Abstracts

NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

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Novel Tracers for Imaging Amyloid Plaques Mei-Ping Kung, Chi-Wan Lee, Zhi-Ping Zhuang, Masahiro Ono, Catherine Hou, Karl Ploessl, Hank Kung University of Pennsylvania, Department of Radiology Alzheimer’s disease (AD) is a neurodegenerative disease of the brain characterized by dementia, cognitive impairment and memory loss. Formation and accumulation of aggregates of ␤-amyloid (A␤)peptides in the brain are critical factors in the development and progression of AD. The A␤-plaque-specific imaging agents will be useful for early detection or monitoring the progression and effectiveness of treatment for AD. Recent advances in developing tracers for binding to A␤ plaques suggest that there are specific and saturable binding sites on the aggregates of A␤, that can be selectively labeled and imaged in vivo. Minimum requirements for diagnostic imaging agents of AD based on A␤-plaque-specific binding can be defined as follows: a). High binding affinity to A␤ aggregates: Ki ⬍ 40 nM. b). High binding selectivity: Ki for other sites: ⬎ 100 fold. c). Readily labeled with appropriate radionuclides for imaging. Imaging agents may be labeled by using either one of the two types of isotopes; 99mTc (T 1/2, 6 h; 140 KeV) and 123I (T 1/2, 13 h; 159 KeV) are routinely used for single photon emission computed tomography (SPECT), while 11C (T 1/2, 20 min; 511 KeV) and 18F (T 1/2, 110 min; 511 KeV) are commonly used for positron emission tomography (PET). d). Small (Mol.Wt. ⬍ 650), lipophilic (P.C. ⫽ 10-1000) and neutral molecules e). Good initial brain uptake and desirable pharmacokinetics (high initial uptake ⬎ 0.5% dose/organ at 2 min post iv injection and fast washout at 30 min, less than 30% of initial uptake remaining in the brain of normal mice). f). In vivo stability. Previously, our laboratory has reported two types of iodinated probes, styrylbenzenes (SB, i.e. IMSB) and thioflavins (T2, benzothiazole, i.e.TZDM), for binding to A␤ aggregates (J. Med. Chem. 44: 1905, 2001). In vitro binding studies of these ligands showed excellent binding affinities with K d values of 0.13 and 0.06 nM for aggregates of A␤ 1-40 and 0.73 and 0.14 nM for aggregates of A␤ 1-42, respectively. More importantly, under a competitive-binding assaying condition, two different and distinctive binding sites on A␤ 1-40 and A␤ 1-42 aggregates, which are mutually exclusive, were observed for SB and TZ. Significantly, [ 125I]TZDM crossed intact blood-brain-barrier and localized in the brain of normal mice after an iv injection. For in vivo imaging of A␤ aggregates to succeed, it will be necessary to develop agents that show good brain uptake in vivo. Brain penetration, a key factor for consideration, is usually related to the molecular size, neutrality and lipophilicity. Further refinements of these probes are necessary to improve the brain uptake and washout from the normal brain regions and to achieve a high retention in the regions rich in A␤ plaques. To further refine the properties of the amyloid binding ligands, derivatives of stilbenes containing a p-Me2N-, -SH, -SMe, -OMe or -OH group on one of the benzene rings were prepared and tested. It was noted that radioactive iodine could be attached to the other benzene ring without affecting the binding affinity. The iodinated stilbenes are simple, relatively small, neutral and lipophilic. On the basis of their exquisitely high binding affinity to A␤ 1-40 aggregates (Ki at the range of 0.1-40 nM), they are suitable candidates as A␤ plaque-selective imaging agents. They also showed ability to penetrate the intact blood-brain barrier, an essential pre-requisite for a useful plaque-imaging agent. The labeled stilbenes showed promise to meet the stringent requirements for ␤-amyloid imaging agents. These imaging agents are potentially powerful tools for improving our understanding of the roles of A␤ plaques in the initiation and progression of the disease. It is likely that when these imaging agents become available, they will be used to identify patients in the early phase of AD, who could be benefited by treatment of neuro-protective agents.

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

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[F-18]AFM Is a Specific PET Radiotracer for the Serotonin Transporters: Comparison with [C-11]AFM Yiyun Huang, Dah-Ren Hwang, Raj Narendran, Peter S. Talbot, Sung-A Bae, Zhihong Zhu, Ningning Guo, Elizabeth Hackett, Lawrence S. Kegeles, Marc Laruelle Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, NY, USA Objective We have reported that in baboon studies the PET ligand [C-11]AFM ([C-11]2-[2-[(dimethylamino)methyl]thiophenyl]-5-fluoromethyl-phenylamine) displays superior imaging properties such as favorable kinetics and high level of specific binding for the serotonin transporter (SERT) in all regions of the brain (1). Another unique feature of this SERT ligand is that it can be labeled with either the short-lived radioisotope C-11 or the longer-lived F-18. The goal of this study was to prepare and evaluate [F-18]AFM and compare to the C-11 version. Methods [F-18]AFM was prepared in a two-step radiosynthesis. Biodistribution study was performed in rats. PET imaging experiments were conducted on the same baboons on the same day following sequential administration of [C-11] and [F-18]AFM. Kinetic analysis was performed to compare the binding characteristics of the two tracers. Results [F-18]AFM was produced in ⬎ 98% radiochemical purity from the benzyl chloride precursor in a two-step radiolabeling sequence. Biodistribution study in rats showed that [F-18]AFM enters the brain readily, with peak uptake reaching 1% ID/g in the hypothalamus, thalamus and prefrontal cortex, and 0.3 % ID/g in the cerebellum at 30 min post-injection. Peak region to cerebellum ratio was about 6 for both the hypothalamus and thalamus at 60 min after [F-18]AFM injection. These values are essentially the same as those obtained with [C-11]AFM and indicate a high level of brain uptake and a high degree of [F-18]AFM specific binding for SERT in rat. Imaging experiments were performed on the same baboons with both C-11 and F-18 AFM (n ⫽ 3 for each tracer) on the same day to compare the brain uptake and specific binding of the two tracers. Metabolite analysis indicated that [C-11]AFM and [F-18]AFM have similarly fast rate of metabolism, with parent fraction accounting for 18% of the total plasma activity at 30 min after [C-11]AFM injection. This fraction was 16% for [F-18]AFM at the same time point. For both [C-11] and [F-18]AFM, only polar metabolites were detected in the baboon blood. Brain uptake was high for both tracers (⬃ 0.3% ID for thalamus at 40 min). Kinetic analysis using the cerebellum as the reference region and metabolite-corrected plasma activity as input function returned similar values of equilibrium specific to nonspecific partition coefficient V3“ (expressed as (VTROI – VTCer)/VTCer) in regions of interest for the two labeled compounds. For [C-11]AFM, V3” was 1.71 for the midbrain, 2.16 for the thalamus, 1.20 for the striatum, and 0.64 for the hippocampus. For [F-18]AFM, V3“ was 1.57 for the midbrain, 1.92 for the thalamus, 1.14 for the striatum, and 0.60 for the hippocampus. Taken together, these data confirmed the high level of specific binding in vivo for both [C-11] and [F-18]AFM. Conclusion [F-18]AFM was prepared by a two-step labeling procedure. Studies in rats and baboons demonstrated that [F-18]AFM is a highly specific radiotracer for the in vivo imaging of SERT using PET. Reference 1. Huang Y, Bae SA, Zhu Z, Guo N, Hwang DR, Laruelle M (2001) J Labelled Compd Radiopharm 44:S18-20

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

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[11C]DADAM—A FAST EQUILIBRIUM PET RADIOLIGAND FOR THE SEROTONIN TRANSPORTER Christer Halldin*, Jari Tarkiainen*, Judith Sovago*, Johnny Vercouillie†, Balazs Gulyas*, Denis Guilloteau†, Patrick Emond†, Sylvie Chalon†, Jukka Hiltunen‡, Johan Lundberg*, Lars Farde *Karolinska Institutet, Stockholm, Sweden †INSERM, Tours, France ‡MAP, Helsinki, Finland Objectives Suitable PET radioligands for the serotonin transporter (5-HTT) belonging to a series of diphenyl sulfide derivatives has recently been developed. The binding equilibrium in brain stem occurs at later time points (⬎60 minutes) which is not optimal for a 11C-labeled radioligand such as DASB and MADAM. The deiodinated ADAM (DADAM, N,N-dimethyl-2-(2-aminophenylthio)benzylamine) has a Ki (nM) of 1.96. We report the synthesis of DADAM and its noranalogue, labeling with 11C, PET evaluation in Cynomolgus monkey and labeled metabolites in monkey plasma. Methods: DADAM and the noranalogue for labeling were obtained by the direct coupling of 1-bromo-2-nitrobenzene with N,N-dimethyl-2-thiobenzamide and N-methyl-2-thiobenzamide, respectively. This reaction was followed by reduction of the amide and nitro functions to give DADAM and the nor compound (N-methyl-2-(2aminophenylthio)benzylamine). [11C]DADAM was labeled using [11C]methyl triflate in acetone (incorporation ⬎75%). The total synthesis time including reversed-phase HPLC purification was about 30 minutes giving [11C]DADAM with a radiochemical purity ⬎99%. [11C]DADAM was injected i.v. into a Cynomolgus monkey and examined with PET. Pretreatment experiment were performed with citalopram. Metabolism was measured in monkey plasma by HPLC. The cerebellum, a region almost devoid of 5-HTT, was used as a reference region for free radioligand and nonspecific binding in brain. Specific [11C]DADAM binding to 5-HTT was defined as the difference between radioactivity in the region of interest and the cerebellum. Results: In the baseline PET experiments a high accumulation of radioactivity was obtained in the monkey brain after i.v. injection of [11C]DADAM (4.5% after 15 min). There was a high uptake of radioactivity in the brainstem, thalamus and striatum with a ratio to cerebellum of about 1.6 obtained at 50 minutes. Specific [11C]DADAM binding in brainstem, thalamus and striatum was obtained already at 15 minutes which is much earlier than for [11C]MADAM (70-90 minutes. In the pretreatment experiments citalopram (5 mg/kg) markedly reduced the radioactivity in all examined brain regions. The labeled metabolites in plasma were all polar with 23% unchanged radioligand at 45 minutes. Conclusion: The main advantage with [11C]DADAM compared to radioligands such as [11C]MADAM and [11C]DASB is the early binding equilibrium in brainstem and thalamus which occurs already at 15 minutes which if confirmed in man should allow simplified clinical protocols. [11C]DADAM demonstrate no significant difference to [11C]MADAM with regards to metabolism measured in monkey plasma. [11C]DADAM merits further evaluation as PET radioligand for 5-HTT in human.

