken et al., 1999; Yarbrough et al., 1984).NPA’s affinity for D2high sites is in the 0.07–0.4 nMrange, while its affinity for D2low sites is in the 20–200nM range (Gardner and Strange, 1998; George et al.,1985b; Lahti et al., 1996; Seeman et al., 1985; Sibley etal., 1982). Thus, NPA displays a selectivity of at least50-fold for D2high vs. D2low sites. In baboons, [11C]NPAdemonstrated a rapid brain uptake with selective ac-cumulation in the striatum (Hwang et al., 2000). Thestriatal uptake was decreased to the level of cerebellaruptake following pretreatment with the D2 receptorantagonist haloperidol, indicating that the striatal up-

take of [11C]NPA was saturable and selective for D2-like receptors. Given the 50-fold D2high vs. D2low selec-tivity described above, it is likely that, at tracer dose,[11C]NPA binding corresponds mainly, if not exclu-sively, to binding to D2high sites. Thus, [11C]NPA ap-peared to be a suitable agonist radiotracer to labelD2high sites in vivo with PET. Furthermore, recent ex-periments in mice suggested that the in vivo binding of[3H]NPA was more vulnerable to endogenous competi-tion by DA than that of the antagonist [11C]raclopride(Cumming et al., 2002a).

The aim of this study was to compare the in vivobinding of the D2 receptor agonist radiotracer[11C]NPA with that of the reference D2 receptor antag-onist radiotracer [11C]raclopride, in terms of vulnera-bility to changes in endogenous DA elicited by an acuteamphetamine challenge. The decrease in [11C]NPA and[11C]raclopride striatal specific binding following threedoses of amphetamine (0.3, 0.5, and 1 mg kg–1) wasstudied with PET in three male baboons. In addition,two experiments were repeated, to provide informationabout the reproducibility of the measurement of theamphetamine effect.

MATERIALS AND METHODSGeneral design

A total of 40 PET scans on three male baboons (de-noted A, B, and C) are reported here. These 40 PETscans were acquired in 20 experimental sessions. Eachexperimental session included two PET scans, a base-line scan, and a postamphetamine scan.

The fundamental aim of this study was to evaluatethe dose–effect relationship of amphetamine on[11C]raclopride and [11C]NPA striatal specific binding.Three doses of amphetamine were tested (0.3, 0.5, and1 mg kg–1), for a total of 36 scans: 2 conditions (baselineand postamphetamine) * 3 baboons (A, B, C) * 2 ligands([11C]raclopride and [11C]NPA) * 3 amphetamine doses(0.3, 0.5, and 1 mg kg–1). A secondary aim of the studywas to assess the reproducibility of the measurement ofthe amphetamine effect for each ligand. For this pur-pose, two experimental sessions were repeated (am-phetamine doses of 0.5 mg kg–1 for each tracer) onbaboon A, for a total of four additional scans.

For each animal, the sequence of radiotracer andamphetamine dose was counterbalanced to preventbias in the between-radiotracer comparison. The dosesof amphetamine were selected for the following rea-sons: 0.3 mg kg–1 is a dose commonly used in humanclinical studies (Abi-Dargham et al., 1998; Anand et al.,2000; Drevets et al., 2001; Kegeles et al., 1999; Laruelleet al., 1995, 1996; Martinez et al., 2003; Parsey et al.,2001; Singer et al., 2002); 1 mg kg–1 is a dose associatedwith a near maximal increase in extracellular DA (La-ruelle et al., 1997); and the doses of 0.3, 0.5, and 1 mgkg–1 were previously used in baboons to assess the

COMPARISON OF [11C]NPA AND [11C]RACLOPRIDE 189

vulnerability of [123I]IBZM to changes in endogenousDA (Laruelle et al., 1997).

RadiochemistrySynthesis of [11C]raclopride

[11C]raclopride and [11C]NPA were prepared as pre-viously described (Hwang et al., 2000; Mawlawi et al.,2001).

PET imaging protocol

Experiments were performed according to protocolsapproved by the Columbia-Presbyterian Medical Cen-ter Institutional Animal Care and Use Committee.Fasted animals were immobilized with ketamine (10mg kg–1 i.m.) and anesthetized with 1.8% isoflurane viaan endotracheal tube. The animals’ vital signs weremonitored every 10 min and temperature was keptconstant at 37°C with heated water blankets. An i.v.perfusion line was used for hydration and injection ofradiotracers and nonradioactive drugs. A catheter wasinserted in a femoral artery for arterial blood sampling.The head was positioned at the center of the field ofview as defined by embedded laser lines. PET imagingwas performed with ECAT EXACT HR� scanner (Sie-mens/CTI, Knoxville, TN). In 3D mode, this cameraprovides in-plane resolutions of 4.3 mm, 4.5 mm, 5.4mm, and 8.0 mm full width at half maximum at dis-tances of 0, 1, 10, and 20 cm from the center of the fieldof view, respectively (Brix et al., 1997). A 10-min trans-mission scan was obtained prior to radiotracer injec-tion for attenuation correction. Activity was injectedi.v. over 30 sec as a bolus. The injected mass andradioactivity had upper limits of 6 �g and 6 mCi, re-spectively. Emission data was collected in 3D mode for60 min and 90 min as successive frames (18 and 21frames) of increasing duration for [11C]raclopride and[11C]NPA, respectively.

Input function measurements

Arterial samples were collected every 10 sec for thefirst 2 min and every 20 sec for the next 2 min with anautomated blood sampling system and manually there-after at various intervals. A total of 30 samples werecollected per experiment. Following centrifugation (10min at 1,800g), plasma was collected and activity mea-sured in 0.2-mL aliquots on a gamma counter (Wallac1480 Wizard 3M Automatic Gamma Counter, Perkin-Elmer, Boston, MA).

For [11C]raclopride, six samples (2, 6, 12, 20, 40, and60 min) were further processed by protein precipitationusing acetonitrile followed by HPLC (Phenomenex C18analytical column) to measure the fraction of plasmaactivity representing unmetabolized parent compound(Mawlawi et al., 2001). For [11C]NPA, five samples (1,4, 12, 40, and 80 min) were further processed by HPLCto determine the fraction of activity associated with the

unmetabolized parent compound. Ascorbic acid (0.01 gmL–1 blood) was added to these blood samples to sta-bilize the [11C]NPA. After centrifugation, plasma (0.5mL) was pipetted into a centrifuge tube containingmethanol (1.0 mL). The contents of the tube weremixed vigorously and centrifuged (14,000g for 4 min).The liquid phase was separated from the precipitates.Activity in 0.1 mL of the liquid phase was counted andthe rest was analyzed by HPLC. The HPLC eluate wasfraction-collected in 12 counting tubes (2.0 mL each).The column was eluted with a mixture of acetonitrileand aqueous 0.1 M ammonium formate at a flow rate of2 mL min–1. Before plasma sample analysis, the reten-tion time of the parent radiotracer was established byinjection of a small amount of the tracer and detectionof the elution time using the Bioscan gamma detector.

The parent fraction was calculated as the ratio ofactivity in the fractions containing the parent to thetotal activity collected. The parent fractions were fittedto the sum of two exponentials. The smallest exponen-tial of the fraction of the parent curve, �par, was con-strained to the difference between �cer (the terminalrate of washout of the cerebellar activity) and �tot (thesmallest elimination rate constant of the total plasma)(Abi-Dargham et al., 1999). The input function wasthen calculated as the product of the total counts andthe interpolated fraction parent at each time point. Themeasured input function values were fitted to a sum ofthree exponentials from the time of peak plasma activ-ity and the fitted values were used as inputs for kineticanalyses. The clearance of the parent compound (CL, Lh–1) was calculated as the ratio of the injected dose tothe area under the curve of the input function (Abi-Dargham et al., 1994; Rowland and Tozer, 1989).

For the determination of the plasma free fraction (f1),triplicate 200 �L aliquots of plasma collected beforeinjection were mixed with radiotracer, pipetted intoultrafiltration units (Centrifree; Amicon, Danvers,MA), and centrifuged at room temperature (20 min at4,000 rpm). At the end of centrifugation, plasma andultrafiltrate activities were counted and f1 was calcu-lated as the ratio of ultrafiltrate to total activity con-centrations (Gandelman et al., 1994).

Image analysis

A magnetic resonance image (MRI) of each baboon’sbrain was obtained for the purpose of identifying theregions of interest (ROIs) (T1-weighted axial MRI se-quence, acquired parallel to the anterior–posteriorcommissure, TR 34 msec, TE 5 msec, flip angle of 45°,slice thickness 1.5 mm, zero gap, matrix 1.5 � 1 � 1mm voxels). The striatum and cerebellum ROIs weredelineated on the MRI based on the criteria defined inthe article by Riche et al. (1988).

PET emission data were attenuation-corrected usingthe transmission scan and frames were reconstructedwith filtered backprojection, using a Shepp filter (cutoff

190 R. NARENDRAN ET AL.

0.5 cycles/projection rays). Reconstructed image fileswere then processed using the image analysis softwareMEDx (Sensor Systems, Sterling, VA). An image wascreated by summing all the frames and this summedimage was used to define the registration parametersfor use with the MRI, using the between-modality au-tomated image registration (AIR) algorithm (Woods etal., 1993). Registration parameters were then appliedto the individual frames for registration to the MRdataset. Regional boundaries were transferred to theindividual registered PET frames and time–activitycurves were measured and decay-corrected. Right andleft striata were averaged. For a given animal the sameregional boundaries were used for all experiments.Brain activity was corrected for the contribution ofplasma activity assuming a 5% blood volume (Mintunet al., 1984).

Derivation of binding parametersOutcome measures

The regional tissue distribution volume (VT, mL g–1)was defined as the ratio of the ligand concentration ina region (CT, �Ci g–1) to the concentration of unmetabo-lized ligand in arterial plasma (CA, �Ci mL–1) at equi-librium:

VT �CT

CA. (1)

The concentration of D2 receptors is negligible in thecerebellum (Hall et al., 1994). Therefore, only free andnonspecifically bound radiotracer were considered tocontribute to VT in the cerebellum (VT CER), and VT CER

was assumed to be equal to the nondisplaceable distri-bution volume (V2). The striatal VT (VT STR) included V2

and the specific binding distribution volume, or bindingpotential (BP). Assuming that V2 was equal in bothregions, BP was derived as the difference between VT

STR and VT CER. Under the assumption of a competitiveinteraction between the radiotracer and DA, BP wasrelated to receptor parameters and endogenous DA by:

VT STR � VT CER � BP � f1 �BMAX

K�D� f1 �

BMAX

KD�1 �FDA

KI� , (2)

where f1 is as defined above, Bmax is the concentrationof D2 receptors (nanomoles per g of tissue), KD is the invivo equilibrium dissociation constant of the radio-tracer (nanomoles per ml of brain water), KD� is KD inthe presence of the competitor DA, FDA is the freeconcentration of endogenous DA in the vicinity of thereceptors, and KI is the inhibition constant of DA forthe binding of the radiotracer (Laruelle et al., 1994a,1998).

The preferred outcome measure for endogenous com-petition experiments is the equilibrium specific to non-

specific partition coefficient (V3�). V3� was calculated asthe ratio of BP to VT CER. V3� was related to receptorparameters and endogenous DA by:

VT STR � VT CER

VT CER� V�3 � f2 �

BMAX

K�D� f2 �

BMAX

KD�1 �FDA

KI� , (3)

where f2 is the free fraction in the nonspecific distribu-tion volume of the brain (f2 � f1/V2) (Laruelle et al.,1994a).

The relative change in V3� elicited by amphetamine(V3�) was calculated as the difference between V3�measured in the postamphetamine condition (V3�AMPH)and V3� measured in the baseline condition on that day(V3�BASE), and expressed in percentage of V3�BASE.

V �3 � 100 �V �3 AMPH � V�3 BASE

V�3 BASE(4)

Equation 3 shows that the increase in FDA resultingfrom the challenge, translates into a decrease in V3�.Equation 3 also indicates that the attribution ofchanges in V3� solely to changes in FDA requires theassumption that all other parameters of this equationremain constant, including the nonspecific binding (f2).The effect of amphetamine on CL, f1, f2, V2, and BP wasalso expressed relative to preamphetamine value mea-sured the same day.

Kinetic analysis

Kinetic derivation of [11C]raclopride and [11C]NPAVT were performed using kinetic analysis and the ar-terial input function. A one-tissue compartment model(1TCM) was used for the cerebellum for both tracers.[11C]Raclopride striatal VT was derived using a two-tissue compartment model (2TCM). [11C]NPA striatalVT was derived using a 1TCM, as previous analyses of[11C]NPA striatal uptake revealed that the 2TCM doesnot significantly improve the goodness of fit comparedto that of the 1TCM and produces less robust estimatesof striatal [11C]NPA VT (Hwang et al., 2004).

