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stimation of Baseline Dopamine D2 Receptorccupancy in Striatum and Extrastriatal Regions inumans with Positron Emission Tomography with

18F] Fallyprideatrizia Riccardi, Ron Baldwin, Ronald Salomon, Sharlet Anderson, Mohammad S. Ansari, Rui Li,enoit Dawant, Amy Bauernfeind, Dennis Schmidt, and Robert Kessler

ackground: This study examined whether positron emission tomography (PET) studies with [18F] fallypride performed before and after-methyl-para-tyrosine (AMPT) administration can be used to estimate baseline dopamine (DA) D2 receptor occupancy in striatal andxtrastriatal regions.

ethods: Six normal subjects underwent PET with [18 F] fallypride before and after administration of AMPT. The DA D2 receptor bindingotentials (bp) were calculated with the reference region method. Percent changes in bp in striatal and extrastriatal regions were calculatedith both region-of-interest analysis and on a voxel by voxel basis with parametric images of DA D2 receptor levels.

esults: The results of the current study indicate that AMPT treatment significantly increased the bp in the caudate, putamen, ventraltriatum, and substantia nigra. A trend level increase was seen in the medial thalamus.

onclusions: This study demonstrates that PET with [18F] fallypride can be used to estimate baseline DA D2 receptor occupancy in striatal

nd extrastriatal regions.

ey Words: [18F] fallypride, �-methyl-para-tyrosine (AMPT)hallenge, dopamine, dopamine D2 receptors, humans, PET

-methyl-para-tyrosine (AMPT) is a competitive inhibitor oftyrosine hydroxylase, the rate limiting step in dopamine(DA) synthesis. Short-term AMPT administration produces

artial depletion of cerebral DA (1). When combined with singlehoton emission-computed tomography or positron emissionomography (PET) studies of striatal DA D2 receptor levels, anssessment of baseline occupancy of striatal DA D2 receptors byndogenous DA can be made in humans (1– 4). These methodsave been used to demonstrate increased baseline occupancy oftriatal DA D2 receptors in schizophrenia (5). Baseline occupancyf DA D2 receptors has been interpreted as a measure of baselineA release (6). Abnormal dopaminergic neurotransmission inortex, limbic regions, substantia nigra, and thalamus is believedo play an important role in a number of psychiatric disorders7–10). Development of a method to measure baseline occu-ancy of DA D2 receptors by endogenous DA in limbic regions,ubstantia nigra, and the thalamus as well as in cortex wouldrovide an important new measure of dopaminergic neurotrans-ission in extrastriatal brain regions believed to be involved insychiatric disorders. The purpose of the current study was toxamine whether PET [18F] fallypride studies performed beforend after AMPT depletion could be used to estimate baseline DA

2 occupancy by endogenous DA in striatal and extrastriatalegions. [18F] fallypride is a high-affinity DA D2 receptor radioli-

rom the Departments of Radiology (PR, RB, MSA, RL, RK), Psychiatry (RS, SA,AB, DS, RK), and Computer Science (RL, BD), Vanderbilt University, Nash-ville, Tennessee.

ddress reprint requests to Robert M. Kessler, M.D., Department of Radiol-ogy, Vanderbilt University Medical Center, 1268-B RRB, Nashville, TN37232-2675; E-mail: robert.kessler@vanderbilt.edu.

eceived September 13, 2006; revised March 6, 2007; accepted March 22,

2007.

006-3223/08/$34.00oi:10.1016/j.biopsych.2007.03.022

gand that can be used to estimate both DA D2 receptor levels andd-amphetamine–induced DA release in striatum and extrastriatalregions (11–13). The sensitivity of [18F] fallypride to extracellularDA levels suggested that it would be a suitable radioligand forstudying baseline occupancy of striatal and extrastriatal DA D2

receptors.