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

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Radiolabelling and in vivo Evaluation of Two Analogues of SB-207710 as Potential PET Radioligands for the 5-HT4 Receptor in the Porcine Brain Christine Parker*, Laurent Martarello*, Jan Passchier*, Julian Matthews*, Stephen Knibb*, Maria Wishart*, Dirk Bender†, Donald Smith†, Albert Gjedde†, Antony Gee* *GlaxoSmithKline ACCI Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2GG, UK †PET Centre, Bygning 10C, Aarhus Kommunehospital, 800 Aarhus C, Denmark Numerous in vitro autoradiographical studies have been performed with selective radioligands to ascertain the CNS distribution of the 5-HT4 receptor in various species, including rat, guinea pig, and human (1-4). SB-207710 labelled with [125I] and [123I] has been reported to be a selective ligand for the 5-HT4 receptor in vitro (1,2) and in vivo (5). In these reports SB-207710 showed a high level of binding in the basal ganglia, hippocampus, and substantia nigra. However, to date no succesful 5-HT4 ligand has been reported for use in PET. This study aimed to investigate the feasibility of imaging the 5-HT4 receptor in the living porcine brain using two PET radiolabelled analogues of SB-207710. [11C]SB-207145 was produced by N-methylation of the corresponding desmethyl precursor with sodium hydride and [11C]MeI in DMF at 110°C for 5 min followed by HPLC purification. The [18F]fluorobutyl analogue of SB-204070 was synthesized by reacting 1,4-dibromobutane with dry [18F]KF, K2CO3 and Kryptofix in acetonitrile followed by alkylation of the corresponding desbutyl precursor with sodium hydride and [18F]1-bromo-4-fluorobutane in DMF at 110°C, followed by HPLC purification. In a pilot study [18F]fluorobutyl-SB-204070 and [11C]SB-207145 were evaluated in anesthetized Yorkshire pigs (40kg). PET scans were performed over a 90 minute period post iv tracer injection using a Siemens 961 camera. Blood samples were obtained from the femoral artery to determine the arterial input function and to measure the rate of metabolism of the parent compound. After initial brain uptake of [18F]fluorobutyl-SB-204070, the ligand was retained in the striatum, however, increasing levels of radioactivity in the skull and extracranial tissues was observed for the remainder of the scanning period, consistent with the rapid metabolism and defluorination of the parent radiotracer or associated metabolite(s). [11C]SB-207145 readily entered the brain with highest uptake and retention observed in the 5-HT4 rich striatum. Based on these results [11C]SB-207145 was further evaluated in Yorkshire porcines under baseline conditions (n⫽2) and after pretreatment with the selective 5-HT4 antagonist SB-204070 (0.5 mg/kg, n⫽2) 5 minutes prior to injection of [11C]SB-207145. Regional time-activity curves (TACs) were generated for striatum, cortices and cerebellum. Data were normalised for dose injected and expressed as percentage dose injected per litre (% ID/L). The analysis of [11C]SB-207145 and its metabolites in plasma were performed using radio-HPLC. [11C]SB-207145 rapidly entered the brain reaching peak regional tissue concentrations at approximately 10 min post injection followed by a slow washout from tissue (Fig 1). Fig. 1. Selected tissue TAC’s for [11C]SB-207145 in the pig brain The observed rank order of regional brain concentrapre (left panel) and post (right panel) 0.5 mg/kg SB-207040. tions was striatum ⬎ thalamus (data not shown) ⬎ cortical regions ⬎ cerebellum, consistent with reported 5-HT4 receptor densities and localisation determined by tissue section autoradiography in animals and man. Post administration of 0.5 mg/kg SB-204070, the concentration of [11C]SB-207145 in striatum was reduced to levels found in cortical regions (Fig 1). In both scans the tracer concentration was lowest in cerebellum, a brain region known to possess very low levels of 5-HT4 receptors. Radio-HPLC analysis revealed that [11C]SB-207145 was rapidly metabolised in arterial plasma, representing approximately 50, 20 and 5% of the total plasma radioactivity at 2.5, 4 and 10 min post tracer administration. Compared with [11C]SB-207145, the metabolite(s) were hydrophilic, eluting at the solvent front of the reversed phase HPLC chromatograms. In conclusion, [18F]fluorobutyl-SB-204070 and [11C]SB-207145 were evaluated as putative 5-HT4 PET ligands in the pig brain. Retention of [18F]fluorobutyl-SB-204070 in the 5-HT4 receptor-rich striatum was observed, however, apparent defluorination of the parent or associated metabolites caused an increasing signal in the skull and extracranial tissues, notibly higher than in brain tissue. Brain uptake of [11C]SB-207145, was characterised by a regional distribution consitent with known 5-HT4 receptor localisation in animal and in man. The striatal binding was blocked by pretreatment with 0.5 mg/kg SB-204070, a known selective 5-HT4 antagonist. As such [11C]SB-207145 shows promise as a ligand for in vivo characterisation of 5-HT4 receptor function in man using PET. References 1. Brown M, et al. Br. J. Pharmacol. (1993) 110, 10P 2. Mulligan R.S., et al. J. Labelled Cpd. Radiopharm. (1999) 42 Suppl. 1, S417-S419. 3. Jakeman L.B., et al. Neuropharmacology (1994) 33 (8), 1027-1038. 4. Bonaventure P., et al. Synapse (2000) 36, 35-46. 5. Pike V.W., et al. J Nucl. Med. (2000) 5 Suppl, 121P.

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

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PET Studies of CNS Substance P (NK1) Receptors in Living Human and Monkey Brain with [18F]SPA-RQ: A Novel Tracer for Substance P Receptor Quantitation H. D. Burns*, M. Goldberg*, D. Sciberras*, J. Hietala, O. Solin†, O. Eskola†, A. Laakso†, T. Gro¨ nroos†, S. Forsback†, J. Bergman†, M. Haaparanta†, M. Bergstrom‡, M. Ogren‡, B. Långstro¨ m‡, D. Brooks§, S. Luthra§, N. Turjanski§, T. G. Hamill*, C. Ryan*, W-s. Eng*, S. M. Sanabria-Boho´ rquez*, A. Coimbra*, R. Gibson*, K. Petty*, S. Reines*, R. J. Hargreaves* *Merck Research Laboratories, Department of Pharmacology & Imaging Research, West Point, PA, 19486 USA †Turku PET Centre and Department of Psychiatry, Turku University Central Hospital, Turku, Finland ‡Uppsala University PET Centre, Uppsala, Sweden §Imaging Research Solutions Ltd.,Cyclotron Building, Hammersmith Hospital, UK Introduction The Substance P (NK 1 ) receptor is widely distributed in the central and peripheral nervous systems and is currently a target of intensive research on its physiological functions. MK-869, a selective Substance P (NK 1 receptor) antagonist (SPA) that penetrates the blood-brain barrier, is in clinical development and has been reported to be useful for the treatment of chemotherapy induced emesis (1) and depression (2). We have labeled another SPA, [ 18 F]SPA-RQ (Substance P Antagonist Receptor Quantifier), at high specific activity with the short-lived, positron emitting radionuclide, 18 F and demonstrated its utility for quantifying Substance P (NK 1 ) receptors in vivo and NK 1 receptor occupancy by SPAs.

Methods [ 18F]SPA-RQ, (S,S)-[2-[ 18F]Fluoromethoxy-5-(5-Trifluoromethyltetrazol-1-yl)benzyl]-(2-phenylpiperidin-3-yl)-amine, was prepared by [ 18F]fluoroalkylation of a deprotonated phenolic hydroxyl group with [ 18F]FCH 2Br in DMF followed by removal of the BOC protecting group. [ 18F]FCH 2Br was prepared from [18F]F- and dibromomethane and purified via preparative gas chromatography. After fluoroalkylation, DMF was removed by applying a stream of helium over the surface of the solution in the heated reaction. The t-BOC protecting group was removed by treatment with TFA at room temperature and the TFA was evaporated by passing a stream of helium through the reaction vessel. The resulting [ 18F]SPA-RQ was purified via gradient preparative HPLC. The final product was formulated in ethanolic D-glucose solution buffered to pH 7. Specific activity was determined using either analytical HPLC or LC/MS to measure the mass concentration in the final solution. Biodistribution studies were conducted in Guinea pigs and autoradiographic studies were conducted using brain slices from Guinea pigs with detection using a FUJI 5000 phosphorimaging system. High specific activity (⬎ 500 GBq/␮mol) was used in biodistribution and autoradiographic studies in guinea pig, as well as in phosphorimaging autoradiographic studies of Rhesus monkey brain slices. PET imaging studies were conducted in Rhesus monkey and in normal healthy young human subjects before and after treatment with potent, selective SPAs.

Conclusion Preclinical and clinical validation experiments have demonstrated that [ 18 F]SPA-RQ is a valuable tool for NK 1 receptor quantitation in living human and non-human primates, as well as for determining the relationship of NK 1 receptor occupancy to dose and/or plasma levels of SPAs in clinical trials of novel treatments for chemotherapy induced nausea and depression. 1. Navari RM, et al. N-Engl-J-Med, 340(3): 190-5, 1999. 2. Kramer M, et al. Science, 281:1640-1645, 1998.