In the 1TCM, K1 (mL g–1 min–1) and k2 (min–1) arethe rate constants governing the transfer of the ligandsin and out of the brain, respectively. In the 2TCM, K1

and k2 are the rate constants governing the transfer ofthe ligands in and out of the nondisplaceable compart-ment (free and nonspecific binding), while k3 (min–1)and k4 (min–1) describe the rate of association anddissociation to and from the receptors, respectively.

In the 1TCM, VT was derived from kinetic parame-ters as:

VT �K1

k2. (5)

COMPARISON OF [11C]NPA AND [11C]RACLOPRIDE 191

In the 2TCM, VT was derived from kinetic parametersas:

VT �K1

k2�1 �

k3

k4� . (6)

For both [11C]raclopride and [11C]NPA, BP and V3�were derived using Eqs. 2 and 3, respectively. Kineticrate constants were derived by nonlinear regressionusing a Levenberg-Marquardt least-squares minimiza-tion procedure (Levenberg, 1944) implemented inMATLAB (MathWorks, Natick, MA) as previously de-scribed (Laruelle et al., 1994b). Given the unequal sam-pling over time (increasing frame acquisition time frombeginning to end of the study), the least-squares min-imization procedure was weighted by the length offrame acquisition time.

Amphetamine plasma levels

Amphetamine plasma levels were measured in onearterial sample obtained at 50 min after amphetamineinjection. Amphetamine was quantified as its N-hep-tafluorobutyryl derivative via gas chromatography/mass spectroscopy utilizing a capillary column withmass spectrometer with simultaneous ion monitoringin the negative chemical ionization mode and reactantgas methane/ammonia (95:5) (Reimer et al., 1993). Fortechnical reasons, amphetamine level was not obtainedin four experiments.

Statistical analysis

Five outcome measures were derived for each scan:CL, f1, V2, BP, and V3�.

The variability in baseline outcome measures wasevaluated first, testing for between-tracer and be-tween-animal differences. Between-tracer comparisonsof outcome measures under baseline condition wereperformed using repeated measured ANOVA (RMANOVA), with tracer as repeated condition, n � 9 pairsof observations). Between-baboon differences in out-come measure at baseline were evaluated with ANOVAfor each tracer (n � 3 per group).

Second, the effect of amphetamine on the outcomemeasures was evaluated for each tracer using RMANOVA, with the outcome measure as dependent vari-able, the baseline and postamphetamine conditions asrepeated condition, and the amphetamine dose as co-factor (n � 9 pairs of observations). The significancelevels of the condition and the dose*condition interac-tion were reported.

When a significant effect of amphetamine was ob-served for at least one of the two tracers, a third-levelanalysis was performed to test for between-tracer dif-ference in this amphetamine effect. This evaluationwas performed using RM ANOVA, with the amphet-amine effect on the outcome measure as dependent

variable (i.e., CL, f1, V2, BP, or V3�), the tracer asrepeated condition, and the baboon and amphetaminedose as cofactors (n � 9 pairs of observations). Thesignificance levels of the tracer condition, the dose fac-tor, the dose*condition interaction, the baboon factorand the baboon*condition interaction were reported(only first-order interactions were tested).

A two-tailed P � 0.05 was selected as the significancelevel for all tests.

RESULTS

Data were collected over an 18-month period. BaboonA was studied over a 16-month period, with an averageof 70 days between experiments (n � 8, six primaryexperiments and two repeat experiments). The experi-mental sequence for baboon A was as follows, where Nand R designate [11C]NPA and [11C]raclopride experi-ments, respectively, and 0.3, 0.5, and 1.0 designate thedoses of amphetamine (mg kg–1): N/0.5, N/0.5 (repeat),R/0.3, N/0.3, R/0.5, R/0.5 (repeat), R/1.0, N/1.0. BaboonB was studied over a 10-month period, with an averageof 58 days between experiments (n � 6), acquired un-der the following sequence: N/1.0, R/0.3, R/0.5, N/0.5,R/1.0, N/0.3. Baboon C was studied over a 4-monthperiod, with an average of 25 days between experi-ments (n � 6), acquired under the following sequence:N/0.5, R/0.5, N/0.3, R/0.3, R/1.0, N/1.0.

Dose–response studyInjected dose and masses

The mean injected dose for [11C]raclopride was4.83 � 0.65 mCi and 5.20 � 0.60 mCi for the baselineand postamphetamine conditions, respectively (RMANOVA, P � 0.07). The mean injected mass for [11C]ra-clopride was 1.68 � 1.00 �g and 2.71 � 2.50 �g for thebaseline and postamphetamine conditions, respectively(RM ANOVA, P � 0.13). Thus, in spite of a trend forhigher [11C]raclopride injected doses for the postam-phetamine condition in comparison to the baseline con-dition, there was no significant differences in the in-jected masses between the two conditions. Note that, ifanything, a larger mass during the postamphetaminescan would result in an overestimation of the amphet-amine effect on [11C]raclopride binding.

The mean injected dose for [11C]NPA was 3.71 � 1.17mCi and 3.89 � 1.33 mCi for the baseline (n � 9) andpostamphetamine (n � 9) conditions, respectively. Themean injected mass for [11C]NPA was 1.52 � 1.23 �gand 1.49 � 1.67 �g for the baseline and postamphet-amine conditions, respectively. [11C]NPA injecteddoses and masses for baseline and postamphetaminescans did not differ significantly (RM ANOVA, P � 0.31and P � 0.94, respectively).

Plasma clearance (CL)

Under baseline conditions, [11C]NPA plasma CL

(36.1 � 9.0 L h–1, n � 9) was significantly faster than

192 R. NARENDRAN ET AL.

[11C]raclopride plasma CL (20.9 � 4.7 L h–1, n � 9, RMANOVA, P � 0.004).

Amphetamine significantly increased [11C]racloprideCL, without a significant dose-by-condition interaction(24.7 � 5.7 L h–1, n � 9, RM ANOVA; [11C]racloprideCL as dependent variable; baseline vs. amphetaminecondition, P � 0.018; dose*condition interaction, P �0.13). Amphetamine did not significantly alter[11C]NPA CL (38.8 � 9.1 L h–1, n � 9, RM ANOVA;[11C]NPA CL as dependent variable; baseline vs. am-phetamine condition, P � 0.13; dose*condition interac-tion, P � 0.89).

The difference between [11C]raclopride CL (19 �23%) and [11C]NPA DCL (9 � 15%) did not reach sig-nificance (RM ANOVA; CL as dependent variable;tracer condition, P � 0.27; dose factor, P � 0.36;dose*tracer interaction, P � 0.43; baboon factor, P �0.91; baboon*tracer interaction, P � 0.45).

Plasma free fraction (f1)

Under baseline conditions, [11C]NPA f1 (5.3 � 0.7%,n � 9) was significantly lower than [11C]raclopride f1

(9.5 � 1.8%, n � 9, RM ANOVA, P 0.001). Between-baboon differences in f1 were observed at trend level,for both [11C]raclopride (ANOVA, P � 0.061) and[11C]NPA (ANOVA, P � 0.094).

Amphetamine did not significantly alter [11C]raclo-pride f1 (9.2 � 1.7%, n � 9, RM ANOVA; [11C]raclopridef1 as dependent variable; baseline vs. amphetaminecondition, P � 0.63; dose*condition interaction, P �0.15). Amphetamine did not significantly alter[11C]NPA f1 (5.9 � 1.1%, n �9, RM ANOVA; [11C]NPAf1 as dependent variable; baseline vs. amphetaminecondition, P � 0.14; dose*condition interaction, P �0.68).

Nondisplaceable distribution volume

Figures 1 and 2 show the cerebellar and striatal timeactivity curves measured in the same experiments un-der baseline and postamphetamine conditions, follow-ing injections of [11C]raclopride and [11C]NPA, respec-tively. Table I lists the values of [11C]raclopride and[11C]NPA cerebellar distribution volumes (V2) underbaseline and postamphetamine conditions in baboonsA, B, and C.

Under baseline conditions, [11C]NPA V2 (4.51 � 1.55mL g–1, n � 9) was significantly higher than [11C]ra-clopride V2 (0.69 � 0.32 mL g–1, n � 9, RM ANOVA,P 0.001). Significant between-baboon differenceswere noted in [11C]raclopride V2 (ANOVA, P 0.001),with baboon B showing lower V2 than the two otherbaboons. No significant between-baboon differences in[11C]NPA V2 were observed (ANOVA, P � 0.10).

Amphetamine did not significantly alter [11C]raclo-pride V2 (RM ANOVA; [11C]raclopride V2 as dependentvariable; baseline vs. amphetamine condition, P �

0.33; dose*condition interaction, P � 0.70). Amphet-amine significantly reduced [11C]NPA V2 (RM ANOVA;[11C]NPA V2 as dependent variable; baseline vs. am-phetamine condition, P � 0.013, dose*condition inter-action, P � 0.94).

The difference between [11C]raclopride V2 and[11C]NPA V2 did not reach significance (RM ANOVA;

Fig. 1. [11C]Raclopride time–activity curves normalized for theinjected dose in striatum (closed circles) and cerebellum (open circles)measured during the pair of experiments depicted in Fig. 3 (baboonA). A: Control experiment. B: Postamphetamine (0.5 mg kg–1) exper-iment. Points are measured values, lines are values fitted to a one-tissue compartment (cerebellum) or two-tissue compartment (stria-tum). In this pair of experiments, amphetamine reduced striatal[11C]raclopride V3� by 26%.

COMPARISON OF [11C]NPA AND [11C]RACLOPRIDE 193

V2 as dependent variable; tracer condition, P � 0.15;dose factor, P � 0.66; dose*tracer interaction; P � 0.31;baboon factor, P � 0.81; baboon*tracer interaction, P �0.81).

Binding potential (BP)

Table II lists the values of [11C]raclopride and[11C]NPA BP (mL g–1) under baseline and postamphet-amine conditions in baboons A, B, and C.

Under baseline conditions, [11C]NPA BP (5.73 � 3.11mL g–1, n � 9) was significantly higher than [11C]ra-clopride BP (2.17 � 1.36 mL g–1, n � 9, RM ANOVA,P � 0.001). Significant between-baboon differences

were noted in [11C]raclopride BP (ANOVA, P 0.001),with baboon B showing lower BP than the two otherbaboons. Significant between-baboon differences werenoted in [11C]NPA BP (ANOVA, P � 0.01), with baboonC showing higher BP than the two other baboons.

Amphetamine significantly reduced [11C]racloprideBP, with no significant dose effect (RM ANOVA;[11C]raclopride BP as dependent variable; baseline vs.amphetamine condition, P � 0.014, dose*condition in-teraction, P � 0.87). Amphetamine significantly re-duced [11C]NPA BP, with no significant dose effect (RMANOVA; [11C]NPA BP as dependent variable; baselinevs. amphetamine condition, P � 0.003, dose*conditioninteraction, P � 0.90).

When both tracers were included in the analysis, aneffect of dose on BP was observed at trend level.[11C]NPA BP was significantly higher than [11C]ra-clopride BP, and this effect was observed irrespectiveof the dose. No between-baboon differences or baboon-tracer interactions were noted (RM ANOVA; BP asdependent variable; tracer condition, P � 0.009; dosefactor, P � 0.08; dose*tracer interaction, P � 0.31;baboon factor, P � 0.20; baboon*tracer interaction, P �0.39).

The [11C]NPA BP to [11C]raclopride BP ratios aregiven in Table III. No significant differences in the[11C]NPA BP to [11C]raclopride BP ratios were ob-served between the doses (ANOVA, P � 0.99).

Specific to nonspecific equilibrium partitioncoefficient (V3�)

Figure 3 displays parametric [11C]raclopride and[11C]NPA V3� maps measured in the same baboon un-der baseline and postamphetamine (0.5 mg kg–1) con-ditions. Table IV lists the values of [11C]raclopride and[11C]NPA V3� under baseline and postamphetamineconditions in baboons A, B, and C.

Under baseline conditions, [11C]NPA V3� (1.21 �0.28, n � 9) was significantly lower than [11C]raclo-pride V3� (3.02 � 1.21, n � 9, RM ANOVA, P 0.001).Significant between-baboon differences were noted in[11C]raclopride V3� (ANOVA, P 0.001), with baboon Cshowing higher [11C]raclopride V3� than the two otherbaboons. Significant between-baboon differences werenoted in [11C]NPA V3� (ANOVA, P 0.001), with ba-boon C showing higher [11C]NPA V3� than the twoother baboons.