Methods and Materials

This study was performed under approval from the VanderbiltUniversity Institutional Review Board. Six normal subjects, threewomen (ages 23–38 years, mean age of 28 years) and three men(ages 23–32 years, mean age of 26 years), without any history ofpsychiatric, neurological, or medical illness were recruited. Thinsection T1 weighted magnetic resonance imaging (MRI) scans(1.2–1.4 mm slice thickness, in plane voxel size of 1 � 1 mm)were performed with a 1.5 T GE scanner. The PET studies wereperformed with a GE Discovery LS PET scanner with a three-dimensional emission acquisition and a transmission attenuationcorrection. The [18F] fallypride PET scans (5.0 mCi, specificactivity � 2,000 Ci/mmol) were performed before and afterAMPT administration (71.4 mg/kg p.o. administered in six doses)over 26 hours (1,2,3). Serial PET scans were obtained for 3.5hours. Female subjects were studied in the follicular phase of themenstrual cycle. To minimize the risk of crystalluria during AMPTadministration, subjects were encouraged to drink fluids, intra-venous fluids (0.45% saline) were infused until midnight after thesecond PET study, and sodium bicarbonate (1.2 grams p.o.) wasgiven after the third, fifth, and final AMPT doses. Subjects’ urinewas tested daily to detect crystal formation. Subjects were askedto rate restlessness, anxiety, speed of thinking, happiness, dis-comfort, and sleepiness on a scale of one to five, and motorneurological examinations were performed before and afterAMPT administration.

Blood samples were collected for determination of ho-movanillic acid (HVA) plasma levels. Serial PET scans were

coregistered to each other and to thin section MRI scans, (and

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242 BIOL PSYCHIATRY 2008;63:241–244 P. Riccardi et al.

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eoriented to the anterior commissure–posterior commissure lineith a mutual information rigid body algorithm) (14). Regions of

nterest (ROIs), caudate, putamen, ventral striatum, medial thal-mus amygdala, temporal cortex, and substantia nigra wereelineated on MRI scans of the brain and transferred to theoregistered PET scans (15). Regional DA D2 receptor bindingotential (bp) and parametric images of DA D2 receptor bp werealculated with the reference region method (16,17,18,19). TheseOIs demonstrate excellent intersubject coefficients of variation,anging from 6.8% to 15.9% (13). Percent change images werealculated on a pixel by pixel basis. With an elastic deformationlgorithm (20), mean parametric images of changes in [18F]allypride bp were calculated. Probability maps were calculatedith two-tailed t tests on a voxel by voxel basis (4 � 4 � 4 mmoxels). Clusters were delineated with a cutoff of p � .05 for eachoxel, and clusters were corrected for multiple comparisons withhe method of Forman (21) as implemented in AFNI (22). Onlylusters with a significance level of p � .001 corrected forultiple comparisons (cluster size � 173 voxels) were examined.

esults

A repeated measures analysis of variance of the ROI data withreatment status, region, and side as factors revealed significantffects of treatment [F (1) � 927.69, p � .0001], region [F (6) �253.27, p �. 0001], and region � treatment [F (6) � 49.2, p �0001] but no overall effect of side [F (1) � 1.37, p � .243],reatment � side [F (1) � .17, p � .68], or treatment � side �egion [F (6) � .1.66, p � .13] interactions. Because no overallffects of side, side � treatment, or side � treatment � regionere seen, the right- and left-sided regions were combined for

urther analysis. Given the significant effects of treatment andegion, paired two-tailed t tests were used to examine the effectf treatment on each region. These tests demonstrated significantncreases in bp after AMPT treatment for the caudate, putamen,entral striatum, and substantia nigra; a trend level increase waseen in the medial thalamus (p � .09). No significant effects ofreatment were seen in the amygdala or temporal cortex (Table 1).

Parametric images revealed a single, significant large cluster652 voxels, p � .001, corrected for multiple comparisons) ofncreased bp, subcortically, that involved the putamen, caudate,nd ventral striatum extending into the region of the hypothala-us and substantia nigra bilaterally (Figure 1). On the right, this

luster extended into the subthalamic region and superiorly intohe inferomedial portion of the thalamus just at the level of andnterior to the posterior commissure. No significant clusters wereeen in cortex.

None of the six subjects reported in the current study requiredreatment for side effects of AMPT. All six subjects were mildly tooderately somnolent and had mild slowing of rapid repetitive

Table 1. AMPT Induced Increases in Regional Binding P

RegionBaseline Binding

PotentialPos

Caudate 35.45 (4.44)Putamen 39.73 (6.11)Ventral Striatum 21.87 (4.21)Substantia Nigra 2.43 (.20)Amygdala 3.55 (.29)Temporal Cortex 1.64 (.18)Medial Thalamus 4.50 (.67)

Levels of significance levels are shown with paired two-ta

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movements. Three subjects had mild dysphoria, and three sub-jects had mild akathisia and restlessness. All six subjects experi-enced some slowing of mentation.