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

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Validation of [F-18]1(3-fluoropropyl)-4-[(4-cyanomethoxy)methyl]piperidine ([F-18]FPS) as an Effective PET Tracer for Imaging Sigma-1 Receptors in the Primate Brain Rikki N. Waterhouse, Mark Slifstein, Peter Talbot, Filip Dumont, Abida Sultana, Raj Narendran, Elizabeth Hackett, Yasuhiko Sudo, Marc Laruelle Department of Psychiatry, Columbia University, New York, NY USA Introduction Recently, sigma receptors have been considered to play a role in processes of memory and cognition, depression, and cocaine addiction. Several sigma-1 receptor antagonists are being evaluated in clinical trials as potential anti-depressant agents. Postmortem analyses have reported a reduction of sigma receptors in the frontal cortex in schizophrenic brains compared to controls, indicating that these sites mayb play some role in schizophrenia. A selective sigma-1 PET ligand is needed to explore these hypotheses in living subjects, and to serve as a tool in drug dose response versus occupancy studies of new sigma-1 drugs. We present here an extended pre-clinical evaluation of the novel sigma-1 selective PET tracer [F-18]FPS (log P7.4 ⫽ 2.8; KD ⫽ 0.8 ⫾ 0.03 nM), including PET evaluation in baboons, analysis of regional brain metabolites in rats, and determination of human radiation dose estimates and toxicity parameters. Methods [F-18]FPS was synthesized as previously described (Collier et al. J. Labelled Cpd. Radiopharm., 1996, 785-794). Emission data was collected (120 min, 3D mode, PET ECAT EXACT HR⫹) in two baboons (n ⫽ 2 for each) following bolus iv administration of high specific activity (0.4-3 Ci/mmol) [F-18]FPS. Blocking experiments were performed by pre-administration of haloperidol (0.5 mg/kg iv, n ⫽ 3). Arterial blood samples were collected. PET data were co-registered to anatomical MRIs, and eleven regions including cerebellum, cingulated cortex, frontal cortex, hippocampus, midbrain, occipital lobe, parietal lobe, striatum, temporal lobe, thalamus and the centrum semioval were analyzed. Time activity curves were formed in each region and both full kinetic modeling with non-linear regression and Logan graphical modeling were applied for cross validation. A whole body biodistribution study in male rats was performed (time points: 5, 15, 60, 240, and 880 min), and the tissue radioactivity concentrations (%ID/g) were used to generate approximate radiation dose estimates for human organs. Acute toxicity analyses at doses of 200, 2000, and 20,000 times the maximum expected human mass dose were carried out in rats, rabbits and dogs to determine an initial baseline safety margin for the a proposed mass dose limit for human studies (2.8 ␮g/dose). Results Plasma metabolite analysis showed the formation of polar metabolites only over the course of the study, a plasma clearance of 116 L/h, and a high plasma free fraction (54 ⫾ 7%). Regional VT values ranged from 111-197 ml/g, and were highest in the cingulate cortex, hippocampus, temporal cortex and brainstem with lower values in other cortical regions, striatum, cerebellum and white matter. Blocking experiments showed reproducible 88-93% reduction of VTs in all regions, and values ranged from 10.5-17.7 ml/g. This result indicates that the degree of non-specific binding is low, supporting that regional VT values can be used as effective outcome measures. Through in vitro binding analysis in baboon brain tissue membranes, we confirmed the presence of saturable [F-18]FPS binding sites in both white and grey matter. To support that the radioactivity in the brain is due to intact [F-18]FPS, a brain metabolite study was performed at 60 minutes in rats. The recovery of radioactivity was 82% for whole blood, and 88-90% for all brain tissues. The percent parent, as determined by HPLC analysis, was 76 ⫾ 15, 94 ⫾ 1, 94 ⫾ 3, and 97 ⫾ 1 in the plasma, cerebellum, frontal cortex, and midbrain, respectively (n ⫽ 3). Human radiation dose estimates revealed that most organs would to receive around 0.01-0.02 mGy/MBq. The adrenal gland (0.07 mGy/MBq, 0.26 rads/mCi) was identified as the critical organ, and the total body dose was moderate (0.013 mGy/MBq, 0.048 rad/mCi). Based on a maximum human mass dose of 2.8 ␮g, extended acute iv toxicity studies in male and female rats and rabbits, including post-mortem histopathological examination of 14 organs, demonstrated a safety margin of ⬎2000-fold (rats and rabbits). Limited toxicity studies were performed in dogs and a safety margin of at least 175 times the maximum administered dose was found in that case. Conclusions Taken together, these studies confirm the suitability of [F-18]FPS for imaging sigma-1 receptors in the primate CNS, and provide evidence that further examination of this tracer through PET studies in humans is warranted. However, due to the lack of a reference region devoid of sigma receptors, binding potential would not likely be a feasible outcome measure, as a blocking experiment would be required for each determination. We propose instead that regional VT values would be appropriate, since the degree of non-specific binding of this tracer is low. This research was funded by NIMH RO1 NS40402-02

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

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PET IMAGING OF ALPHA4BETA2 NICOTINIC ACETYLCHOLINE RECEPTORS IN HUMAN BRAIN WITH 2-[18F]FLUORO-A-85380: COMPARISON OF RECEPTOR BINDING AND PHARMACOKINETICS IN HUMANS AND NON-HUMAN PRIMATES. Alexey G. Mukhin*, Svetlana I. Chefer*, Andrew G. Horti*, Carlo Contoreggi*, Varughese Kurian*, Phyllis Friello*, Monique Ernst†, John A. Matochik*, Anrew Hall*, Bruce D. Vaupel*, Olga A. Pavlova*, Andrei O. Koren‡, Edythe D. London‡, Alane S. Kimes* *Brain Imaging Center, Intramural Research Program, NIDA, 5500 Nathan Shock Drive, Baltimore, MD 21224, USA †NIMH, 9000 Rockville Pike, Bethesda, MD 20892 ‡University of California, Los Angeles, CA 90024 Neuronal alpha4beta2 nicotinic acetylcholine receptors (nAChRs) mediate a variety of brain functions and have been implicated in a number of neuropathologies, including Alzheimer’s and Parkinson’s diseases, epilepsy, and schizophrenia. In addition, nAChRs are the primary target of nicotine, a tobacco component that is believed to promote dependence. Therefore, noninvasive imaging of nAChRs might prove crucial for understanding these disorders and for developing diagnostic tools and therapies targeted at nAChRs. The recent identification of 2-[18F]fluoro-A-85380 as a potential radioligand for in vivo imaging of nAChRs, followed by the in vitro and in vivo characterization of this compound, made it possible to image the nAChRs in human brain with PET. Total accumulation of 2-[18F]fluoro-A-85380 in the brain of healthy non-smoker subjects (ca. 2.5% of injected dose) was 2.5 times higher than that in Rhesus monkey brain. Such uptake allowed visualizing nAChRs up to 5 h after a bolus injection of 1.6 MBq/kg (0.043mCi/kg) of 2-[18F]fluoro-A-85380 (sp. act. 185-740 GBq/mmol; 5,000-20,000 Ci/mmol). Consistent with the distribution pattern of nAChRs in human brain, the greatest accumulation of radioactivity was observed in the thalamus, superior colliculus and pons; intermediate in the cerebellum and cortex; and lowest in white matter. This pattern of radioactivity accumulation was similar to that observed in Rhesus monkey brain. However, with regard to other brain structures, accumulation of radioactivity in human pons and cerebellum was higher compared with that in the monkey. The increased accumulation of radioactivity in human vs. monkey brain was probably due to a greater concentration of radioactivity in plasma and a larger fraction of radioactivity represented by the parent compound in humans. The fraction of the parent compound bound with plasma protein (ca. 20%) was almost identical in humans and monkeys. Metabolism of 2-[18F]fluoro-A-85380 was slower in humans. Thus, at 6 h after radioligand injection, radioactivity from the unchanged compound constituted ca.50% of total plasma radioactivity in humans vs. less than 25% in the monkey. Furthermore, metabolic profiles in humans and monkeys blood were quite different. In human plasma, a major portion of radioactivity from metabolites was represented by derivatives more lipophilic than the parent compound, whereas in monkey plasma - by more hydrophilic ones. After 2-[18F]fluoro-A-85380 administration to humans, ca. 90% of radioactivity eliminates from the body in urine (effective half-life ca. 4h). High accumulation of radioactivity in the urinary bladder renders it a critical organ. The effective dose equivalent for the urinary bladder wall was estimated as 0.19 mSv/MBq (0.7 rem/mCi) with a voiding interval of 2.4 h. Other organs with high radioactivity accumulation were kidneys and liver, each having less than a half of the urinary bladder absorbed dose. These results suggest the possibility of using doses up to 3.7 MBq/kg (0.1 mCi/kg) of the radioligand in a single bolus administration, which might allow measuring radioactivity in human brain structures at times up to 6-7 h. Our previous studies with 2-[18F]fluoro-A-85380 in Rhesus monkeys using different paradigms of administration (bolus, bolus plus infusion, and continuous infusion with variable rate) demonstrate the utility of this ligand for the quantitation of receptor binding in vivo. The binding potential (BP) values calculated for thalamus, cortex, striatum, and cerebellum were, respectively, ca. 2, 0.4, 0.3, and 0.03 in Rhesus monkey. While it was possible to estimate the BP value for the thalamus using cerebellum (CB) as a reference region, accurate measurements of BP in the cortex and striatum required determination of the non-displaceable volume distribution (VDnds) from a separate study where specific binding was blocked by pre-administration of 1 mg/kg s.c. of cytisine. Attempts to calculate the VDnds from the control studies data using the three compartment (two tissue) model failed. For example, even in studies with nearly complete blockade of specific binding by cytisine, time-activity curves for any brain structures were fitted best with the three-compartment model, suggesting the presence of a so-called “diffusion boundary” for the radioligand in brain tissue. Comparison of kinetics of brain radioactivity accumulation after administration of 2-[18F]fluoro-A-85380 in human with that in monkey suggests that BP values for this radioligand in human thalamus, cortex, and CB are 2, 0.6, and 0.5, respectively. More accurate estimation of these values will require direct measurement of VDnds in human brain, potentially from the blocking studies. To summarize, 2-[18F]fluoro-A-85380 is a promising ligand for studying nAChRs in human brain. Optimization of the use of this radioligand for quantitative analysis of the receptor binding with PET in humans is the goal of our future studies.

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

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WHOLE-BODY BIODISTRIBUTION, RADIATION-ABSORBED DOSE, AND BRAIN SPECT IMAGING WITH [I-123]5-I-A-85380 IN HEALTHY HUMAN SUBJECTS Masahiro Fujita†, Christopher van Dyck*, Masanori Ichise†, Sami Zoghbi†, Gilles Tamagnan*, Ali Bozkurt*, Nick Seneca†, Alexey G. Mukhin‡, Bruce Vaupel‡, Andrew G. Horti‡, Andrei O. Koren§, Shaukat Khan, Michele Early ¶, Alane S. Kimes‡, Edythe D. London§, John Seibyl ¶, Ronald Baldwin*, Robert Innis† *Yale University †NIMH ‡NIDA §UCLA ¶

Institute for Neurodegenerative Disorders

[ 123 I]5-Iodo-3-(2(S)-2-azetidinyl-methoxy)pyridine (5-I-A-85380) is a new SPECT tracer with high affinity and low toxicity developed to image ␣4␤2 nicotinic acetylcholine receptors (nAChRs). Our previous studies in baboons showed quick metabolism of the tracer and difficulties to apply a 3-compartment model to the brain data [1, 2]. Objectives: I) To study biodistribution of radioactivity after intravenous administration of [ 123 I]5-I-A-85380 and to estimate radiation-absorbed doses (biodistribution study), and II) to develop a method to measure ␣4␤2 nAChR densities in healthy human subjects (brain kinetic study). Methods: I) Biodistribution study: In 10 subjects (five men and five women, age: 34⫾4 years (mean ⫾ SD)), following intravenous administration of 98⫾6 MBq [ 123 I]5-I-A-85380, serial whole body images were acquired with the IRIX whole-body imager over 24 h and the image data were corrected for attenuation. Radioactivity in urine was also measured for 24 h. Estimates of radiation-absorbed doses were calculated using MIRDOSE 3.1 with the Cloutier’s dynamic bladder model and the ICRP 30 gastro-intestinal tract model. II) Brain kinetic study: [ 123 I]5-I-A-85380 (381⫾91 MBq) was administered intravenously to healthy subjects (age: 38⫾13) in three ways: 1) bolus only (n⫽6), 2) bolus plus constant infusion (B/I) with B/I ⫽ 4 –11 h adjusted based on kinetic parameters obtained in a bolus study of each subject (n⫽6), and 3) 20 min constant infusion (slow bolus) (n⫽7). The study duration was 5-8 h, 14 h, and 8 h in these paradigms, respectively. Brain data were acquired with Prism 3000XP and scatter correction was applied with transmission dependent convolution subtraction. The concentration of the parent compound was measured in arterial (in bolus and slow bolus) and venous (in all studies) plasma after sodium azide denaturation followed by HPLC analysis. In bolus and slow bolus studies, specific (V3⬘: specific binding/total (free plus protein bound) parent) and total (VT⬘: total binding/total parent) distribution volumes (mL/mL) were calculated with nonlinear least squares analysis using metabolite-corrected arterial input function for the thalamus, each cerebral cortex, cerebellum, pons, and basal ganglia. In addition, in slow bolus studies, a standard arterial input function was created from the data of all subjects. In each slow bolus study, distribution volumes were estimated by nonlinear least squares analysis by scaling the standard input function based on the total parent concentration in venous plasma at 250 min without using arterial data obtained in individual study. Results: I) Biodistribution study: Exponential curve fitting indicated that 81⫾2% of the injected activity was excreted in urine. The whole brain activity at the time of the initial whole body imaging at 14 min was 5.0% of the injected dose. The estimates of radiation-absorbed doses are shown in Table 1. II) Brain kinetic study: Although B/I studies showed stable thalamic activities with mean change of 2.2⫾1.1%/h after 9 h indicating that equilibrium was