Amphetamine significantly reduced [11C]racloprideV3�, with no significant dose effect (RM ANOVA; V3� asdependent variable; baseline vs. amphetamine condi-tion, P 0.001, dose*condition interaction, P � 0.25).Amphetamine significantly reduced [11C]NPA V3�, withno significant dose effect (RM ANOVA; V3� as depen-dent variable; baseline vs. amphetamine condition, P 0.001; dose*condition interaction, P � 0.12).

With both tracers included in the analysis, a signif-icant effect of dose on V3� was observed. [11C]NPA

Fig. 2. [11C]NPA time–activity curves normalized for the injecteddose in striatum (closed circles) and cerebellum (open circles) mea-sured during the pair of experiments depicted in Fig. 3 (baboon A).A: Control experiment. B: Postamphetamine (0.5 mg kg–1) experi-ment. Points are measured values, lines are values fitted to a one-tissue compartment. In this pair of experiments, amphetamine re-duced striatal [11C]NPA V3� by 46%.

194 R. NARENDRAN ET AL.

V3� was significantly higher than [11C]racloprideV3�, and this effect was observed irrespective of thedose used (Fig. 4). No between-baboon differences orbaboon-tracer interactions were noted (RM ANOVA;V3� as dependent variable; tracer condition, P � 0.03;dose factor, 0.02; dose*tracer interaction, P � 0.82;baboon factor, P � 0.98; baboon*tracer interaction, P �0.83). After removal of the nonsignificant baboon fac-tor, the significance of the remaining factors was asfollows: tracer condition, P � 0.01; dose factor, P �0.005; dose*tracer interaction, P � 0.76.

The [11C]NPA V3� to [11C]raclopride V3� ratios aregiven in Table III. No significant differences in the[11C]NPA V3� to [11C]raclopride V3� ratios were ob-served between the doses (ANOVA, P � 0.72). The[11C]NPA V3� to [11C]raclopride V3� ratios were notsignificantly different from the [11C]NPA BP to[11C]raclopride BP ratios (RM ANOVA, amphetamine

effect as dependent variable; outcome measures as re-peated conditions, P � 0.73).

Plasma amphetamine levels

Amphetamine plasma levels were linearly correlatedwith dose (r2 � 0.95, P 0.001, n � 14). Doses of 0.3,0.5, and 1.0 mg kg–1 produced plasma levels of 73 � 12ng mL–1 (n � 5), 126 � 16 ng mL–1 (n � 5), and 270 �33 ng mL–1 (n � 4), respectively. No differences inamphetamine plasma levels were observed during the[11C]raclopride experiments compared to the [11C]NPAexperiments (RM ANOVA, tracer condition, P � 0.94).

Physiological parameters

No significant differences were observed in the meanexperimental systolic blood pressure, diastolic bloodpressure, heart rate, respiratory rate, and body tem-perature between the [11C]raclopride and [11C]NPAscans under baseline conditions (RM ANOVA, tracercondition; systolic blood pressure, P � 0.73; diastolicblood pressure, P � 0.75; heart rate, P � 0.08; respi-ratory rate P �0.80; body temperature, P � 0.54) andunder postamphetamine conditions (RM ANOVA, sys-tolic blood pressure, P � 0.78; diastolic blood pressure,P � 0.90; heart rate, P � 0.81; respiratory rate, P �0.44; body temperature, P � 0.72).

TABLE I. Effect of amphetamine on [11C]raclopride and [11C]NPA nondisplaceable distribution volume (V2, mL g�1)

Amphetaminedose Baboon

[11C]Raclopride V2 (mL g�1) [11C]NPA V2 (mL g�1)

Baseline scanPostamphetamine

scan V2 Baseline scanPostamphetamine

scan V2

0.3 A 0.70 0.75 6% 3.08 3.16 3%B 0.20 0.15 �26% 3.95 3.52 �11%C 1.00 0.87 �13% 6.27 5.02 �20%

Mean � SD 0.63 � 0.40 0.59 � 0.39 �11 � 16% 4.43 � 1.65 3.90 � 0.98 �9 � 11%0.5 A 0.81 0.81 0% 2.84 2.50 �12%

B 0.27 0.26 �5% 4.31 4.01 �7%C 0.99 0.83 �16% 7.74 6.91 �11%

Mean � SD 0.69 � 0.38 0.63 � 0.32 �7 � 8% 4.96 � 2.51 4.47 � 2.24 �10 � 2%1.0 A 0.75 0.82 9% 4.23 3.99 �6%

B 0.41 0.47 16% 4.37 3.39 �23%C 1.04 0.91 �12% 3.80 3.16 �17%

Mean � SD 0.73 � 0.31 0.74 � 0.23 4 � 14% 4.14 � 0.30 3.51 � 0.43�15 � 9%

TABLE II. Effect of amphetamine on [11C]raclopride and [11C]NPA binding potential (BP, mL g�1)

Amphetaminedose Baboon

[11C]Raclopride BP (mL g�1) [11C]NPA BP (mL g�1)

Baseline scanPostamphetamine

scan BP Baseline scanPostamphetamine

scan BP

0.3 A 1.75 1.52 �13% 2.78 2.01 �28%B 0.48 0.29 �38% 4.42 2.69 �39%C 3.68 2.08 �44% 10.57 5.61 �47%

Mean � SD 1.97 � 1.61 1.30 � 0.91 �32 � 16% 5.92 � 4.10 3.44 � 1.91 �38 � 10%0.5 A 1.72 1.27 �26% 2.89 1.39 �52%

B 0.72 0.44 �40% 4.27 2.10 �51%C 4.22 2.31 �45% 11.29 5.87 �48%

Mean � SD 2.22 � 1.80 1.34 � 0.94 �37 � 10% 6.15 � 4.50 3.12 � 2.40 �50 � 2%1.0 A 1.90 1.07 �44% 4.42 1.89 �57%

B 1.43 0.83 �42% 4.93 1.84 �63%C 3.68 2.16 �41% 5.98 2.38 �60%

Mean � SD 2.33 � 1.19 1.35 � 0.71 �42 � 1% 5.11 � 0.79 2.04 � 0.30 �60 � 3%

TABLE III. [11C]NPA to [11C]raclopride BP and V �3 ratios

Amphetaminedose n

[11C]NPA BP/[11C]raclopride BP

[11C]NPA V �3/[11C]raclopride V �3

0.3 3 1.40 � 0.61 1.46 � 0.450.5 3 1.44 � 0.48 1.42 � 0.301.0 3 1.42 � 0.09 1.24 � 0.28Average all doses 9 1.42 � 0.39 1.37 � 0.32

COMPARISON OF [11C]NPA AND [11C]RACLOPRIDE 195

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196 R. NARENDRAN ET AL.

Repeat experiments

For both tracers the experimental session involvingan amphetamine dose of 0.5 mg kg–1 was repeated onbaboon A. The repeat sessions with [11C]NPA and[11C]raclopride took place 14 and 42 days after the teststudies, respectively. Table V provides the values ofbaseline and postamphetamine outcome measures V2,BP, and V3� measured during the test and repeat ses-sions for each ligand. The reproducibility of V3� wasbetter than that of BP.

DISCUSSION

The fundamental aim of this study was to comparethe effect of amphetamine on [11C]NPA and [11C]raclo-pride in vivo specific binding. Experiments were per-formed in the same baboons, under carefully controlledconditions, and in counterbalanced sequence. The re-

sults demonstrate that the in vivo binding of [11C]NPAis more affected than that of [11C]raclopride by changesin endogenous DA. Under the experimental conditionsof this study, the amphetamine-induced increase insynaptic DA resulted in a 42% larger decrease in[11C]NPA binding compared to [11C]raclopride binding.

Rationale for a radiolabeled agonistas imaging agent

The decrease in [11C]raclopride or [123I]IBZM specificbinding following amphetamine administration hasbeen well validated as a noninvasive measure of thechange in DA extracellular levels produced by the chal-lenge. First, the decrease in ligand binding measuredwith PET or SPECT is linearly related to the increasein extracellular DA measured with microdialysis. Thisrelationship was demonstrated in nonhuman primatesfor both [123I]IBZM (Laruelle et al., 1997) and [11C]ra-clopride (Breier et al., 1997; Tsukada et al., 1999).Second, several studies confirmed that the decrease inligand binding is mediated by DA. DA depletion,achieved with alpha-methyl-para-tyrosine, resulted insignificant blunting of the effect of amphetamine on[123I]IBZM specific binding (Laruelle et al., 1997). Pre-treatment with the DA transporter inhibitorGBR12909 (an intervention known to attenuate am-phetamine-induced DA release) significantly reducedamphetamine-induced decrease in [11C]raclopride spe-cific binding (Villemagne et al., 1999). Finally, meth-amphetamine-induced decrease in striatal [11C]raclo-pride specific binding was considerably reduced inpatients with Parkinson’s disease (Piccini et al., 2003).Together, these data validate the use of amphetamine-induced reduction in [123I]IBZM or [11C]raclopride BPas an indirect measure of the changes in synaptic DAinduced by the challenge. This test has been success-fully used as a probe of presynaptic DA function inhealth and disease (Abi-Dargham et al., 1998; Anand etal., 2000; Breier et al., 1997; Laruelle et al., 1996;Parsey et al., 2001; Singer et al., 2002).

Two important characteristics of these endogenouscompetition studies with radiolabeled antagonists have

TABLE IV. Effect of amphetamine on [11C]raclopride and [11C]NPA specific to nonspecific equilibrium partition coefficient (V �3, unitless)

Amphetaminedose Baboon

[11C]Raclopride V �3 [11C]NPA V �3

Baseline scanPostamphetamine

scan V �3 Baseline scanPostamphetamine

scan V �3

0.3 A 2.50 2.04 �18% 0.90 0.64 �30%B 2.38 1.97 �17% 1.12 0.77 �32%C 3.68 2.38 �35% 1.69 1.12 �34%

Mean � SD 2.85 � 0.72 2.13 � 0.22 �24 � 10% 1.24 � 0.40 0.84 � 0.25 �32 � 2%0.5 A 2.12 1.57 �26% 1.02 0.56 �46%

B 2.65 1.69 �36% 0.99 0.53 �47%C 4.25 2.78 �35% 1.46 0.85 �42%

Mean � SD 3.01 � 1.11 2.01 � 0.66 �32 � 6% 1.16 � 0.26 0.64 � 0.18 �45 � 3%1.0 A 2.51 1.30 �48% 1.04 0.47 �55%

B 3.50 1.74 �50% 1.13 0.54 �52%C 3.55 2.36 �34% 1.57 0.75 �52%

Mean � SD 3.19 � 0.58 1.80 � 0.53 �44 � 9% 1.25 � 0.28 0.59 � 0.15 �53 � 2%

Fig. 4. Mean � SD [11C]raclopride V3� (open bars) and [11C]NPAV3� (closed bars) measured in the same three baboons followingvarious doses of amphetamine (0.3, 0.5, and 1 mg kg–1). Repeatedmeasure ANOVA revealed a significant tracer factor (P � 0.01), dosefactor (P � 0.005), and no significant dose*tracer interaction (P �0.76).

COMPARISON OF [11C]NPA AND [11C]RACLOPRIDE 197

emerged over the years. First, it has been demon-strated that relatively large increases in extracellularDA are associated with relatively modest effects on[123I]IBZM or [11C]raclopride binding (Breier et al.,1997; Laruelle et al., 1997; Tsukada et al., 1999). Sec-ond, only about half of radiolabeled antagonist in vivospecific binding appears to be vulnerable to endogenouscompetition. Irrespective of the nature of the challenge,the reduction in radiotracer binding seems to be lim-ited by a ceiling effect at about 40% (see table 7 inLaruelle, 2000).

Both phenomena might be related to the fact thatendogenous competition studies have been carried outso far with radiolabeled antagonists. D2 receptors areconfigured in interconvertible states of high or lowaffinity for agonists. The high-affinity sites (D2high

sites) are G-protein-coupled, whereas the low-affinitysites (D2low sites) are those uncoupled with G-proteins.In vitro, �50% of D2 receptors are configured in thehigh-affinity state (George et al., 1985a; Richfield et al.,1989b; Seeman and Grigoriadis, 1987; Sibley et al.,1982; Zahniser and Molinoff, 1978). Antagonists, suchas [123I]IBZM or [11C]raclopride, bind with equal affin-ity to both states. The agonist DA is not expected tocompete efficiently with [123I]IBZM or [11C]racloprideat the D2low sites. This factor would leave only about50% of the antagonist binding susceptible to endoge-nous competition by DA (and less than that if onestakes into account that a fraction of the high-affinitysites are already occupied by DA in the baseline state).This model suggests that the ideal radiotracer for en-dogenous competition studies would be a D2 receptoragonist (Laruelle, 2000). A radiolabeled agonist wouldpreferentially, if not exclusively, bind to the D2high

sites. Therefore, endogenous DA should be more potentat reducing the in vivo binding of a radiolabeled ago-nist.