Plasma HVA levels were reduced by an average of 70.6 �15.2% (SD) at the time of the post-AMPT study. Correlation of thechange in plasma HVA levels with the increase in putaminal bprevealed an r � .61 that failed to reach significance for the groupstudied (p � .19).

Discussion

The results of this study indicate that [18F] fallypride PETstudies performed before and after AMPT depletion of cerebralDA can be used to estimate the baseline occupancy of DA D2

receptors in the caudate, putamen, ventral striatum, and substan-tia nigra, In the medial thalamus, five of six subjects demon-strated an increase in bp, but the overall effect only reached atrend level in this initial group. These changes are probably notdue to upregulation of DA D2 receptors, because animal studies

Figure 1. Parametric image analysis revealed a single, large cluster of signif-icantly elevated [18F] fallypride bps involving the caudete (A, B) and puta-men (A, B, C) bilaterally, the right thalamus (A), hypothalamus bilaterally(C), substania nigra bilaterally (D) and the dorsal midbrain (D).

tial (means and SD)

PT Bindingential

PercentChange

SignificanceLevel

(4.71) 8.83% (5.42) .001(6.00) 11.20% (4.53) .001(5.19) 10.64% (6.61) .001(.20) 12.69% (6.40) .001(.29) 1.00% (5.91) .466(.19) .11% (2.8) .434(.54) 3.83% (4.91) .09

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P. Riccardi et al. BIOL PSYCHIATRY 2008;63:241–244 243

ndicate that DA depletion for 2 days does not cause receptorpregulation (1,23). The changes in striatal bp in this study areimilar to those reported for [11C] raclopride with similar origher doses of AMPT (2– 4). The current results, however, differrom those of Fujita (24), who reported increased temporalortical bp for [123I] epidepride (25) after a 6-g/36-hour schedulef AMPT administration.

The baseline occupancy of DA D2 type receptors in theubstantia nigra is statistically not different from that in theutamen (p � .789 two-tailed t test) despite microdialysis datahat show that the extracellular levels of DA in primate substantiaigra are 5- to 10-fold lower in the substantia nigra than in theutamen (26,27). This apparent discrepancy might be related tohree factors. First, DA D2 type receptors in the substantia nigrare autoreceptors in the high-affinity state (28,29), whereas DA

2 receptors in the striatum are in the high- and low-affinitytates (30). Second, recent PET studies in baboons (31) andostmortem studies in human brain (32) demonstrate a highraction of DA D3 receptors in the substantia nigra that have0-fold higher affinity for DA than high-affinity state D2 receptors33). Fallypride has nearly identical affinities for the DA D2 and

3 receptor, (i.e., 30 pmol/L and 31 pmol/L; Kessler, unpub-ished data). A third factor might be the predominance of the DA

2s in the nigra, although the effect of this is unclear, because theffinities of fallypride for the D2s versus D2l are unknown (34).hese observations might explain the greater unmasking of DA

2 receptors after AMPT in the nigra than in the striatum despitehe considerably lower levels of extracellular DA.

Parametric image analysis demonstrated significant changesn bp only in subcortical regions consistent with the findings forhe ROI analysis. In addition to the regions showing significanthanges on ROI analysis, parametric image analysis demon-trated significant increases in bp in the hypothalamus. Noignificant changes were seen in cortical regions with both ROInd parametric image analyses.

In conclusion, this study demonstrates that [18F] fallypride PETtudies performed before and after AMPT administration can besed to assess baseline occupancy of DA D2 type receptors intriatum and subcortical extrastriatal regions.

Funding for this research was provided by a National Insti-utes of Health (NIH) grant entitled, “PET Imaging of Extrastria-al Dopamine Levels,” National Institute of Mental Health 5R01H60898-03, and supported in part by grant M01 RR-00095

rom the National Center for Research Resources, NIH.The authors have no financial conflicts of interest to disclose

n regard to this paper.We wish to thank Janine Belote, Michelle Costner, and Sarah

oore for technical assistance and Joann Fields for her excellentssistance in the preparation of this manuscript.

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