Table 1. Radiation Dose Estimates (Gy/MBQ) Urine voiding intervals (h)

Urinary bladder wall

Lower large intestine wall

Upper large intestine wall

Effective dose (SV/MBQ)

2.4 4.8

713 1405

704 724

634 644

261 301

achieved in all brain regions, the brain data showed poor statistics with 25⫾4% and 22⫾4 % COV in pixel values in the frontal cortex and the cerebellum, respectively. After a bolus injection, parent concentration in arterial plasma decreased quickly to 6⫾2% of the peak at 8 min. In two of six bolus studies, 3-compartment fitting did not converge to give V3⬘ and only VT⬘ was identified. In all slow bolus studies, V3⬘ was identified as shown in Table 2. Nondisplaceable distribution volume, calculated in each subject and fixed among all brain regions, was 11⫾2 mL/mL, with COV, 8.2⫾5.2%. At 250 min, the difference in parent concentration between arterial and venous plasma was 5⫾12%. Both V3⬘ and VT⬘ values estimated

Table 2. Specific Distribution Volume (ml/ml)

V3⬘ COV (%)

Thalamus

Pons

Cerebellum

Frontal cx.

Parietal cx.

Temporal cx.

Putamen

378 1.11.1

216 3.12.0

113 6.84.6

6.52.7 9.79.7

6.42.1 10.010.0

7.12.1 4.24.4

14.52.4 3.42.7

using the standard input function correlated well with those obtained from individual arterial data (r 2 ⫽0.99 for both, with difference ⫽ 11⫾8 and 5⫾4% for V3⬘ and VT⬘, respectively). Conclusions: [ 123 I]5-I-A-85380 is a promising SPECT agent to image ␣4␤2 nAChR in humans, with favorable dosimetry and high brain uptake. With slow bolus injection, V3⬘ was identified in the thalamus, each cerebral cortex, cerebellum, pons, and basal ganglia, and the distribution was consistent with the known distribution of ␣4␤2 nAChRs in humans. V3⬘ values were successfully estimated by a standard arterial input function and venous data at 250 min. Supported by NIDA & NCI P50 DA84733. [1] Fujita, et al., J Nucl Med 2000; 41:1552-1560. [2] Zoghbi, et al. Nucl Med Biol 2001; 28: 91-96.

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

NOVEL TRACERS

Occupancy of Cortical and Substantia Nigra DA D2 Receptors by Typical and Atypical Antipsychotic Drugs Robert Kessler, M-Sib Ansari, Rui Li, Myung Lee, Dennis Schmidt, Benoit Dawant, Herbert Meltzer Vanderbilt University Medical Center Previous PET and SPECT studies have consistently demonstrated that typical antipsychotic drugs (APD’s) such as haloperidol must block about 70 percent of striatal dopamine D2 receptors (DA D2r) to produce therapeutic effects. Atypical APD’s such as clozapine and quetiapine, olanzapine and risperidone, produce therapeutic effects with a much lower incidence of extrapyramidal motor side effects. While olanzapine and risperidone have been shown to occupy at least 65-70 percent of striatal DA D2r at therapeutic doses, clozapine and quetiapine produce therapeutic effects at significantly lower levels of striatal DA D2r occupancy, 30-65 percent. It has been suggested that an atypical profile of APD can be achieved by a number of mechanisms, including a low affinity for the DA D2r and a high ratio of 5HT2A: DA D2r affinity. Animal studies have indicated that clozapine and other atypical APD’s unlike typical APD’s have selective effects on cortical and limbic dopaminergic neurotransmission compared to dorsolateral striatum. Previous PET and SPECT studies using [125I]epidepride, [11C] FLB457, and [76Br] FLB457 have produced conflicting results regarding the issue of preferential occupancy of cortical DA D2 by atypical APDs. The purpose of the present study was to determine if the selective therapeutic effects of atypical APDs are mediated by preferential occupancy of extrastriatal DA D2r. PET studies of striatal and extrastriatal DA D2r were performed using [18F] fallypride (4-5 mCi, specific activity greater than 2,000 Ci/mmol) a GE Advance PET scanner. Serial PET scans were obtained for 150-250 minutes after injection and were coregistered to high resolution T1 weighted gradient echo MR images (1.2 mm. thick). Estimation of regional receptor levels was performed using the reference region of Lammertsma. Schizophrenic subjects (ages 18-50) who were off medication (n⫽7), treated with haloperidol (n⫽6), clozapine (n⫽5), quetiapine (n⫽6), olanzapine (n⫽4), or risperidone (n⫽2) were studied. The mean regional values for the off-medication schizophrenic subjects were used to compute regional receptor occupancy for the treated subjects. Haloperidol produced a relatively uniform receptor occupancy in nearly all regions studied, i.e. occupancies of 76 percent in putamen, 79 percent in anterior thalamus, 70 percent in temporal cortex, 78 percent in ventral striatum and 76 percent in amygdala. Compared to haloperidol, olanzapine and risperidone demonstrated similar regional occupancies in most regions, i.e. 69-75 percent in the putamen, anterior thalamus, temporal cortex, ventral striatum, and amygdala. Clozapine and quetiapine demonstrated different levels and patterns of DA D2 receptor occupancy. Clozapine showed significantly lower occupancy in the putamen than haloperidol, i.e. 50 versus 76 percent, but not significantly different occupancy in the temporal cortex 63 versus 70 percent. Clozapine produced DA D2r occupancies of 55 percent in ventral striatum, 62 percent in anterior thalamus and 57 percent in amygdala. Quetiapine treated subjects were studied two hours following the last dose. Quetiapine produced a mean DA D2r occupancy of 31 percent in the putamen, 41 percent in the anterior thalamus, 46 percent in the temporal cortex, 36 percent in the ventral striatum, and 41 percent in the amygdala. Like clozapine, the occupancy in temporal cortex relative to putamen was significantly greater than seen with haloperidol. DA D2r occupancy was also measured in the ventral midbrain at a level corresponding to the substantia nigra and ventral tegmental region. The occupancy in the region of the substantia nigra was 60 percent for haloperidol versus 76 in putamen, producing a ratio of putamenal to nigral occupancies of 1.27. The lower occupancy in the substantia nigra may be related to its small size. Clozapine, had a mean nigral occupancy of 20 percent, which was not statistically different from untreated subjects. The putamenal: nigral ratio of DA D2r occupancies was 2.5 for clozapine. Unlike clozapine, quetiapine had a putamenal: nigral occupancy ratio of 1.07, resulting from a nigral occupancy of 29%. Olanzapine and risperidone had nigral occupancies of 43 and 41 percent respectively producing putamenal: nigral occupancy ratios of 1.67 and 1.75. Correlating the putamenal: nigral ratios for all five APD’s with the ratio of 5HT2:DA D2r affinities produced a correlation coefficient of 0.95, n⫽0.01. The results of this study suggest that atypical APD’s have different effects on cortical and nigral DA D2r than typical APD’s. These effects appear to be mediated by low affinity for the DA D2r and by a high 5HT2A: DA D2 ratio. As recent animal studies have shown that the substantia nigra is involved in integration of motor behaviors. The relative sparing of nigral DA D2r may contribute to the lower incidence of motor side effects seen with atypical APDs.

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

NOVEL TRACERS

Development of a radiotracer for in vivo imaging of the norepinephrine transporter Alan A. Wilson*, David Patrick Johnson†, Paul David Mosley†, Doug Hussey*, Nathalie Ginovart*, Sylvain Houle* *PET Centre, Centre for Addiction and Mental Health and Department of Psychiatry, University of Toronto, Toronto †Eli Lilly, Indianapolis, Indiana Despite a growing recognition that the norepinephrine transporter (NET) is a site of action of many old and new antidepressant drugs, there have been few reports on attempts to develop radiotracers for imaging NET in vivo. Haka and Kilbourn reported the synthesis of [ 11C]-nisoxetine which demonstrated modest specific binding in mice (1). Kung’s group synthesised an iodinated derivative of tomoxetine which showed no specific binding in vivo in rat brain and very high lung uptake (2). [ 11C]-Desipramine has also been reported but no in vivo data was included (3). We report here the radiosynthesis of (S,S)-[ 11C]-MeNER (Figure), an analogue of the selective NET inhibitor reboxetine, and explore its in vivo binding properties in the rat brain. (S,S)-[ 11C]-MeNER was synthesised by alkylation of its normethyl precursor with [ 11C]-iodomethane and base in an HPLC sample loop (4) in good radiochemical yield (20-40%, uncorrected) from [ 11C]-CH 3I and at high specific activity (30-60 GBq/␮mole) in 25 min from end-of-bombardment. Its enantiomer, (R,R)-[ 11C]-MeNER, was similarly prepared from the corresponding phenolic precursor. Upon tail vein injection of (S,S)-[ 11C]-MeNER in rats moderate brain uptake was observed (0.6%/organ) with retention of radioactivity in NET rich regions such as hypothalamus and cortex and a faster washout of radioactivity in the striatum, a region poor in NET. Hypothalamus to striatum ratios of 2.5 to 1 were reached at 60 min post injection. In contrast, no regional retention of radioactivity was found upon injection of the enantiomeric radiotracer (R,R)-[ 11C]-MeNER. Treatment of rats (all 2 mg/kg) with the NET binding drugs, reboxetine or desipramine, followed by injection of (S,S)-[ 11C]-MeNER, abolished ⬎95% specific binding in hypothalamus or other brain regions as compared to striatum. In contrast, treatment of rats with DASB (a SERT selective inhibitor), L-deprenyl (a selective MAO type B inhibitor) or GBR 12909 (a DAT selective inhibitor) had no significant effect on the regional distribution of radioactivity following injection of (S,S)-[ 11C]-MeNER. HPLC analysis of rat plasma showed that (S,S)-[ 11C]-MeNER was quickly metabolised with only 50% unchanged radiotracer at 30 min post-injection and 20% after 60 min. Radioactive metabolites were all hydrophilic. HPLC analysis of rat brain extracts showed that greater than 95% of radioactivity in the brain was unchanged (S,S)-[ 11C]-MeNER. (S,S)-[ 11C]-MeNER shows promise for imaging NET in humans by positron emission tomography. References (1) (2) (3) (4)