Rationale for the selection of NPA

The development of [11C]NPA as a PET imagingagent provided the opportunity to test this model. NPAis a potent and full DA D2 receptor agonist. Adminis-tered in rodents, it induces a typical DA agonist re-sponse, i.e., increased locomotion and stereotypies (Gu-lat-Marnay et al., 1985; Menon et al., 1978; Yarbroughet al., 1984). These effects are unaltered by alpha-methyl-para-tyrosine pretreatment, which is consis-

tent with a direct agonist effect (Schoenfeld et al.,1975). At low doses, NPA inhibits locomotor response,which is consistent with a preferential stimulation ofautoreceptors at these doses (Argiolas et al., 1982;Yarbrough et al., 1984). NPA fully inhibits firing of theDA cells in the substantia nigra, an electrophysiologi-cal response typical of DA agonists and inhibited byhaldol (Martin et al., 1990; Yarbrough et al., 1984).NPA stimulates [35S]-GTP S binding in CHO cells ex-pressing D2 receptors, an agonist functional assay,with an intrinsic activity of 114% relative to DA (Gard-ner and Strange, 1998; Rinken et al., 1999). The bind-ing of NPA to D2 receptors is best fitted by a two-sitemodel, and the high-affinity sites are converted to low-affinity sites in the presence of GTP (Gardner andStrange, 1998; Grigoriadis and Seeman, 1985; Seemanet al., 1985). In vitro, the selectivity of NPA for D2high

vs. D2low sites has been reported as 96 in bovine ante-rior pituitary membranes (Sibley et al., 1982), 58 in ratstriatal membranes (Seeman et al., 1985), 664 in por-cine anterior pituitary membranes (George et al.,1985b), and 208 and 42 in CHO cells expressing humanD2 receptor (Gardner and Strange, 1998; Lahti et al.,1996). NPA affinity for D1 receptors ranges from 340nM to �1 �M, conferring appropriate D2/D1 selectivity(Andersen, 1988; Baldessarini et al., 1991; Ross andJackson, 1989; Sunahara et al., 1991; Tiberi et al.,1991). NPA affinity of 480 nM for adrenergic �2 recep-tors, and no appreciable affinity (IC50 � 1,000) for thefollowing receptors: adrenergic beta and �1 receptors,serotonin 5-HT1 and 5HT2, DA transporters, adenosinereceptors, glutamate receptors (NMDA, kainate, aspar-tate, glycine site), GABAA, GABAB, benzodiazepine re-ceptors, peptide receptors, or ion channel receptors(Baldessarini et al., 1994).

Besides these pharmacological properties, previousin vivo experiments using [3H]NPA suggested that thiscompound would provide not only a good imagingagent, but also an effective probe to report on fluctua-tions in endogenous DA. In rodents, reduction in DAlevels following reserpine, 6-OHDA, and -butyrolac-tone increased [3H]NPA in vivo binding (Cumming etal., 2002b; Ross and Jackson, 1989; van der Werf et al.,1983). Conversely, increases in DA levels following am-phetamine and methylphenidate significantly de-creased [3H]NPA in vivo binding (Cumming et al.,2002b; Kohler et al., 1981; Ross and Jackson, 1989).

TABLE V. Reproducibility of amphetamine effect on [11C]raclopride and [11C]NPA V2, BP and V �3

Ligand Session

V2 (mL g�1) BP (mL g�1) V �3 (unitless)

Baselinescan

Postamphetaminescan V2

Baselinescan

Postamphetaminescan BP

Baselinescan

Postamphetaminescan V �3

[11C]Raclopride Test 0.81 0.81 0% 1.72 1.27 �26% 2.12 1.57 �26%Repeat 0.72 0.76 6% 1.61 1.27 �21% 2.24 1.68 �25%

[11C]NPA Test 2.84 2.50 �12% 2.89 1.39 �52% 1.02 0.56 �46%Repeat 3.18 3.60 13% 3.25 1.92 �41% 1.02 0.53 �48%

198 R. NARENDRAN ET AL.

Based on this literature, NPA was selected for thedevelopment of a radiolabeled D2 receptor agonist PETimaging agent. The authors previously reported theradiolabeling procedure, as well as initial imaging re-sults (Hwang et al., 2000). In a second set of studies,they further characterized [11C]NPA as an imagingagent and developed a model-based method to derive[11C]NPA BP and V3� (Hwang et al., 2004). In the studyreported here, the effects of amphetamine on [11C]ra-clopride and [11C]NPA in vivo binding were comparedin baboons.

In our experience, [11C]NPA has been routinely pre-pared with a radiochemical yield of 2% at the end-of-synthesis with good specific activity (�1 mCi/nmol),radiochemical purity (�95%), and chemical purity(�90%). When stabilized with ascorbic acid at a con-centration of 0.1 mg/mL, the tracer solution in 10%ethanol in saline is stable for more than 2 h. In addi-tion, NPA has been tested at pharmacological doses inPhase II clinical trials in patients with schizophreniaand Parkinson’s disease (Cotzias et al., 1976; Moura-dian et al., 1991; Tamminga et al., 1986). Thus, the factthat that [11C]NPA can be reliably produced and thatNPA has been safely administered to humans makes ita interesting candidate radiotracer for human PETstudies.

Methodological considerations

This study was designed to establish the amphet-amine dose–effect relationship for both [11C]racloprideand [11C]NPA. Because of the between-animal variabil-ity in the magnitude of this effect (Laruelle et al.,1997), it was important to study the dose–responserelationship with both tracers in the same animals.This requirement induced the concern that tolerance orsensitization to the amphetamine effect might developduring the course of the study. To alleviate this con-cern, the amphetamine doses and radiotracers wereadministered in counterbalanced order.

To decrease the effects of between-experiment fac-tors that might affect D2 receptor availability measure-ments, a baseline scan was obtained on each experi-mental day, and the effects of amphetamine werecomputed relative to the baseline results obtained onthe same day. The arterial time–activity curve wasmeasured in each experiment, and outcome measureswere derived using kinetic modeling based on the ar-terial input function. The reasons for avoiding analyt-ical methods that do not require arterial input functionmeasurement to derive V3� were two-fold. First, thesemethods can be associated with V3�-dependent biasesthat have the potential to increase or decrease truebetween-conditions differences (Slifstein et al., 2000).Second, and foremost, the measure of the input func-tion made it possible to check the validity of the fun-damental assumption implied in the use of V3� as out-

come measure, i.e., the invariance of V2 betweenconditions.

V3� is the ratio of the specific (BP) to nondisplaceable(V2) distribution volumes at equilibrium (Eq. 3). Com-pared to BP, the ratio of specific binding to plasmaparent concentration at equilibrium (Eq. 2), V3� is lessaffected by experimental errors associated with themeasurement of the input function and might thus beassociated with a lower test–retest variability. In thisstudy, two experiments were repeated to sample thereproducibility of the amphetamine effects in the con-ditions of this study. The excellent reproducibility ofV3� observed here (Table V) was similar to that of[123I]IBZM V3� previously reported in baboons (Laru-elle et al., 1997), as well as to that of [123I]IBZM V3�previously reported in healthy human subjects (Keg-eles et al., 1999). As in previous studies, the lack ofsignificant changes in V3� between the first and sec-ond experiments suggests that one exposure to am-phetamine does not induce long-lasting potentiation orattenuation of this response. In this study, the repro-ducibility of BP was slightly lower, but nonethelessacceptable. The lower reproducibility of BP comparedto V3� supported the choice of V3� as the primaryoutcome measure for these experiments.

Yet, to exclusively ascribe changes in V3� to changesin receptor parameters (Bmax/KD) implies that V2 is notaffected by the experimental factors under study (Eq.3), a reasonable assumption for most within-subjectdesigns. Similarly, the use of BP as defined here im-plies that f1 is invariant (Eq. 2). Amphetamine did notsignificantly affect [11C]raclopride V2, but induced asignificant decrease in [11C]NPA V2. This result wasunexpected, and the mechanisms by which amphet-amine might affect the nonspecific binding of [11C]NPAare unclear. If replicated, this phenomenon might limitthe usefulness of [11C]NPA used in combination withamphetamine. More importantly, this observation im-plied that the use of V3� to quantify the change in[11C]NPA Bmax/KD induced by amphetamine was asso-ciated with an underestimation of the true effect (Eq.3). On the other hand, no significant effect of amphet-amine on f1 was detected, which validated the use ofBP as an unbiased outcome measure to quantify thechange in Bmax/KD induced by amphetamine (Eq. 2).

In conclusion, the use of BP as primary outcomemeasure in this study was preferable because it wasunbiased, while the use of V3� was preferable becauseit was more reproducible. Given the results, the choicewas not critical, as the use of either outcome measureled to similar conclusions, namely, that the effect ofamphetamine on [11C]NPA Bmax/KD was larger than on[11C]raclopride Bmax/KD. Because V3� is the preferredoutcome measure in the endogenous competition liter-ature, the remainder of the discussion will primarilyfocus on this measure.

COMPARISON OF [11C]NPA AND [11C]RACLOPRIDE 199

[11C]Raclopride �V3�: comparisonwith previous results

The magnitude of [11C]raclopride V3� observed inthis study (–24%, –32%, and –44% at 0.3, 0.5, and 1 mgkg–1, respectively) was consistent with previous obser-vations in baboons, although some variability is notedin the literature. Price et al. (1998) reported [11C]raclo-pride V3� of –29% (n � 1) and –39% (n � 1) followingamphetamine doses of 0.6 and 1.6 mg kg–1, respec-tively. Drevets et al. (1999) reported [11C]racloprideV3� in the putamen of –11% (n � 4), –17% (n � 4), and–29% (n � 4) in baboons following amphetamine dosesof 0.3, 0.6, and 1 mg kg–1, respectively. In rhesus mon-keys, Carson et al. (1997) reported [11C]raclopride V3�of –10% (n � 4) and –21% (n � 4) following doses of 0.2and 0.4 mg kg–1, respectively. The [11C]raclopride V3�observed in this study is also similar to the [123I]IBZMV3� previously observed in baboons: –20 � 5%, –28 �8%, and –38 � 11% following amphetamine doses of0.3, 0.5, and 1 mg kg–1, respectively (n � 3 per dose)(Laruelle et al., 1997).

The [11C]raclopride V3� observed in anaesthetizedbaboons at the dose of amphetamine most often used inclinical studies (–24% at 0.3 mg kg–1) was about twicethe [11C]raclopride V3� observed in healthy humansfollowing the same dose of amphetamine. For example,Martinez et al. (2003) observed a [11C]raclopride V3�of –10.3 � 7.2% in 14 healthy controls following am-phetamine 0.3 mg kg–1. Similar results were reportedby Drevets et al. (2001). The difference between ba-boons and humans might be due, at least partly, tospecies differences in amphetamine disposition. In thisstudy, plasma amphetamine levels, measured 50 minfollowing amphetamine 0.3 mg kg–1, were 73 � 12 ngmL–1. Plasma amphetamine levels measured in hu-mans, using the same laboratory method, at 40 minfollowing injection of the same per kilo dose, were sig-nificantly lower (40 � 13 ng mL–1, n � 14; Martinez etal., 2003).

[11C]NPA �V3�: comparison with[11C]raclopride �V3�

In this study, the effect of amphetamine was signif-icantly larger on [11C]NPA binding than on [11C]raclo-pride binding. This result was observed using both BPand V3� as outcome measure, and was expressed bycalculating the [11C]NPA to [11C]raclopride BP andV3� ratios (Table III). Importantly, these ratios wereconstant across amphetamine doses, an observationconsistent with the model developed in the Appendix.The small decrease in [11C]NPA V2 observed followingamphetamine, especially at the large dose, implies that[11C]NPA V3� is slightly underestimated. For this rea-son, the values derived with BP were selected as ref-erence values, and it was concluded that the effect ofamphetamine on [11C]NPA specific binding was largerthan on [11C]raclopride specific binding by 42%.