Haka, MS, et al. Nucl. Med. Biol., 16, 771. (1989) Chumpradit, S, et al. J Med Chem, 35, 4492. (1992) Van Dort, ME, et al. Nucl Med Biol, 24, 707. (1997) Wilson, AA, et al. Nucl Med Biol, 27, 529. (2000)

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

BASIC BIOLOGY

Temporal characterisation of amphetamine induced dopamine release assessed with [ 11C] raclopride in anaesthetised rodents Gavin Houston*, Sue Hume†‡, Paul Grasby* *MRC Cyclotron Unit, Hammersmith Hoispital, Du Cane Road, London, UK †Imaging Research Solutions Limited, MRC Cyclotron Unit, Hammersmith Hoispital, Du Cane Road, London, UK Introduction In recent years Positron Emission Tomography (PET) and Single-Photon Emission Computed Tomography (SPECT) imaging techniques have been increasingly used to provide an index of synaptic dopamine (DA) concentration in vivo. Previous investigations have reported a temporal mismatch between the DA release measured by microdialysis and the observed decrease in tracer binding 1,2. A sustained reduction in radiotracer binding (up to 5.5 hrs post administration) extending beyond the restoration of baseline extra-synaptic DA levels (typically 2.5 hrs post administration) has been reported. Such observations are inconsistent with the simple receptor occupancy model. The aim of this investigation was to characterise the change in [ 11C] raclopride specific binding in rodents following an amphetamine challenge as a function of time. Methods In the first experimental series, 22 isoflurane anaesthetised male Sprague Dawley (SD) rats were scanned using RATPET. Saline alone (n⫽6) or an amphetamine dose (1mg/kg (n⫽6), 2mg/kg (n⫽6), 4mg/kg (n⫽4)) was administered (i.p.) 30 minutes prior to [ 11C] raclopride injection. Binding potentials (BP), estimated for left and right striata with a 4 parameter reference tissue compartmental model, were correlated with percentage change in extracellular DA measured by microdialysis under the same anaesthetic (amphetamine 1mg/kg, 2mg/kg or 4mg/kg). In the second experimental series comprised 16 SD rats scanned under isoflurane anaesthesia using quad-HIDAC 3. 4mg/kg amphetamine sulphate was administered (i.p.) at one of three points times prior to [ 11C] raclopride administration: 30 mins (n⫽3), 4 hours (n⫽4) & 24 hours (n⫽4). Five animals were pre-dosed with to saline to obtain a measure of baseline binding. Image regions were sampled using a stereotaxic volume of interest template projected onto images sampled at 20-60 minutes. Striatal specific binding was expressed as the striata/cerebellum ratio. Results The left hand panel shows a linear correlation of the mean percentage change in binding potential and increase in extracellular DA concentration following the three amphetamine doses tested. Data are presented as the mean ⫾ sem. The right hand panel shows specific binding ratios in the striatum (left & right averaged). The mean and standard deviation for each group is depicted with the mean percentage change relative to baseline [ 11C] binding. Statistically significant decreases in raclopride binding were observed at 30 minutes (p ⬍ 0.001, two tailed t-test) and 4 hours (p⬍ 0.005, two tailed t-test). At 24 hours after amphetamine, the mean ratio had returned to baseline (p ⬎ 0.98, two tailed t-test). Discussion [ 11C] raclopride binding was assessed at three time point following amphetamine administration. Times were selected based on the following basis: 30 minutes to scan during the peak of DA release as determined by microdialysis; 4 hours when extra-cellular DA concentrations are known to have normalised (but has previously been reported to exhibit sustained reduction in binding 1,2), and 24 hours when binding would be expected to return to baseline levels assuming no change in the receptor population. The linear relationship observed between DA concentration and BP measurements 30 mins after amphetmaine supports a competitive occupancy model. Should this correlation remain at four hours the 18% decrease in raclopride binding would suggest some 30 fold elevation of extracellular DA. However, microdialysis measurements in rats maintained under isoflurane anaesthesia recovered by 2.5 hours (data not shown) suggesting either a mismatch between intra- & extra-synaptic DA concentrations or a non-competitive component of changes in radiotracer BP. Long term changes in receptor population due to internalisation, previously postulated as a possible mechanism for sustained BP changes, can be discounted by observation of full recovery of [ 11C] binding at 24 hours. 1. Laurelle M, Iyer RN, Al-Tikriti MS et al. Synapse 25:1 (1997) 2. Carson RE, Channing MA, Vuong B-K et al. Physiological Imaging of the Brain with PET Academic Press (2001) 3. Bloomfield PM, Myers R, Hume SP et al.. Phys Med Biol. 42:389-400 (1997) 4. Laruelle M. J Cereb Flow Metab 20:423-451 (2000)

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

BASIC BIOLOGY

The effect of natural rewards and abused drugs on the dopamine system: similarities and differences Alain Dagher*†, Isabelle Boileau*†, Dana Small*†‡§, Chawki Benkelfat†, Jean-Marc Asaad†, Robert Pihl†, Marco Leyton† *McConnell Brain Imaging Centre, Montreal Neurological Institute †McGill University ‡Northwestern University School of Medicine The anhedonia hypothesis of drug addiction posits that drugs of abuse target the same neural systems as natural rewards such as food and sex (1). The most likely candidate brain region is the mesolimbic dopamine system. In animals, dopamine is released in the nucleus accumbens (NAC) in response to food, sexual mates, and to almost all drugs of abuse. Different theories propose that dopamine acts: by mediating the hedonic impact of a reinforcing stimulus, by promoting associative learning about the stimulus, or by serving as an incentive to the consumption of the stimulus. The anhedonia hypothesis has been criticized on the grounds that food-induced dopamine release in the NAC is only present with novel foods, and that dopamine blockade or depletion do not impair the hedonic response to food (2, 3). There are two predictions one can make from the anhedonia hypothesis: (1) dopamine release in response to natural reinforcers will correlate with the degree of pleasure experienced; (2) natural reinforcers and drugs of abuse will release dopamine in the same brain areas. We sought to test these two hypotheses in a series of three experiments in human subjects. Dopamine release was measured in healthy human volunteers using positron emission tomography (PET) and [11C]raclopride. All subjects underwent two scans, labeled “activation” and “baseline”, in counterbalanced fashion. Experiment 1: eight males were scanned following either d-amphetamine (0.3 mg/Kg p.o. one hour before [11C]raclopride injection) or placebo. Experiment 2: seven males were scanned following either alcohol (1ml/kg of 95% USP ethanol over 15-minutes 30 minutes prior to [11C]raclopride injection) or orange juice. Experiment 3: seven individuals (2 males) were scanned after an overnight fast following either a favourite meal (consumed 30 minutes prior to [11C]raclopride) or no food intake. All subjects also underwent anatomical MRI. All studies were analyzed with statistical parametric maps of the change in [11C]raclopride binding potential (BP) as previsouly described (4) and regions of interest drawn on the MRI. Experiment 1: Compared to placebo, d-amphetamine increased extracellular DA levels, as measured by decreased [11C]raclopride BP. Significant effects were seen in the ventral but not dorsal striatum (Fig. 1). Self-reported drug-induced ‘drug wanting’ correlated with the change in BP in the ventral striatum (r⫽0.83, p⫽0.01), but not in dorsal putamen or caudate. Changes in [11C]raclopride BP did not correlate with mood. Experiment 2: The t-statistical maps showed bilateral reductions in [11C]raclopride BP in the NAC in the alcohol compared to the alcohol-free condition (Fig. 1). ROI analysis confirmed this finding with a mean difference in the NAc of -20.1% (p ⫽ 0.001). There were smaller differences in the caudate (-6.2%, p ⫽ 0.049) and putamen (-7.0%, p ⫽ 0.016). Experiment 3: Statistical parametric maps of change in [11C]raclopride BP showed a significant reduction in the post-feeding condition bilaterally in the dorsal putamen caudate nucleus (Fig. 1). There were no changes elsewhere in the striatum, including the NAC. We performed stepwise linear regression analyses of subjective ratings (meal pleasantness, hunger or satiety) and BP change. Pleasantness ratings predicted 82% of the variance of the change in BP in the dorsal caudate (F(1,6) 23.1; p ⫽ 0.005). No relationship was observed between change in BP and hunger or satiety ratings in this region (p ⫽ 0.3 and 0.8 respectively). In the dorsal putamen, pleasantness accounted for the majority of the variance (87%{F(1,6) 34.5; p ⫽ 0.002). However, here hunger ratings also significantly predicted BP change, accounting for an additional 8% of the variance (F(2,6) 9.3; p ⫽ 0.04). Satiety ratings did not contribute significantly (p ⫽ 0.5). Thus, we found that food and two drugs of abuse, alcohol and amphetamine, all promoted dopamine release in human subjects. Moreover, the amount of dopamine release following a meal correlated with the self-rated pleasantness of the meal. While these results fit with the anhedonia hypothesis of Wise, there was an important double-dissociation between food and drugs. Food caused dopamine release in the dorsal but not the ventral striatum, consistent with animal studies showing that the dorsal striatal dopamine mediates feeding behaviour (5). Conversely, drugs promoted dopamine release in the ventral but not the dorsal striatum. 1.R. A. Wise, Curr Opin Neurobiol 6, 243-51 (1996). 2.K. C. Berridge, Neuroscience & Biobehavioral Reviews 20, 1-25 (1996). 3.J. D. Salamone, M. S. Cousins, B. J. Snyder, Neuroscience & Biobehavioral Reviews 21, 341-59 (1997). 4.J. A. Aston, et al., Neuroimage 12, 245-256 (2000). 5.M. S. Szczypka, et al., Neuron 30, 819-28 (2001).

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

BASIC BIOLOGY

Specific Binding of [11C]Raclopride and N-[3H]Propyl-Norapomorphine to Dopamine Receptors in Striatum of Living Mouse: Occupancy by Endogenous Dopamine and GTP-free G-Protein Paul Cumming*, Albert Gjedde*, John Hilton†, Robert C. Dannals†, Ursula Scheffel†, Dean F. Wong† *Center of Functionally Integrative Neuroscience, Aarhus University, Aarhus, Denmark †Division of Nuclear Medicine, Department of Radiology, Johna Hopkins Medical Institutions, Baltimore, Maryland, USA According to the ternary complex model of G-protein-linkage to receptors, agonists increase the affinity of the receptors for the G-protein. The model predicts that an endogenous agonist’s constant of inhibition towards an agonist radioligand is lower than towards an antagonistic radioligand. We hypothesised that competition from endogenous dopamine in striatum of living mice should have a greater effect on the binding of the D2-3 partial agonist N-[3H]propylnorapomorphine ([3H]NPA), than on the binding of the D2-3 antagonist [11C]raclopride. The baseline binding potential (pB(0)), defined as the ratio of bound-tounbound ligand in the absence of competition from endogenous dopamine, was measured in mouse striatum for [11C]raclopride (pB (0) ⫽ 8.5) and, simultaneously for [3H]NPA (p’B(0) ⫽ 5.3). The baseline was established by treatment with alpha-methyl-p-tyrosine (AMPT) and reserpine. Relative to these baseline values, in saline-treated mice, the pB of [3H]NPA declined 52%, while the pB of [11C]raclopride declined only 30%, indicating greater sensitivity of the former compound to inhibition by synaptic dopamine. Furthermore, amphetamine decreased the pB of [3H]NPA to a greater extent (73%) than that of [11C]raclopride (43%), both relative to the reserpine condition. For both radioligands, the occupancy of the dopamine receptors by endogenous agonist obeyed Michaelis-Menten kinetics over a wide range of agonist concentrations established by the pharmacological treatments The graph below summarizes the decline of binding potentials of raclopride and NPA under different pharmacological challenges (panel A), the relationship between inhibited and non-inhibited binding potentials of NPA and raclopride (panel B), the relationship between occupancy (␴ a) and normalized concentration (␹ a) of the putative endogenous inhibitor (dopamine) determined with Figure 1. Calculated dopamine concentrations and occupanNPA or raclopride (panel C), and the ratio between the nor- cies. malized NPA- and raclopride-derived concentrations (␹⬘a /␹ a) of this inihibitor as a function of its normalized NPA-derived concentration (␹⬘a) (panel D): The apparent inhibition constant of endogenous dopamine depended on the dopamine occupancy and declined to a value 1.66 times greater for [3H]NPA than for [11C]raclopride at its highest occupancies. The results are consistent with the hypothesis that agonist binding is more sensitive than antagonist binding to competition from endogenous dopamine. Dopamine agonist ligands may therefore be superior therefore to benzamide antagonist ligands for the estimation of dopamine receptor occupancy by endogenous synaptic dopamine. Figure 2 shows the relationships between the calculated normalized concentrations and occupancies of G-protein as a function of the concentration of the putative endogenous inhibitor (dopamine): In panels A-D, ␹ g and ␴ g refer to G-protein concentration Figure 2 and occupancy, respectively, and ␹⬘a and ␴⬘a to dopamine concentration and occupancy, respectively, determined with NPA. The analysis of the effect of dopamine occupancy on the inhibition of [3H]NPA binding indicated a limited supply of G-protein with a maximum ternary complex fraction of 40% of maximum agonist binding capacity.

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

BASIC BIOLOGY

5-HT7 and Possibly 5-HT6, but not 5-HT1A, 5-HT2A or 5-HT4 Blockers, Reduce the Fluoxetine-Mediated Increase in R-[C-11]Rolipram Binding in Rat Brain Miran Kenk, Celia M. Lourenco, Sylvain Houle, Jean N. DaSilva Centre for Addiction and Mental Health PET Centre, University of Toronto, 250 College St., Toronto, Canada M5T 1R8; [email protected] Introduction Phosphodiesterase-4 (PDE4) is the main enzyme that hydrolyses cAMP produced following stimulation of many G-protein coupled receptors. PDE4 is encoded by 4 distinct genes, generating 4 subtypes PDE4A-D, all of which bind the inhibitor R-rolipram saturably, reversibly and with high affinity (Kd 1-2 nM). Previous studies demonstrated that cAMP regulates PDE4 activity and density, by altering protein phosphorylation and gene expression (1). The cAMP signal transduction pathway is disrupted in several neuropsychiatric disorders. Takahachi et al. (2) have recently reported that chronic treatments with antidepressants, including selective serotonin reuptake inhibitors (SSRI) fluoxetine and sertraline, increased the expression levels of both mRNA and protein of PDE4A and B in rat frontal cortex. We have developed R-[ 11C]rolipram as a probe to study PDE4 before and after treatment using PET (3). Dose-response effects of pre-treatment with SSRI fluoxetine or sertraline on R-[ 11C]rolipram binding were evaluated in rat brain. Establishment of which serotonin receptor subtype is responsible for the fluoxetine-mediated increase in R-[ 11C]rolipram binding was also carried out in this study. Methods R-[ 11C]Rolipram was prepared as described previously (4). Dose-response studies were carried out with fluoxetine (0.5-30 mg/kg) or sertraline (1-40 mg/kg). Rats were then injected with R-[ 11C]rolipram 3 h later, killed at 45 min post-injection, and dissected as recently reported (3). Non-specific binding was determined following co-injection of a saturating dose of R-rolipram (1 mg/kg (3)). Blocking of serotonin receptor subtypes was achieved using various selective or non-selective antagonists 30 min before fluoxetine treatment, at various doses (WAY 100635 3 mg/kg; ritanserin 2.5 mg/kg; SDZ 205557 5-20 mg/kg; Ro 04-6790 5-20 mg/kg; olanzapine 3-10 mg/kg; SB 269970 (0.5-20 mg/kg); risperidone 0.5-20 mg/kg; clozapine 1-50 mg/kg). Results The dose producing the highest effect on R-[ 11C]rolipram uptake across brain regions was 10 mg/kg for fluoxetine and 40 mg/kg for sertraline. Both SSRI increased similarly R-[ 11C]rolipram retention (22-51%) in comparison to controls, and this increase was due to specific binding to PDE4. High doses of selective 5-HT1A, 5-HT2A or 5-HT4 antagonists (WAY 100635, ritanserin and SDZ 205557, respectively), previously shown to be centrally active, had no effect on the fluoxetine-meditated increase in R-[ 11C]rolipram binding. In comparison to fluoxetine, a significant reduction was obtained with both the selective 5-HT7 antagonist SB 269970 and non-selective 5-HT6 antagonist olanzapine in various brain regions. Non-selective antagonists risperidone (5HT7) and clozapine (5-HT6&7) had minimal effect on fluoxetine-mediated increase in R-[ 11C]rolipram binding. However, even though olanzapine is a potent antagonist at 5-HT6 receptor (5), like risperidone and clozapine, it also displays effects at many other neuroreceptor systems. Further studies are currently in progress. Conclusions These results indicate that 5-HT7 and possibly 5-HT6, but not 5-HT1A, 5-HT2A or 5-HT4 receptors, are involved in the increase in R-[ 11C]rolipram binding observed after fluoxetine treatment. (Supported by Eli Lilly Canada) 1. Houslay M.D., Sullivan M., Bolger G.B. Adv. Pharmacol. 44: 225-342 (1998) 2. Takahashi M., Terwilliger R. et al. J. Neurosci. 19: 610-618 (1999) 3. Lourenco C.M., Houle S., et al. Nucl. Med. Biol. 28: 347-358 (2001) 4. DaSilva, J.N., Lourenco C.M. et al. J. Label. Compd. Radiopharm. 44: 373-384 (2001) 5. Bymaster F.P., Falcone J.F. et al. Eur. J. Pharmacol. 430: 341-349 (2001)

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METHODOLOGY

The Development of a Motion Correction System in Neurological PET using List Mode Data Acquisition and a Polaris Tracking System Peter M. Bloomfield*, Terry J. Spinks*, Johnny Reed†, Leonard Schnorr*, Anthony M. Westrip*, Lefteris Livieratos*, Roger Fulton‡, Terry Jones§ *Imaging Research Solutions Ltd., Hammersmith Hospital, London, UK †CPS Inc., 810 Innovation Drive, Knoxville, Tennessee, USA ‡Royal Prince Alfred Hospital, Sydney, NSW, Australia §Imaging Science and Biomedical Engineering, Manchester University, Manchester, UK Introduction With the development of higher resolution positron emission tomographs, there is increasing need to measure and correct for patient motion during acquisition. Correcting for the motion prior to image reconstruction allows the inherent tomograph resolution to be realised and potentially to minimise errors in the measurement of time-activity curves in small structures. This paper describes the development and implementation of a system to monitor motion during a PET acquisition and then correct for the motion post acquisition. Methods The ECAT Exact3D has the ability to acquire data in list mode (Spinks et al, 2000). During list mode acquisition, the emission events (prompt and random) are written directly to the acquisition disk. At 1 msec intervals a time tag is inserted into the event stream. The state of the 4 gating inputs is inserted into bits 27-30 of the time tag. There are two hardware components to the motion tracking system. The first is a Polaris tracking system (Northern Digital, Waterloo, Ontario, Canada), which uses a near infrared source to accurately track the location of four reflective markers, which are attached to the subject’s head via a cap. The second piece of hardware was developed in-house to insert a 4 bit random number, in the range 1-15, into the list mode event stream. The hardware generates 4 TTL output pulses, which are fed into the gating inputs on the tomograph. Both the Polaris system and pulse generator are controlled through standard serial communications. The complete motion tracking system is controlled using a standard laptop PC operating under Suse Linux 7.2, which is networked. The motion tracking acquisition is initiated remotely from the tomograph study protocol executive prior to the start of the transmission scan. During acquisition, the Polaris is polled for the current marker location, the random number is inserted into the list mode event stream, and both these values are written to a test file on disk. The default Polaris polling rate is 5 samples/second. Sorting of the list mode data is completed off-line post acquisition. The first task aligns the list mode data with the motion tracking data. The random number in the list mode time tag is extracted and compared to that recorded in the motion tracking file. An exact match between these two sets of data indicates the start of the motion tracking information for the list mode data. Data are corrected on an event by event basis for both normalisation and dead time, prior to correcting for motion. Once the correct position is determined the appropriate sinogram location is then modified. Once the frame time is complete the sinogram is written to disk, prior to further defined frames being sorted if required. A calibration acquisition is required to relate the centre of the Polaris field of view (FOV) to the transaxial centre of a crystal ring (close to one end of the axial FOV) in the tomograph. This defines the space into which the transmission and emission data are moved prior to image reconstruction. Results and Discussion The initial results presented are for a Hoffman brain phantom. Two acquisitions were completed for the phantom centred in the tomograph field of view. The first was made with the phantom stationary, the second involved rotating about (10o), and moving in and out (10 mm.) along, the axis of the tomograph. Images of (a) the stationary phantom, (b) the moving phantom when no motion correction is made, and (c) the motion corrected phantom are shown in figures 1-3. Comparison of the figures 1 and 3 shows clear evidence of the efficacy of the motion correction scheme implemented. The range of motion was also probably greater than would be experienced with a patient. There is a visual impression that the corrected image is smoother than that for no motion. This might be due to under sampling of the motions during acquisition. It is intended to quantify the effect with a line source phantom. Conclusions A system has been developed and implemented to firstly monitor accurately the position of the subject during acquisition, and secondly correct for this motion post acquisition. It is often overlooked, but the ability to monitor subject motion during acquisition is as important as the ability to correct for this motion. We have demonstrated that the system can record motion accurately and that when this information is combined with list mode acquisition, an accurate correction for motion can be applied. Reference Spinks, T.J., Jones, T., Bloomfield, P.M. et al, Phys. Med. Biol., 2000, 45 2601-2618