The NPA V3� observed in this study were alsohigher than the [123I]IBZM V3� previously reported inbaboons (Laruelle et al., 1997). Figure 5 illustrates theeffect of amphetamine on V3� of the agonist [11C]NPA(Khigh � 0.2 nM) and of the antagonists [11C]raclopride(KD � 1.2 nM) and [123I]IBZM (KD � 0.4 nM). At eachdose, [11C]NPA V3� was larger than [11C]racloprideV3� and [123I]IBZM V3�. Conversely, the effect ofamphetamine on both antagonists was similar. Theseresults imply that the pharmacological property of theligand (agonist vs. antagonist) is more important thanits affinity as a predictor of V3�.

In the context of a simple competition model, thisobservation means that DA is more potent at inhibiting[11C]NPA binding compared to [11C]raclopride binding(in other terms, that the in vivo IC50 of DA for[11C]NPA binding is lower than that for [11C]raclopridebinding). The IC50 of DA is related to the KI of DA by:

KI �IC50

�1 �L

KD� , (7)

where L is the free concentration of the radiotracer andKD is the affinity of the radiotracer. Since both radio-ligands are given at tracer doses, L is much smaller

Fig. 5. Effect of amphetamine on V3� for [11C]NPA (triangles, thisstudy), [11C]raclopride (circles, this study) and [123I]IBZM (squares,data from Laruelle et al., 1997). Each point is the mean of threeexperiments in baboons. For clarity, the error bars are omitted.Curves are fit to a saturation curve, V3� � (Emax*dose)/ED50�dose.This figure illustrates that the effect of amphetamine on V3� of theagonist [11C]NPA (Khigh � 0.2 nM) is larger than its effect on V3� of theantagonists [11C]raclopride (KD � 1.2 nM) and [123I]IBZM (KD � 0.4nM). Conversely, the effect of amphetamine on both antagonists issimilar. The results imply that the pharmacological property of theligand (agonist vs. antagonist) is more important than its affinity aspredictor of V3�.

200 R. NARENDRAN ET AL.

than KD, the term L/KD tends toward zero, and the IC50

of DA is equal to the KI of DA. The difference in DAIC50 between the two ligands implies that these ligandsbind to sites that differ in terms of their affinity for DA.Because both DA and [11C]NPA are agonists, the sim-plest explanation is that these two agonists preferen-tially bind to D2high sites, whereas [11C]raclopridebinds with equal affinity to all D2 receptors, irrespec-tive of their G-protein-coupling status.

The results of this study are consistent with recentexperiments in mice that indicated that the in vivobinding of [3H]NPA was more vulnerable to endoge-nous competition by DA compared to that of [11C]raclo-pride (Cumming et al., 2002a). In mice, amphetamine(10 mg kg–1, i.p.) reduced [3H]NPA and [11C]raclopridebinding by 44% and 24% compared to saline-treatedmice, respectively. Thus, in mice, the amphetamine[3H]NPA to [11C]raclopride V3� ratio is 44/24 � 1.80, avalue higher than that observed here in baboons (1.42).

It should be noted that, at the time of initiation ofthis study, little information was available about theminimum scanning time needed to derive time-invari-ant parameters with [11C]NPA. Therefore, a prolongedscanning time (90 min) was selected for this study.Subsequent characterization of [11C]NPA revealed that50 min of scanning time was adequate to derive time-invariant parameters in baboons (Hwang et al., 2004).To verify that the difference in the amphetamine effectmeasured between [11C]raclopride and [11C]NPA wasnot an artifactual result due to the scanning time dif-ference (60 min for [11C]raclopride vs. 90 min for[11C]NPA), the [11C]NPA data were also analyzed usingonly the first 60 min of data. This analysis yieldedresults similar to the one presented in this article. Forexample, the comparison of V3� for [11C]racloprideand [11C]NPA (using 60 min of data for both) providedthe following results: tracer condition, P � 0.02; dosefactor, P � 0.006; dose*tracer interaction, P � 0.72.

Ceiling effect

Another important result of this study is that, like[11C]raclopride V3�, [11C]NPA V3� appears to be as-sociated with a ceiling effect. The increase in DA ex-tracellular concentration following 1 mg kg–1 of am-phetamine is close to the maximal release that can beelicited in the brain; further increase in the dose ofamphetamine does not translate into a substantial fur-ther increase in DA extracellular concentration (Laru-elle et al., 1997). Following this dose of amphetamine,the average synaptic concentration of DA should reachthe micromolar range, concentrations that are an orderof magnitude higher than the affinity of DA for D2high

sites, which is in the 5–50 nM range (Endres et al.,1997; Richfield et al., 1986, 1989a; Seeman, 1993; Sib-ley et al., 1982). Yet a significant proportion of sitesremained available to the binding of [11C]NPA follow-ing amphetamine 1 mg kg–1 dose (about 40%).

This result might indicate that a substantial portionof [11C]NPA binding corresponds to binding to D2low

sites. This scenario is unlikely, given a 50-fold D2high

vs. D2low selectivity. More likely, this result suggeststhat a substantial proportion of D2high is protected fromsignificant occupancy by DA in vivo. These protectedsites might correspond to internalized receptors, al-though whether or not internalized D2 receptors areable to couple to G-proteins is unclear. Alternatively,these sites might be localized far from DA terminals, aproposition supported by electron microscopy studies(Hersch et al., 1995; Levey et al., 1993; Yung et al.,1995). Given these uncertainties, D2 receptors belong-ing to this protected pool will be simply designated hereas nonsynaptic D2 receptor sites.

Extended D2 receptor model

The model of D2 receptor in vivo pharmacologicalstates previously proposed (Laruelle, 2000) has to berefined in the light of the data of the present study. Theprevious model, as well as the updated model, arepresented in Figure 6. In the previous model, it wasassumed that 50% of D2 receptors were present inD2high and D2low states, an estimate derived from invitro measurements. Accounting for the fact that 10%of the total receptor pool were already occupied by DAin the baseline state (Abi-Dargham et al., 2000), 90% ofD2 receptors were available to [11C]raclopride binding(40% being D2high sites, 50% being D2low sites). Underthis model, 44% (40/90) of [11C]raclopride bindingwould be susceptible to competition by endogenous DA.This number was consistent with the data available atthe time, and was confirmed in this study, where themaximal effect of amphetamine on [11C]raclopride V3�was –44 � 9% (Table IV).

This model made two predictions for the results ofthe present study. First, it predicted that 100% of[11C]NPA binding would be susceptible to competitionby endogenous DA. Second, it predicted that, at anygiven dose of amphetamine, [11C]NPA V3� would be2.25 times larger than [11C]raclopride V3� (see Appen-dix for the derivation of this prediction). These predic-tions were not confirmed here. First, it was observedthat 60% rather than 100% of [11C]NPA binding wassusceptible to competition by endogenous DA. Second,the [11C]NPA to [11C]raclopride V3� ratio was 1.42rather than 2.25.

Integrating both observations, and accounting forthe fact that 10% of the receptors are occupied by DA atbaseline leads to the following estimates (see Appen-dix): 73% of the receptors are D2high sites; 10% of thereceptors are D2high sites that are occupied by endoge-nous DA at baseline; 38% are D2high sites not occupiedby endogenous DA at baseline and vulnerable to occu-pancy by endogenous DA (“synaptic”), 25% are D2high

sites that are protected from significant occupancy byendogenous DA (“nonsynaptic”), and 27% are in the

COMPARISON OF [11C]NPA AND [11C]RACLOPRIDE 201

low-affinity state (the proportion of synaptic vs. non-synaptic low-affinity sites being unknown). This modelrests on several assumptions, which are listed in theAppendix. The most critical assumptions are that theoccupancy of D2 receptors by [11C]NPA is negligible(tracer conditions), and that the fraction of [11C]NPAbinding corresponding to the low-affinity state is neg-ligible. Both of these assumptions are testable by sat-uration studies.

In addition, the model makes it possible to estimatethe in vivo affinity of [11C]NPA for D2high sites in thepresence of baseline levels of DA (Khigh�). This deriva-tion is based on the observed values of [11C]NPA and[11C]raclopride BP in this study. Under the assumptionthat, in tracer conditions, the binding of [11C]NPA toD2low is negligible, Eq. 2 can be rewritten for [11C]NPAas

BPNPA � f1 �Rhigh

K�high, (8)

where Rhigh is the concentration of D2high sites. In thisdataset, the average [11C]raclopride Bmax/KD� ratio was23 (calculated as BP/f1, or 2.17/0.095, Eq. 2) and theaverage [11C]NPA Rhigh/K�high ratio was 109 (5.73/0.053). From these numbers, the following equivalenceis produced:

4.74 �Bmax

K�D RAC�

Rhigh

K�high NPA(9)

Substituting in Eq. 9 [11C]raclopride KD� by its value (1nM, Kohler et al., 1985) and Rhigh by 0.73Bmax, pro-duces a K�high value for NPA of 0.15 nM. This K�high

value is consistent with that reported in vitro (Gardnerand Strange, 1998; George et al., 1985b; Lahti et al.,1996; Seeman et al., 1985; Sibley et al., 1982). Satura-tion experiments with [11C]NPA are warranted to testthis prediction.

Clinical implications

The use of [11C]NPA in endogenous competition stud-ies would potentially provide two critical benefits com-pared to [11C]raclopride. First, because [11C]NPA ismore sensitive than [11C]raclopride to endogenous com-petition, it might provide a superior probe to report onacute changes in synaptic DA concentration. Second, asthe baseline binding of [11C]NPA is affected by theaffinity for agonists of the available receptors, the useof [11C]NPA might enable researchers to control forthis important parameter in clinical studies.

In healthy humans, [11C]raclopride striatal V3� fol-lowing amphetamine 0.3 mg kg–1 is –10.3 � 7.2% (Mar-tinez et al., 2003). The test/retest variability of [11C]ra-clopride striatal V3� measurement is � 6.3% � 4.1%(Mawlawi et al., 2001). Therefore, the signal-to-noiseratio (SNR) of the amphetamine effect on [11C]raclo-pride V3� is 10.3/6.3 � 1.63, resulting in significantuncertainties being associated with individual mea-surements of V3� and in reduced power for clinicalstudies. Assuming that the increased sensitivity of[11C]NPA to endogenous competition observed here ispredictive of the situation in humans, the same dose ofamphetamine would induce a 10.3*1.42 � 14.6% reduc-tion in [11C]NPA V3�, and an improved SNR from 1.6 to2.3. Thus, studies with [11C]NPA might be associatedwith enhanced accuracy in the measurement of theamphetamine effect and in improved power to detectbetween-group differences.

On the other hand, [11C]NPA V3� itself is lower than[11C]raclopride V3� (Table IV). It follows that the test/retest variability of [11C]NPA V3� measurement mightbe higher than that of [11C]raclopride V3�. A compari-son of the test/retest variability of [11C]raclopride and[11C]NPA V3� measurements is thus required before

Fig. 6. Original (A) and extended (B) pharmacological model of D2receptors in vivo, as derived from the data of endogenous competitionexperiments. The original model, proposed by Laruelle (2000), as-sumed that D2 receptors are divided into two affinity states for ago-nists (D2high and D2low sites), with each site contributing to 50% of thepopulation. Under baseline conditions (prior to the challenge), 10% ofthe sites are occupied by DA, and not available to [11C]raclopride. Itfollows that D2 receptors are divided into three compartments: D2highalready occupied by DA (10% of total D2 receptors, black box), D2highnot occupied by DA at baseline (40%, shaded box), and D2high (50%,white box). Thus, 90% of the sites are available to [11C]raclopridebinding. Of these, only 44% (40/90) are vulnerable to occupancy by DAfollowing the challenge. The extended model is derived from theresults of the present study. As in the original model, D2 receptors aredivided between D2high and D2low sites and it is assumed that 10% ofthe D2high sites are already occupied by DA at baseline. A fraction(40%) of D2high sites appears to be protected from significant occu-pancy by DA, so that D2 receptors are further divided into synapticand nonsynaptic compartments. The differential effect of amphet-amine on [11C]NPA and [11C]raclopride observed here (0.70) suggeststhat 71% of the receptors are D2high (Eq. 29). Thus, the followingproportions are calculated for the extended model: 10% of the recep-tors are in Rhigh and occupied by DA at baseline (black box); 38% areRhigh, and vulnerable to endogenous competition (synaptic compart-ment, shaded box); 25% are Rhigh, and protected from significantoccupancy by endogenous DA (nonsynaptic compartment, white boxon the right); 27% are Rlow (white box on top). The proportion ofsynaptic vs. nonsynaptic Rlow cannot be calculated. See the Appendixfor mathematical derivation of the model and assumptions.