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GAS PHASE PRODUCTION OF 11CD3I. SYNTHESIS AND BIOLOGICAL EVALUATION OF S-[N-METHYL-D3-11C]CITALOPRAM AND S-[N-METHYL-11C]CITALOPRAM Jacob Madsen*, Betina Elfving†, Kim Andersen‡, Gitte Moos Knudsen†, Lars Martiny* *PET & Cyclotron Unit 3982, Copenhagen University Hospital, Blegdamsvej 9, 2100 Copenhagen Ø, Denmark †Neurobiology Research Unit, Rigshospitalet ‡H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark Introduction Citalopram is a selective serotonin reuptake inhibitor (SSRI) with a high affinity for serotonin reuptake sites, kd ⫽ 0.7nM in rats at 20°C.(1) With a medium lipofilicity, LogP ⫽ 3.6, citalopram thus fulfill some of the criteria for being a suitable Positron emission tomography (PET)-ligand candidate. However, the carbon-11 labelled analog of S-citalopram, S-[N-methyl-11C]citalopram 2 has in human studies not proven successful as a PET-ligand, because a very low signal to noise ratio was observed in these studies(2). In ex vivo rat studies, however, S-[N-methyl-11C]citalopram reaches an acceptable thalamus to cerebellum ratio of 1.8. From clinical studies, desmethylcitalopram is known as the quantitatively major metabolite from citalopram (3) and, therefore, we speculated that metabolism of the carbon-11 labelled methylgroup may cause formation of nonselective labelled metabolites, that do not readily leave the brain, but remain as non-specific binding signals. Therefore we hypothezised, that substitution of 11CH3I with 11CD3I in the synthesis of carbon-11 labelled S-citalopram could reduce the demethylation rate through a kinetic isotope effect and thereby delaying metabolism and non-specific binding signal. Results and Discussion Recently, it has been reported that 11CH3I can be produced from 11CH4 with high specific activity in a gas phase iodination reaction.(4) We have extended this method to include the synthesis of 11CD3I, also in high specific activity. 11CH4 is produced by bombardment of a gas target containing N2/H2. In a similar fashion 11CD4 is produced when the target gas is H2/D2. In separate runs 11CH3I and 11CD3I have been used in N-alkylation reactions with S-desmethylcitalopram oxalate 1 to afford the carbon-11 labelled ligands, S-[N-methyl11C]citalopram 2 and S-[N-methyl-D3-11C]citalopram 3 (Scheme 1). The total synthesis time was 45 min. and the specific activity of the final product was 1-3.5 Ci/␮mol (37-130 Scheme 1. Synthesis of two radiolabeled ligands, 2 and 3. GBq/␮mol) EOS. The radiochemical yield was 34% (decay corrected, and based on trapped 11CH3I) Biological evaluation In 6 male Sprague-Dawley rats 5-10 MBq of S-[N-methylD3-11C]citalopram or S-[N-methyl-11C]citalopram in 6-8% EtOH were injected in the tail vein. After 60 min, the rats were decapitated. Seven brain regions were dissected, weighed and the radioactive content of the tissue was determined in a gamma counter to calculate the ratios between the ROI’s and the reference region, cerebellum. The ratios were determined as [Bq/g tissue(ROI)]/[Bq/g cerebellum], Figure 1. Our findings with S-[N-methyl-11C]citalopram 2 was in good agreement with literature values. No significant difference was found in the ratios between the selected ROI’s and cerebellum when S-[N-methyl-11C]citalopram 2 and S-[Nmethyl-D3-11C]citalopram 3 were compared.

Figure 1. Distribution of 1 and 2 between ROI’s and cerebellum in rat brains.

Conclusion We have developed an efficient procedure to produce 11CD3I in a gas phase reaction with high specific activity. The system is easily setup to produce the desired ligands, by changing the target gas. S-[N-methyl-11C]citalopram 2 and S-[N-methyl-D3-11C]citalopram 3 were synthesized in high specific activity and evaluated in ex vivo rat studies. No significant differences were observed in the ROI/cerebellum ratios for the two ligands. References (1) Plenge P. and Mellerup E. T. J Neurochem 56: 248 (1991) (2) Hume S.P. et. al. Nucl. Med. Biol. 19:8 851 (1992) (3) Olesen O. Et. al. Pharmacology 59:6 298 (1999) (4) Larsen P. et. al Appl. Radiat. Isot. 48:2 153 (1997)

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A PET EXAMINATION OF [11C]MADAM IN MONKEY AND HUMAN BRAIN—A SELECTIVE RADIOLIGAND FOR THE SEROTONIN TRANSPORTER Johan Lundberg*, Christer Halldin*, Jari Tarkiainen*, Judith Sovago*, Johnny Vercouillie†, Balazs Gulyas*, Denis Guilloteau†, Patrick Emond†, Sylvie Chalon†, Jukka Hiltunen‡, Lars Farde* *Karolinska Institutet, Stockholm, Sweden †INSERM, Tours, France ‡MAP, Helsinki, Finland Objectives The serotonin system is of central interest in relation to human brain functions. The role of the serotonin transporter (5-HTT) in the treatment of psychiatric disorders such as depression is significant. The diphenyl sulfide derivative MADAM (N,N-dimethyl2-(2-amino-4-methylphenylthio)benzylamine) is a highly potent and selective compound for the 5-HTT (Ki5-HTT ⫽ 0.013 ⫾ 0.003 nM, KiDAT ⫽ 840 ⫾ 100 nM, KiNET ⫽ 699 ⫾ 80 nM). We report the PET-examination of MADAM labeled with 11C in two different positions in cynomolgus monkeys and the regional distribution in five healthy human subjects. Methods Authentic MADAM compound and the required precursors were synthesized. MADAM was labeled with carbon-11 in two different positions: in the methyl group of the phenyl ring, [p-11C-methyl]MADAM and of the tertiary amino moiety, [N-11Cmethyl]MADAM. [11C]Methyl iodide or [11C]methyl triflate was used to react in a Stille coupling reaction with the Bu3SnADAM precursor using a palladium (0) catalyst or to react in an N-methylation with the N-desmethyl-MADAM. [p-11Cmethyl]MADAM and [N-11C-methyl]MADAM were injected i.v. into Cynomolgus monkeys and examined with PET. Pretreatment experiments were performed with citalopram and GBR 12909. Metabolism was measured in monkey plasma by HPLC. The regional brain distribution of [N-11C-methyl]MADAM was then examined in five healthy human subjects. The cerebellum, a region almost devoid of 5-HTT, was used as a reference region for free radioligand and nonspecific binding in brain. Specific [11C]MADAM binding to 5-HTT was defined as the difference between radioactivity in the region of interest and the cerebellum. Results [p-11C-methyl]MADAM and [N-11C-methyl]MADAM were prepared in sufficient yields for PET studies. After i.v. injection of any of the two 11C-labeled MADAM there was a high and comparable accumulation of radioactivity in the monkey brain (3-5% after 15 min). There was high uptake of radioactivity in the brainstem, thalamus and striatum with a ratio to cerebellum of about 1.6-2.1 obtained at 75-85 minutes. In the pretreatment experiments citalopram (5 mg/kg) markedly reduced the radioactivity in all examined brain regions but the DAT ligand GBR 12909 (10 mg/kg) had no evident effect. In human subjects radioactivity was three to four times higher in the brainstem, thalamus and striatum at 90 minutes when compared with the cerebellum. For the neocortical regions the ratio was about two. Specific [11C]MADAM binding approached equilibrium conditions at 70-90 minutes. There was no significant effect on the rate or pattern of labeled metabolites in plasma which were all polar (15-20% and 30-40% unchanged radioligand at 45 minutes in monkey and human, respectively). Conclusion There is no support for radioactive metabolites entering the brain as [p-11C-methyl]MADAM and [N-11C-methyl]MADAM demonstrate no significant difference with regards to brain kinetics or metabolism in monkey. The results support that [11C]MADAM is a suitable selective radioligand for PET-imaging of the 5-HTT in man and for determination of drug induced 5-HTT occupancy in relation to clinical drug treatment.

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N,N-dimethyl-2-(2-amino-4-methylphenylthio)benzylamine or MADAM is a serotonin transporter ligand with high affinity and specificity: in vitro characterization Sylvie Chalon*, Jari Tarkiainen†, Lucette Garreau*, Hakan Hall†, Patrick Emond*, Johnny Vercouillie*, Lars Farde†, Jean-Claude Besnard*, Christer Halldin†, Denis Guilloteau* *INSERM U316, Tours, France †Karolinska Institute, Stockholm, Sweden The serotonin transporter (SERT) which actively clears serotonin from the synaptic cleft is a key-protein in the regulation of serotoninergic transmission. Alterations of the SERT are thought to be involved in several neurological and psychiatric disorders such as neurodegenerative diseases, depression and dementia. Exploration of this molecular target could therefore be of interest for help in the diagnosis and treatment of these diseases. Recently, several compounds from the diphenyl sulfide family have been proposed as potent ligands of the SERT. Among these ligands we described the N,N-dimethyl-2-(2-amino-4-methylphenylthio)benzylamine or MADAM, which can be used as an in vitro probe when labelled with 3H, as well as a PET scintigraphic tracer when labelled with 11C. We present here the pharmacological properties of [3H]MADAM in vitro on rat and human cerebral tissues. In vitro saturation and competition studies were performed on cerebral membranes from rat cortex. After Scatchard analysis of saturation experiments, the Kd and Bmax of [3H]MADAM were found to be 60 ⫾ 9 pM and 543 ⫾ fmol/mg protein (mean ⫾ SD), respectively. The pharmacological profile of specific [3H]MADAM binding was studied using drugs known to bind to the SERT, dopamine and noradrenaline transporters. The rank order of potency of these drugs, assessed by measurement of Ki values, was MADAM ⫽ ADAM ⬎ paroxetine ⬎ citalopram ⬎ PE2I ⬎ nisoxetine ⬎ GBR12935 ⫽ desipramine. The localization of [3H]MADAM throughout the rat and human post-mortem brain was studied by binding on cerebral sections followed by autoradiographic analysis. Rat cerebral sections were incubated for 60 min at 22°C with 500 pM of [3H]MADAM in phosphate buffer pH 7.4 either alone (total binding) or in the presence of 1 microM paroxetine (non-specific binding). Exposure was carried out for 4 weeks on sensitive films, and regional absorbance measured and converted in fmol/mg tissue on identified anatomical regions using the Biocom analyser software. The localization of [3H]MADAM binding was consistent with the repartition of SERT in rat brain regions such as the frontal cortex, latero-dorsal thalamus, superior colliculi and raphe nuclei. This binding was totally abolished in the presence of paroxetine. Human post-mortem cerebral sections of 100-microM thickness were incubated for 60 min at 22°C in phosphate buffer pH 7.4 either alone (total binding) or in the presence of 10 microM fluoxetine (non-specific binding). Exposure was carried out on phosphor image plates and analysed using the Fuji application software Science Labs. The highest densities of [3H]MADAM binding were seen in the anterior cingulate cortex and the superior colliculus, with slightly lower densities in the caudate and putamen. The only neocortical region with substantial [3H]MADAM binding was, in addition to the cingulate cortex, the insular cortex. The labelling of SERT with [3H]MADAM to the regions mentioned above was to a large extent abolished by the addition of excess of fluoxetine. These findings demonstrated that [3H]MADAM bound in vitro to the SERT with a very high affinity and specificity. The affinity of [3H]MADAM (60 pM) was more than 2 times better than those described for the most used in vitro radioactive probes of the SERT such as [3H]paroxetine and [3H]citalopram. Competition studies showed that [3H]MADAM had around 1000-fold selectivity for the SERT over the dopamine and noradrenaline transporters. This selectivity was similar to that obtained for other recently described SERT ligands such as ADAM (2-((2-((dimethylamino)methyl)phenyl)thio)-5-iodophenylamine) and DASB (3-amino-4-(2-dimethylaminomethyl-phenylsulfanyl)-benzonitrile). In vitro binding studies on rat and human brain sections showed a widespread occurrence of [3H]MADAM binding throughout cerebral regions, fitting well with known SERT localization. Highest levels of [3H]MADAM were found cerebral regions richest in SERT such as the raphe nuclei, superior colliculi, and cortex, with a low non-specific background. From these whole findings it can be concluded that (1) MADAM could be useful for further in vitro and in vivo studies of the SERT, especially in animal models involving SERT abnormalities, (2) MADAM could be useful for in vivo study of the distribution, pharmacology and pathophysiology of the SERT in the human brain.