202 R. NARENDRAN ET AL.

adopting [11C]NPA for endogenous competition studies.The present study was not designed for this purpose.Yet, since baseline V3� was measured three times ineach animal, the coefficient of variation (%CV) of thismeasurement can be computed. No significant differ-ences were observed between the reproducibility ofbaseline [11C]raclopride V3� (%CV of 12.8% � 6.7%) andthat of baseline [11C]NPA V3� (6.9% � 0.4%, P � 0.20).The determination of the reproducibility of [11C]NPAmeasurements in humans is needed to further addressthis point.

The second potential benefit of [11C]NPA is at thelevel of the interpretation of the data. In endogenouscompetition studies, changes in endogenous DA aremeasured through their effects on D2 receptor avail-ability. Using [11C]raclopride or [123I]IBZM, between-subject differences in affinity of D2 receptors for DA atbaseline are not distinguishable from between-condi-tions changes in DA release. For example, three studiesreported that the reduction in [123I]IBZM or [11C]raclo-pride binding induced by amphetamine was elevated inschizophrenia (Abi-Dargham et al., 1998; Breier et al.,1997; Laruelle et al., 1996). In theory, increased dis-placement of [123I]IBZM or [11C]raclopride by DAmight result from larger increase in DA synaptic con-centration following amphetamine (i.e., presynapticfactors), from increased affinity of D2 receptors for DA(i.e., postsynaptic factors), or from some combination ofpre- and postsynaptic mechanisms.

According to the original model (Fig. 6), the use of[11C]NPA would have enabled the investigators to re-solve this issue, as changes in affinity of availablereceptors for agonists will translate to changes in base-line [11C]NPA binding. For example, should the highereffect of amphetamine on [11C]raclopride in schizophre-nia be secondary to a higher proportion of D2high sites,the baseline binding of [11C]NPA would be increasedrelative to the baseline [11C]raclopride binding in thiscondition. However, according to the extended model,the use of [11C]NPA (Fig. 6) will not enable the inves-tigators to completely resolve the pre- vs. postsynapticissue. If, as the data suggest, a proportion of D2high

sites are not accessible to DA (nonsynaptic compart-ment), between-conditions changes in the amphet-amine effect could still be due to changes in the distri-bution of D2high sites between synaptic andnonsynaptic compartments. It is thus important toidentify the nature of the factors that seem to protectsome of the D2high sites from significant occupancy byDA released from the terminals.

CONCLUSION

In this study, the effect of amphetamine on the invivo binding of the radiolabeled D2 receptor agonist[11C]NPA and of the D2 receptor antagonist [11C]raclo-pride were compared with PET in three baboons. Threedoses of amphetamine were studied (0.3, 0.5, and 1 mg

kg–1). At each dose, the reduction of [11C]NPA bindingwas significantly larger than that of [11C]raclopridebinding. The magnitude of this difference was compa-rable at each dose and averaged 42%. The higher po-tency of DA at reducing [11C]NPA compared to [11C]ra-clopride binding is consistent with the pharmacologicalproperties of these ligands (agonist vs. antagonist). The42% difference between [11C]NPA V3� and [11C]raclo-pride V3� suggests that 73% of D2 receptors wereconfigured in states of high affinity for agonists. Asubstantial proportion of D2 receptor sites remainedavailable to [11C]NPA binding after the highest dose ofamphetamine, suggesting that about 40% of D2high

sites are protected from occupancy by DA, maybe be-cause of nonsynaptic localization. Overall, the resultsof this study support the proposition that [11C]NPA is asuperior PET radioligand to investigate changes insynaptic DA following pharmacological or behavioralchallenges. Studies in humans are needed to confirmthe results of the baboon studies reported here.

ACKNOWLEDGMENTS

The authors thank Richard Carson, Ph.D., for in-sightful comments, and acknowledge the superb tech-nical assistance of Jennifer Bae, John Castillon, RanoChatterjee, Elizabeth Hackett, Kimchung Ngo, VanPhan, Nurat Quadri, Lyudmila Savenkova, HarryAcosta, and Stanley Dicks.

REFERENCES

Abi-Dargham A, Laruelle M, Seibyl J, Rattner Z, Baldwin RM, ZoghbiSS, Zea-Ponce Y, Bremner JD, Hyde TM, Charney DS, Hoffer PB,Innis RB. 1994. SPECT measurement of benzodiazepine receptorsin human brain with [123-I]iomazenil: kinetic and equilibrium par-adigms. J Nucl Med 35:228–238.

Abi-Dargham A, Gil R, Krystal J, Baldwin R, Seibyl J, Bowers M, vanDyck C, Charney D, Innis R, Laruelle M. 1998. Increased striataldopamine transmission in schizophrenia: confirmation in a secondcohort. Am J Psychiatry 155:761–767.

Abi-Dargham A, Simpson N, Kegeles L, Parsey R, Hwang DR, AnjilvelS, Zea-Ponce Y, Lombardo I, Van Heertum R, Mann JJ, Foged C,Halldin C, Laruelle M. 1999. PET studies of binding competitionbetween endogenous dopamine and the D1 radiotracer [11C]NNC756. Synapse 32:93–109.

Abi-Dargham A, Rodenhiser J, Printz D, Zea-Ponce Y, Gil R, KegelesL, Weiss R, Cooper T, Mann JJ, Van Heertum R, Gorman J, Laru-elle M. 2000. Increased baseline occupancy of D2 receptors by do-pamine in schizophrenia. Proc Natl Acad Sci USA 97:8104–8109.

Anand A, Verhoeff P, Seneca N, Zoghbi SS, Seibyl JP, Charney DS,Innis RB. 2000. Brain SPECT imaging of amphetamine-induceddopamine release in euthymic bipolar disorder patients. Am J Psy-chiatry 157:1108–1114.

Andersen PH. 1988. Comparison of the pharmacological characteris-tics of [3H]raclopride and [3H]SCH 23390 binding to dopaminereceptors in vivo in mouse brain. Eur J Pharmacol 146:113–120.

Andersen PH, Nielsen EB. 1986. The benzazepine, SCH 23390, inhib-its 3H-NPA binding in mouse brain in vivo. Acta Pharmacol Toxicol59:315–318.

Argiolas A, Mereu G, Serra G, Melis MR, Fadda F, Gessa GL. 1982.N-n-propyl-norapomorphine: an extremely potent stimulant of do-pamine autoreceptors. Brain Res 231:109–116.

Baldessarini RJ, Kula NS, Gao Y, Campbell A, Neumeyer JL. 1991.R(–)2-fluoro-N-n-propylnorapomorphine: a very potent and D2-se-lective dopamine agonist. Neuropharmacology 30:97–99.

Baldessarini RJ, Kula NS, Zong R, Neumeyer JL. 1994. Receptoraffinities of aporphine enantiomers in rat brain tissue. Eur J Phar-macol 254:199–203.

COMPARISON OF [11C]NPA AND [11C]RACLOPRIDE 203

Breier A, Su TP, Saunders R, Carson RE, Kolachana BS, deBar-tolomeis A, Weinberger DR, Weisenfeld N, Malhotra AK, EckelmanWC, Pickar D. 1997. Schizophrenia is associated with elevatedamphetamine-induced synaptic dopamine concentrations: evidencefrom a novel positron emission tomography method. Proc Natl AcadSci USA 94:2569–2574.

Brix G, Zaers J, Adam LE, Bellemann ME, Ostertag H, Trojan H,Haberkorn U, Doll J, Oberdorfer F, Lorenz WJ. 1997. Performanceevaluation of a whole-body PET scanner using the NEMA protocol.National Electrical Manufacturers Association. J Nucl Med 38:1614–1623.

Carson RE, Breier A, deBartolomeis A, Saunders RC, Su TP, SchmallB, Der MG, Pickar D, Eckelman WC. 1997. Quantification of am-phetamine-induced changes in [C-11]raclopride binding with con-tinuous infusion. J Cereb Blood Flow Metab 17:437–447.

Cotzias GC, Papavasiliou PS, Ginos JZ. 1976. Therapeutic approachesin Parkinson’s disease: possible roles of growth hormone and soma-tostatin. Res Publ Assoc Res Nerv Ment Dis 55:305–315.

Cumming P, Wong DF, Dannals RF, Gillings N, Hilton J, Scheffel U,Gjedde A. 2002a. The competition between endogenous dopamineand radioligands for specific binding to dopamine receptors. AnnNY Acad Sci 965:440–450.

Cumming P, Wong DF, Gillings N, Hilton J, Scheffel U, Gjedde A.2002b. Specific binding of [(11)C]raclopride and N-[(3)H]propyl-norapomorphine to dopamine receptors in living mouse striatum:occupancy by endogenous dopamine and guanosine triphosphate-free G protein. J Cereb Blood Flow Metab 22:596–604.

Dewey SL, Smith GS, Logan J, Brodie JD, Fowler JS, Wolf AP. 1993.Striatal binding of the PET ligand 11C-raclopride is altered by drugsthat modify synaptic dopamine levels. Synapse 13:350–356.

Drevets WC, Price JC, Kupfer DJ, Kinahan PE, Lopresti B, Holt D,Mathis C. 1999. PET measures of amphetamine-induced dopaminerelease in ventral versus dorsal striatum. Neuropsychopharmacol-ogy 21:694–709.

Drevets WC, Gautier C, Price JC, Kupfer DJ, Kinahan PE, Grace AA,Price JL, Mathis CA. 2001. Amphetamine-induced dopamine re-lease in human ventral striatum correlates with euphoria. BiolPsychiatry 49:81–96.

Endres CJ, Kolachana BS, Saunders RC, Su T, Weinberger D, BreierA, Eckelman WC, Carson RE. 1997. Kinetic modeling of [C-11]raclo-pride: combined PET-microdialysis studies. J Cereb Blood FlowMetab 17:932–942.

Gandelman MS, Baldwin RM, Zoghbi SS, Zea-Ponce Y, Innis RB.1994. Evaluation of ultrafiltration for the free fraction determina-tion of single photon emission computerized tomography (SPECT)radiotracers: �-CIT, IBF and iomazenil. J Pharmaceutical Sci 83:1014–1019.

Gardner B, Strange PG. 1998. Agonist action at D2(long) dopaminereceptors: ligand binding and functional assays. Br J Pharmacol124:978–984.

George SR, Watanabe M, Di Paolo T, Falardeau P, Labrie F, SeemanP. 1985a. The functional state of the dopamine receptor in theanterior pituitary is in the high affinity form. Endocrinology 117:690–697.

George SR, Watanabe M, Seeman P. 1985b. Dopamine D2 receptors inthe anterior pituitary: a single population without reciprocal antag-onist/agonist states. J Neurochem 44:1168–1177.

Ginovart N, Farde L, Halldin C, Swahn CG. 1997. Effect of reserpine-induced depletion of synaptic dopamine on [C-11]raclopride bindingto D-2-dopamine receptors in the monkey brain. Synapse 25:321–325.

Goldman ME, Kebabian JW. 1984. Aporphine enantiomers. Interac-tions with D-1 and D-2 dopamine receptors. Mol Pharmacol 25:18–23.

Grigoriadis D, Seeman P. 1985. Complete conversion of brain D2dopamine receptors from the high- to the low-affinity state fordopamine agonists, using sodium ions and guanine nucleotide.J Neurochem 44:1925–1935.

Gulat-Marnay C, Lafitte A, Schwartz JC, Protais P. 1985. Effects ofdiscriminant and non-discriminant dopamine antagonists on invivo accumulation of 3H-N-propyl-norapomorphine in mouse stria-tum and tuberculum olfactorium. Naunyn Schmied Arch Pharma-col 329:117–122.

Hall H, Sedvall G, Magnusson O, Kopp J, Halldin C, Farde L. 1994.Distribution of D1- and D2-dopamine receptors, and dopamine andits metabolites in the human brain. Neuropsychopharmacology 11:245–256.

Hersch SM, Ciliax BJ, Gutekunst CA, Rees HD, Heilman CJ, YungKK, Bolam JP, Ince E, Yi H, Levey AI. 1995. Electron microscopicanalysis of D1 and D2 dopamine receptor proteins in the dorsalstriatum and their synaptic relationships with motor corticostriatalafferents. J Neurosci 15:5222–5237.

Hwang D, Kegeles LS, Laruelle M. 2000. (–)-N-[(11)C]propyl-norapo-morphine: a positron-labeled dopamine agonist for PET imaging ofD(2) receptors. Nucl Med Biol 27:533–539.

Hwang DR, Narendran R, Huang Y, Slifstein M, Talbot P, Ngo K,Sudo Y, Van Berckel BNM, Kegeles L, Martinez D, Laruelle M.2003. Quantitative analysis of (–)-N-[11C]propyl-norapomorphine([11C]NPA) in vivo binding in nonhuman primates. J Nucl Med45:338–346.

Innis RB, Malison RT, Al-Tikriti M, Hoffer PB, Sybirska EH, SeibylJP, Zoghbi SS, Baldwin RM, Laruelle MA, Smith E, Charney DS,Heninger G, Elsworth JD, Roth RH. 1992. Amphetamine-stimu-lated dopamine release competes in vivo for [123I]IBZM binding tothe D2 receptor in non-human primates. Synapse 10:177–184.

Kegeles LS, Zea-Ponce Y, Abi-Dargham A, Rodenhiser J, Wang T,Weiss R, Van Heertum RL, Mann JJ, Laruelle M. 1999. Stability of[123I]IBZM SPECT measurement of amphetamine-induced striataldopamine release in humans. Synapse 31:302–308.

Kohler C, Fuxe K, Ross SB. 1981. Regional in vivo binding of [3H]N-propylnorapomorphine in the mouse brain. Evidence for labelling ofcentral dopamine receptors. Eur J Pharmacol 72:397–402.

Kohler C, Hall H, Ogren SO, Gawell L. 1985. Specific in vitro and invivo binding of 3H-raclopride. A potent substituted benzamide drugwith high affinity for dopamine D-2 receptors in the rat brain.Biochem Pharmacol 34:2251–2259.

Lahti RA, Mutin A, Cochrane EV, Tepper PG, Dijkstra D, WikstromH, Tamminga CA. 1996. Affinities and intrinsic activities of dopa-mine receptor agonists for the hD21 and hD4.4 receptors. EurJ Pharmacol 301:R11–13.

Laruelle M. 2000. Imaging synaptic neurotransmission with in vivobinding competition techniques: a critical review. J Cereb BloodFlow Metab 20:423–451.

Laruelle M, van Dyck C, Abi-Dargham A, Zea-Ponce Y, Zoghbi SS,Charney DS, Baldwin RM, Hoffer PB, Kung HF, Innis RB. 1994a.Compartmental modeling of iodine-123-iodobenzofuran binding todopamine D2 receptors in healthy subjects. J Nucl Med 35:743–754.

Laruelle M, Wallace E, Seibyl JP, Baldwin RM, Zea-Ponce Y, ZoghbiSS, Neumeyer JL, Charney DS, Hoffer PB, Innis RB. 1994b. Graph-ical, kinetic and equilibrium analysis of [123-I]�-CIT in vivo bind-ing to dopamine transporters in healthy subjects. J Cereb BloodFlow Metab 14:982–994.

Laruelle M, Abi-Dargham A, van Dyck CH, Rosenblatt W, Zea-PonceY, Zoghbi SS, Baldwin RM, Charney DS, Hoffer PB, Kung HF, InnisRB. 1995. SPECT imaging of striatal dopamine release after am-phetamine challenge. J Nucl Med 36:1182–1190.

Laruelle M, Abi-Dargham A, van Dyck CH, Gil R, De Souza CD, ErdosJ, McCance E, Rosenblatt W, Fingado C, Zoghbi SS, Baldwin RM,Seibyl JP, Krystal JH, Charney DS, Innis RB. 1996. Single photonemission computerized tomography imaging of amphetamine-in-duced dopamine release in drug free schizophrenic subjects. ProcNatl Acad Sci USA 93:9235–9240.

Laruelle M, Iyer RN, Al-Tikriti MS, Zea-Ponce Y, Malison R, ZoghbiSS, Baldwin RM, Kung HF, Charney DS, Hoffer PB, Innis RB,Bradberry CW. 1997. Microdialysis and SPECT measurements ofamphetamine-induced dopamine release in nonhuman primates.Synapse 25:1–14.

Laruelle M, Abi-Dargham A, Innis RB. 1998. Imaging receptor occu-pancy by endogenous transmitters in humans. In: Carson R, Daube-Whiterspoon ME, Herscovith P, editors. Quantitative functionalbrain imaging with positron emission tomography. San Diego: Ac-ademic Press.

Levenberg K. 1944. A method for the solution of certain problems inleast squares. Q Appl Math 2:164–168.

Levey AI, Hersch SM, Rye DB, Sunahara RK, Niznik HB, Kitt CA,Price DL, Maggio R, Brann MR, Ciliax BJ. 1993. Localization of D1and D2 dopamine receptors in brain with subtype-specific antibod-ies. Proc Natl Acad Sci USA 90:8861–8865.

Martin LP, Cox RF, Waszczak BL. 1990. Efficacy and potency com-parisons among aporphine enantiomers: effects on dopamine neu-rons in substantia nigra of rat. Neuropharmacology 29:135–143.

Martinez D, Slifstein M, Broft A, Mawlawi O, Hwang DR, Huang Y,Cooper T, Kegeles L, Zarahn E, Abi-Dargham A, Haber SN, Laru-elle M. 2003. Imaging human mesolimbic dopamine transmissionwith positron emission tomography. II. Amphetamine-induced do-pamine release in the functional subdivisions of the striatum.J Cereb Blood Flow Metab 23:285–300.

Mawlawi O, Martinez D, Slifstein M, Broft A, Chatterjee R, HwangDR, Huang Y, Simpson N, Ngo K, Van Heertum R, Laruelle M.2001. Imaging human mesolimbic dopamine transmission withpositron emission tomography. I. Accuracy and precision of D2receptor parameter measurements in ventral striatum. J CerebBlood Flow Metab 21:1034–1057.

204 R. NARENDRAN ET AL.

Menon MK, Clark WG, Neumeyer JL. 1978. Comparison of the dopa-minergic effects of apomorphine and (–)-N-n-propylnorapomor-phine. Eur J Pharmacol 52:1–9.

Mintun MA, Raichle ME, Kilbourn MR, Wooten GF, Welch MJ. 1984.A quantitative model for the in vivo assessment of drug bindingsites with positron emission tomography. Ann Neurol 15:217–227.

Mouradian MM, Heuser IJ, Baronti F, Giuffra M, Conant K, DavisTL, Chase TN. 1991. Comparison of the clinical pharmacology of(–)NPA and levodopa in Parkinson’s disease. J Neurol NeurosurgPsychiatry 54:401–405.

Neumeyer JL, Neustadt BR, Oh KH, Weinhardt KK, Boyce CB,Rosenberg FJ, Teiger DG. 1973. Aporphines. 8. Total synthesis andpharmacological evaluation of (plus or minus)-apomorphine, (plusor minus)-apocodeine, (plus or minus)-N-n-propylnorapomorphine,and (plus or minus)-N-n-propylnorapocodeine. J Med Chem 16:1223–1228.

Parsey RV, Oquendo MA, Zea-Ponce Y, Rodenhiser J, Kegeles LS,Pratap M, Cooper TB, Van Heertum R, Mann JJ, Laruelle M. 2001.Dopamine D(2) receptor availability and amphetamine-induced do-pamine release in unipolar depression. Biol Psychiatry 50:313–322.

Piccini P, Pavese N, Brooks DJ. 2003. Endogenous dopamine releaseafter pharmacological challenges in Parkinson’s disease. Ann Neu-rol 53:647–653.

Price J, Mason NS, Lopresti B, Holt D, Simpson NR, Drevets W,Smith GS, Mathis CA. 1998. PET measurement of endogenousneurotransmitter activity using high and low affinity radiotracers.In: Carson R, Daube-Witherspoon ME, Herscovitch P, editors.Quantitative functional brain imaging with positron emission to-mography. San Diego: Academic Press. p 441–448.

Reimer ML, Mamer OA, Zavitsanos AP, Siddiqui AW, Dadgar D.1993. Determination of amphetamine, methamphetamine and des-methyldeprenyl in human plasma by gas chromatography/negativeion chemical ionization mass spectrometry. Biol Mass Spectrom22:235–242.

Riche D, Hantraye P, Guibert B, Naquet R, Loch’h C, Maziere M.1988. Anatomical atlas of the baboon’s brain in the orbito-meatalplane used in experimental positron emission tomography. BrainRes Bull 20:283–301.

Richfield EK, Young AB, Penney JB. 1986. Properties of D2 dopaminereceptor autoradiography: high percentage of high-affinity agonistsites and increased nucleotide sensitivity in tissue sections. BrainRes 383:121–128.

Richfield EK, Penney JB, Young AB. 1989a. Anatomical and affinitystate comparisons between dopamine D1 and D2 receptors in the ratcentral nervous system. Neuroscience 30:767–777.

Richfield EK, Penney JB, Young AB. 1989b. Anatomical and affinitystate comparisons between dopamine D1 and D2 receptors in the ratcentral nervous system. Neuroscience 30:767–777.

Rinken A, Finnman UB, Fuxe K. 1999. Pharmacological character-ization of dopamine-stimulated [35S]-guanosine 5�(gamma-thio-triphosphate) ([35S]GTPgammaS) binding in rat striatal mem-branes. Biochem Pharmacol 57:155–162.

Ross SB, Jackson DM. 1989. Kinetic properties of the in vivo accu-mulation of [3H](–)-N-n-propylnorapomorphine in the mouse brain.Naunyn Schmied Arch Pharmacol 340:13–20.

Rowland M, Tozer TN. 1989. Clinical pharmacokinetics. Philadelphia:Lea & Febiger.

Schoenfeld RI, Neumeyer JL, Dafeldecker W, Roffler-Tarlov S. 1975.Comparison of structural and stereoisomers of apomorphine onstereotyped behavior of the rat. Eur J Pharmacol 30:63–68.

Seeman P. 1993. Receptor Tables. Vol. 2: drug dissociation constantsfor neuroreceptors and transporters. Toronto: Schizophrenia Re-search.

Seeman P, Grigoriadis D. 1987. Dopamine receptors in brain andperiphery. Neurochem Int 10:1–25.

Seeman P, Watanabe M, Grigoriadis D, Tedesco JL, George SR,Svensson U, Nilsson JL, Neumeyer JL. 1985. Dopamine D2 receptorbinding sites for agonists. A tetrahedral model. Mol Pharmacol28:391–399.

Seeman P, Niznik HB, Guan HC, Booth G, Ulpian C. 1989. Linkbetween D1 and D2 dopamine receptors is reduced in schizophreniaand Huntington diseased brain. Proc Natl Acad Sci USA 86:10156–10160.

Seeman P, Sunahara RK, Niznik HB. 1994. Receptor-receptor link inmembranes revealed by ligand competition — example for dopa-mine D-1 and D-2 receptors. Synapse 17:62–64.

Sibley DR, De Lean A, Creese I. 1982. Anterior pituitary receptors:Demonstration of interconvertible high and low affinity states of theD2 dopamine receptor. J Biol Chem 257:6351–6361.

Singer HS, Szymanski S, Giuliano J, Yokoi F, Dogan AS, Brasic JR,Zhou Y, Grace AA, Wong DF. 2002. Elevated intrasynaptic dopa-

mine release in Tourette’s syndrome measured by PET. Am J Psy-chiatry 159:1329–1336.

Slifstein M, Parsey RV, Laruelle M. 2000. Derivation of [(11)C]WAY-100635 binding parameters with reference tissue models: effect ofviolations of model assumptions. Nucl Med Biol 27:487–492.

Sunahara RK, Guan HC, O’Dowd BF, Seeman P, Laurier LG, Ng G,George SR, Torchia J, Van Tol HH, Niznik HB. 1991. Cloning of thegene for a human dopamine D5 receptor with higher affinity fordopamine than D1. Nature 350:614–619.

Tamminga CA, Gotts MD, Thaker GK, Alphs LD, Foster NL. 1986.Dopamine agonist treatment of schizophrenia with N-propyl-norapomorphine. Arch Gen Psychiatry 43:398–402.

Tiberi M, Jarvie KR, Silvia C, Falardeau P, Gingrich JA, Godinot N,Bertrand L, Yang-Feng TL, Fremeau Jr R, Caron MG. 1991. Clon-ing, molecular characterization, and chromosomal assignment of agene encoding a second D1 dopamine receptor subtype: differentialexpression pattern in rat brain compared with the D1A receptor.Proc Natl Acad Sci USA 88:7491–7495.

Tsukada H, Nishiyama S, Kakiuchi T, Ohba H, Sato K, Harada N.1999. Is synaptic dopamine concentration the exclusive factor whichalters the in vivo binding of [11C]raclopride? PET studies combinedwith microdialysis in conscious monkeys. Brain Res 841:160–169.

van der Werf JF, Sebens JB, Vaalburg W, Korf J. 1983. In vivobinding of N-n-propylnorapomorphine in the rat brain: regionallocalization, quantification in striatum and lack of correlation withdopamine metabolism. Eur J Pharmacol 87:259–270.

Verhoeff NP, Hussey D, Lee M, Tauscher J, Papatheodorou G, WilsonAA, Houle S, Kapur S. 2002. Dopamine depletion results in in-creased neostriatal D(2), but not D(1), receptor binding in humans.Mol Psychiatry 7:233–238.

Villemagne VL, Wong DF, Yokoi F, Stephane M, Rice KC, Matecka D,Clough DJ, Dannals RF, Rothman RB. 1999. GBR12909 attenuatesamphetamine-induced striatal dopamine release as measured by[(11)C]raclopride continuous infusion PET scans. Synapse 33:268–273.

Woods RP, Mazziotta JC, Cherry SR. 1993. MRI-PET registrationwith automated algorithm. J Comput Assist Tomogr 17:536–546.

Yarbrough GG, McGuffin-Clineschmidt J, Singh DK, Haubrich DR,Bendesky RJ, Martin GE. 1984. Electrophysiological, biochemicaland behavioral assessment of dopamine autoreceptor activation bya series of dopamine agonists. Eur J Pharmacol 99:73–78.

Yung KK, Bolam JP, Smith AD, Hersch SM, Ciliax BJ, Levey AI.1995. Immunocytochemical localization of D1 and D2 dopaminereceptors in the basal ganglia of the rat: light and electron micros-copy. Neuroscience 65:709–730.

Zahniser NR, Molinoff PB. 1978. Effect of guanine nucleotides onstriatal dopamine receptors. Nature 275:453–455.

Zhang X, Segawa T. 1989. Selective blockade of dopamine D-1 recep-tor by SCH 23390 affects dopamine agonist binding to 3H-spiperonelabeled D-2 receptors in rat striatum. Jpn J Pharmacol 50:333–345.

APPENDIX

This appendix provides the set of equations used toderive the expanded D2 receptor model.

Derivation of %Rhigh

For simplicity, the binding of endogenous DA atbaseline is initially ignored. The following assumptionsare made: 1) the total number of D2 receptors, Bmax, isthe sum of sites configured as D2high sites, Rhigh, andthe sites configured as D2low sites, Rlow; 2) [11C]raclo-pride binds with the same affinity to both sites (Khigh

RAC � Klow RAC � KD RAC); 3) [11C]NPA binds withhigher affinity to D2high sites compared to D2low sites,and the affinity difference between Khigh NPA and Klow

NPA is such that, at tracer dose, the binding of[11C]NPA to D2low sites is negligible; 4) similarly, the invivo affinity of DA for D2low sites is negligible, such thatDA released from terminals in vivo only binds to D2high

sites; 5) for a given dose of amphetamine, the change inDA during the [11C]raclopride and [11C]NPA experi-ments are equal (i.e., the tracers do not affect amphet-

COMPARISON OF [11C]NPA AND [11C]RACLOPRIDE 205

amine-induced DA release); 6) the occupancy of D2

receptors by these ligands is negligible and does notaffect the occupancy of D2 receptors by DA; and 7) theinteraction between DA and radiotracer binding iscompetitive, and the effects of DA on binding availabil-ity are well approximated by equilibrium binding con-ditions.

Baseline [11C]raclopride BP is given by:

BPRAC BASE �f1Bmax

KD RAC�

f1Rlow

KD RAC�

f1Rhigh

KD RAC. (10)

After amphetamine, [11C]raclopride BP becomes:

BPRAC AMPH �f1Rlow

KD RAC�

f1Rhigh

KD RAC�1 � a�, (11)

where a is the FDA/Khigh DA ratio, FDA is as definedabove, and Khigh DA is the inhibition constant of DA forthe binding of the radiotracer to the D2high sites. Thedifference between [11C]raclopride BP at baseline andfollowing amphetamine is:

BPRAC BASE � BPRAC AMPH �f1Rhigh

KD RAC� a1 � a�. (12)

[11C]Raclopride BP is obtained by dividing Eq. 12 byEq. 10:

BPRAC �Rhigh

BMAX� a1 � a�. (13)

[11C]NPA BP at baseline is given by:

BPNPA BASE �f1Rhigh

KHIGH NPA. (14)

[11C]NPA BP following amphetamine is given by:

BPNPA AMPH �f1Rhigh

KHIGH NPA�1 � a�. (15)

The difference between [11C]NPA BP at baseline andfollowing amphetamine is:

BPNPA BASE � BPNPA AMPH �f1Rhigh

KHIGH NPA� a1 � a�. (16)

[11C]NPA BP is given by:

BPNPA � � a1 � a�. (17)

Dividing Eq. 13 by Eq. 17 leads to a fundamentalequivalence:

BPRAC

BPNPA�

Rhigh

BMAX. (18)

Equation 18 predicts that the ratio of [11C]racloprideBP to [11C]NPA BP will be constant and indepen-dent of the magnitude of the change in FDA (the sameBP ratio will be observed at each dose of amphet-amine). This prediction was confirmed in the data (Ta-ble III). It follows by Eq. 18 that 70% (100*1/1.42) of D2

receptors are D2high sites. Equation 18 also applies tothe [11C]raclopride to [11C]NPA V3� ratio (this can bederived by substitution of V3� for BP and of f2 for f1 inEq. 10–18 above). Indeed, no significant differenceswere observed between the V3� and BP ratios of[11C]raclopride to [11C]NPA (Table III).

Note that the original model postulated that 50% ofD2 receptors are D2high sites (as suggested by in vitrostudies). This hypothesis predicted a [11C]NPA BP/[11C]raclopride BP equal to 2 (under the scenario of nooccupancy of D2 receptors by DA at baseline) or 2.25(under the scenario of 10% baseline occupancy, seebelow), which was not observed. Fundamentally, Eq.18 indicates that the lower the %Rhigh, the higher[11C]NPA BP is compared to [11C]raclopride BP.

Incorporation of synaptic and nonsynapticcompartments into the model

Following the highest dose of amphetamine (1 mgkg–1), the concentration of DA is expected to fully sat-urate Rhigh sites (see Discussion). Yet, [11C]NPA V3�and BP were only –54% and –60%, respectively (dueto the slight decrease in [11C]NPA V2 observed at thisdose, the value of BP is considered more accurate andwill be used in this section). This observation suggeststhat, in vivo, 40% of D2high sites are protected fromsignificant occupancy by DA released from the termi-nals. The terms synaptic and nonsynaptic are usedhere to denote the sites accessible and nonaccessible toDA, respectively.

The population of D2 receptors is divided into fourpools according to affinity states for agonists (high andlow) and physiological compartments (synaptic andnonsynaptic). The concentrations of D2 receptors inthese pools are termed RLS (low, synaptic), RLNS (low,nonsynaptic), RHS (high, synaptic), and RHNS (high,nonsynaptic). All sites are available to [11C]raclopride(Bmax � RLS�RLNS�RHS�RHNS), while only D2high sitesare available to [11C]NPA (Rhigh � RHS�RHNS). Theterms Bmax and Rhigh can be substituted in Eq. 18,leading to:

BPRAC

BPNPA�

RHS � RHNS

RLS � RLNS � RHS � RHNS. (19)

Equation 19 indicates that the 0.70 proportionalityfactor applies to all sites, not just the synaptic ones.Therefore,

206 R. NARENDRAN ET AL.

RHS � RHNS � 0.70�RLS � RLNS � RHS � RHNS�. (20)

RHS � 0.60�RHS � RHNS�. (21)

which leads to %RHS � 42%, %RHNS � 28%, and%Rlow � 30% (the respective proportion of RLS andRLNS can not be derived from the data of this study).

Incorporation of baseline DA in the model

To incorporate baseline DA occupancy of D2 recep-tors, the term a is replaced by a1 at baseline and a1 �a2 following the challenge, where a1 is the FDA/Khigh DA

ratio at baseline, FDA BASE/Khigh DA, and a2 is the addi-tional free DA following the challenge divided by Khigh

DA, (FDA AMPH – FDA BASE)/Khigh DA. At baseline:

BPRAC BASE � f1� RHS

KD�1 � a1��

RHNS � RLS � RLNS

KD� (22)

and following challenge:

BPRAC AMPH

� f1� RHS

KD�1 � a1 � a2��

RHNS � RLS � RLNS

KD� (23)

The difference between [11C]raclopride BP at baselineand following amphetamine is:

BPRAC BASE � BPRAC AMPH

�f1RHS

KD� a2�1 � a1���1 � a1 � a2��, (24)

and BP is given by:

BPRAC �

RHS� a2�1 � a1 � a2��

�RHS � �1 � a1��RHNS � RLS � RLNS��. (25)

Similarly, for [11C]NPA:

BPNPA BASE � f1� RHS

KD�1 � a1��

RHNS

KD�. (26)

The difference between [11C]NPA BP at baseline andfollowing amphetamine is:

BPNPA BASE � BPNPA AMPH

�f1RHS

KD� a2�1 � a1���1 � a1 � a2�� (27)

and [11C]NPA BP is:

BP �

RHS� a2�1 � a1 � a2��

�RHS � �1 � a1�RHNS�(28)

so that the [11C]raclopride to [11C]NPA BP ratioequals:

BPRAC

BPNPA�

RHS � �1 � a1�RHNS

RHS � �1 � a1��RHNS � RLS � RLNS�. (29)

Note that, as in the case of no baseline DA occupancy,this ratio is independent of a2, i.e., of the amphetaminedose. The relationship between the Rhigh/Bmax ratio, theBP ratios, and the baseline DA occupancy (OCC) canbe obtained from Eq. 29 by replacing the various recep-tor pools and a1 by their expressions in terms of Bmax,Rhigh, and OCC, but it is simpler to derive this ratiofrom the apparent occupancies. Let OCC1 be the occu-pancy of D2 receptors by DA at baseline, and OCC2 bethe additional occupancy due to the DA surge followingthe challenge, so that, after the challenge, (OCC1 �OCC2)*Bmax D2 receptors are occupied. Then, since re-ceptors available to [11C]raclopride at baseline are (1-OCC1)*Bmax, [11C]raclopride BP can be expressed as:

BPRAC �OCC2

1 � OCC1. (30)

Similarly, receptors available to [11C]NPA at baselineare [(Rhigh/Bmax) – OCC1)*Bmax, and [11C]NPA BP canbe expressed as:

Fig. 7. The true ratio Rhigh/Bmax as a function of baseline occu-pancy by DA, given observed [11C]raclopride V3�/[11C]NPA V3�equal to 0.70 and that synaptic sites account for 60% of the D2highsites. Ratios are given explicitly for 10, 20, and 30% baseline DAoccupancy. Maximal displayed occupancy is 60%, based on the ob-served ceiling effect. See the Appendix for mathematical derivation.

COMPARISON OF [11C]NPA AND [11C]RACLOPRIDE 207

BPNPA �OCC2

�RHIGH

BMAX� � OCC1

. (31)

The BP ratio is the ratio of these, yielding the rela-tionship:

RHIGH

BMAX�

BPRAC

BPNPA�1 � OCC1� � OCC1. (32)

Note that when there is no baseline occupancy by DA(OCC1 � 0), Eq. 32 simplifies to Eq. 18. If we assume10% baseline occupancy by DA, the 70% observed BPratio indicates that Rhigh is 73% of Bmax. It would thenfollow that RHS is 38%, RHNS is 25%, and Rlow is 27%(Fig. 6).

It should be noted that there is considerable uncer-tainty about the proportion of sites occupied by base-

line DA. The estimate used here (10%) is derived fromacute depletion studies in healthy volunteers, whereacute DA depletion increased [123I]IBZM V3� by 9 � 7%,n � 18 (Abi-Dargham et al., 2000) and [11C]racloprideV3� by 13%, n � 6 (Verhoeff et al., 2002). Larger valueshave been reported in anesthetized baboons (30%, La-ruelle et al., 1997) and anesthetized rhesus monkeys(40%, Ginovart et al., 1997). The point of Eq. 32 is thatthe Rhigh/Bmax ratio cannot be derived from the data ofthe present study without making an assumptionabout baseline occupancy by DA. However, large vari-ations in D2 receptor occupancy by DA at baselineinduce only small deviations from the equivalence ex-pressed in Eq. 18. Figure 7 graphs true Rhigh/Bmax as afunction of D2 receptor occupancy by DA at baseline,given the [11C]raclopride BP/[11C]NPA BP observedin this study (0.70).

208 R. NARENDRAN ET AL.