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Two new C-methyl analogues of WAY-100635–preparation, labeling with carbon11 and evaluation as PET radioligands for brain 5-HT1A receptors in monkey V.W. Pike*, J. Tarkiainen†, S. Marchais‡, J.A. McCarron§, C. Halldin†, J. Sovago†, B. Gulyas†, P. Truong†, H.V. Wikstrom‡, L. Farde† *Molecular Imaging Branch, National Institute of Mental Health, Building 1, B3-10, 1 Center Drive, Bethesda, MD 20892, USA. †Karolinska Institutet, Department of Clinical Neuroscience, Psychiatry Section, Stockholm, S-171 76, Sweden. ‡Department of Medicinal Chemistry, University Center for Pharmacy, University of Groningen, Antonius Deusinglaan 1, NL-9713 AV Groningen, The Netherlands. §MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Ducane Road, London, W12 ONN, UK. Introduction [carbonyl - 11C]WAY-100635 ([ 11C]N-(2-(1-(4-(2-Methoxyphenyl)piperazinyl)-ethyl))-N-(2-pyridinyl)cyclohexanecarboxamide) is an effective radioligand for imaging human brain 5-HT 1A receptors with PET, and is currently the most popular radioligand for PET study of this receptor in clinical research and drug development. However, [carbonyl- 11C]WAY-100635 has some drawbacks for measuring relative regional receptor densities. These include rapid metabolism (acting against definition of an arterial-input function for compartmental modeling) and very low non-specific binding (detracting from the accuracy of applying a simplified reference tissue model). Hence, a search for a PET radioligand without these limitations is warranted. It is known that replacement of the cyclohexyl group in WAY with a bulkier cycloalkyl group can counter primary metabolism by amide hydrolysis. Introduction of a smaller alkyl group proximal to the amide bond might also block or retard metabolism without increasing lipophilicity too much for attainment of favourable radioligand behavior. Here we report the effects of introducing a methyl group at either of two carbon atoms alpha to the amido group on radioligand behavior and metabolism. Methods Lipophilicity (LogP) values for ligands (free base) were calculated by computer program (Pallas). The ligands with a methyl group on either the alpha carbon of the cyclohexyl group (SWAY) or the C2H 4 linker (JWAY) were each prepared from commercially available materials in 4 steps, and fully characterized to assure identity and purity. The two ligands were tested for affinity and intrinsic activity at 5-HT1A receptors in vitro. SWAY was labeled with carbon-11 in its O-methyl group by methylation of the corresponding phenol with [ 11C]iodomethane and JWAY in its carbonyl group by acylation of an amino precursor with [carbonyl- 11C]cyclohexanecarbonyl chloride. Each no-carrier-added radioligand was evaluated by PET experiments in cynomolgus monkey (⬃ 5 kg), which included the analysis of serial plasma samples for radioactive metabolites by gradient HPLC on a reverse phase column. Results LogP values for SWAY and JWAY were calculated to be 3.42 and 3.81, respectively (c.f. 3.28 for WAY-100635). SWAY and JWAY were found to be high affinity antagonists at 5-HT 1A receptors (Ki ⫽ 2.3 and 0.91 nM, respectively). Each 11C-labeled ligand was obtained in high radiochemical purity, chemical purity and specific radioactivity, and in adequate yield for PET experiments. After intravenous injection of [ 11C]SWAY (41 MBq) into monkey, maximal radioactivity uptake in brain was 3% at 4.5 min. At 72 min the radioactivity concentration in 5-HT 1A receptor-rich regions was only fractionally higher than in receptor-devoid cerebellum. [ 11C]SWAY decreased to 50% of the radioactivity in plasma at 45 min. All radioactive metabolites eluted ahead of the radioligand in the HPLC analyses. After intravenous injection of [ 11C]JWAY (54 MBq) alone into a monkey, maximal radioactivity uptake in brain was 4.8% at 2.5 min. This declined to 1.2% of dose at 90 min. The radioactivity concentration in 5-HT 1A receptor-rich brain regions at 90 min was markedly greater than in cerebellum (e.g. 2.58-fold in hippocampus; 2.48-fold in cingulate cortex; 2.39-fold in amygdala; 2.2-fold in frontal cortex; and 2.15-fold in temporal cortex). Cerebellar radioactivity concentration was 14% of its peak value at 90 min. In 5-HT 1A receptor-rich regions, radioactivity concentration minus cerebellar radioactivity concentration reached a maximum at ⬃30 min. In a second PET experiment, the monkey was treated with WAY-100635 (0.5 mg/kg) at 12 min before [ 11C]JWAY injection. At 90 min the ratio of radioactivity concentration in receptor-rich regions to that in cerebellum was greatly reduced (e.g. to 1.42 in hippocampus; 1.22 in cingulate; 1.37 in amygdala; 1.08 in frontal cortex; 1.27 in temporal cortex). [ 11C]JWAY represented 12% of the radioactivity in plasma at 45 min. All radioactive metabolites eluted ahead of the radioligand in the HPLC analyses. Conclusions The methyl groups in SWAY and JWAY do not block metabolism in monkey. Each radioligand is metabolized to give more polar radioactive compounds. SWAY and JWAY readily enter brain. SWAY is not an effective PET radioligand in monkey. [ 11C]JWAY (racemic) gives a sizeable 5-HT 1A receptor-selective signal in monkey and now merits further evaluation as a homochiral radioligand in humans. Acknowledgement The authors are grateful to the Human Frontier Science Program Organization for their support of this research (grant number: RG0235/1998-B), and to Dr. Lise T. Brennun for measurements in vitro.

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Effects of Tryptophan Depletion on [11C]McN5652 Binding in Baboons Ramin V. Parsey*†, John Castrillon*, J. John Mann*†‡ *Department of Neuroscience, New York State Psychiatric Institiute †Department of Psychiatry, Columbia University ‡Department of Radiology, Columbia University Introduction Given several assumptions, PET imaging studies allow us to quantify the binding potential, the ratio of the total number of receptors to the affinity of the ligand to the receptor (B max/K D). One assumption is that the radiotracer is the only compound competing for the binding site of interest at the time of the study. The binding of radioligands to their receptors is dependent on B max and K D, but in some cases binding is influenced by the concentration of the endogenous neurotransmitter in the synapse. For example, some dopamine D2 ligands are sensitive while most D1 ligands are not (Laruelle, JCBF 2000). Amphetamine increases the synaptic concentration of dopamine and causes a decrease in raclopride or IBZM binding. Alpha-methyl-para-tyrosine decreases intrasynaptic dopamine and increases IBZM binding. When sensitive to levels of endogenous neurotransmitter, radioligand binding during states of release and depletion can be informative as to the basal synaptic concentrations of neurotransmitter as well as the releasable stores providing valuable information about the pathobiology of psychiatric and neurological illnesses. Similar methods have not been as easily developed in the serotonergic system. We have previously investigated the role of amphetamine and fenfluramine, powerful secretogogues of serotonin, as well as of other neurotransmitters, on the binding of [11C]WAY 100635 to 5HT1A receptors (Parsey et al., 1999). Now we present data on the effect of depleting synaptic serotonin on the binding of [11C]McNeil 5652 by using the well established tryptophan depletion paradigm in baboons. Methods Three baseline and three tryptophan depleted [11C]McNeil 5652 PET scans were obtained in the same male baboon. Each study was done at least one month apart. On depletions days, five hours prior to the injection of the radiotracer the baboon was lightly anesthetized with ketamine (10 mg/kg i.m.) A nasogastric tube was placed in the stomach and 50 cc of a 3/4 strenght tryptophan depleting shake were introduced (Delgado et al., 1980). Scans were performed under isoflurane anesthesia (1.8% via endotracheal tube) while temperature was kept constant at 37 degrees Celsius by a circulating heated water blanket. The baboon?s head was positioned at the center of the field of view as defined by imbedded laser lines and was scanned for 2 hours after bolus injection of [11C]McNeil 5652. Regions of interest drawn on an MRI scan were transferred to the AIR coregistered frames of PET data and time activity curves were generated. A two compartment kinetic model using the metabolite corrected arterial input function was used to calculate the total volume of distribution (VT ⫽ K1/k2). Binding potential (BP) was defined as VT in the region of interest/VT of the cerebellum, a measure of the free and nonspecifically bound tracer. Results There was a 39% increase in [11C]McNeil 5652 BP in the midbrain (66.0 ⫾ 12.9 mL/g baseline, 92.0 ⫾ 1.58 mL/g post tryptophan depletion, p ⫽0.015). The increase was specific to the midbrain as there were no statistically significant increases in any other brain regions examined: hippocampus, striatum, thalamus, or cingulate cortex (Figure 1). There was no difference in injected mass (14.4 ⫾ 4.6 vs. 12.6 ⫾ 2.0 nmoles) or the cerebellar VT (39.4 ⫾ 18.3 vs. 43.8 ⫾ 2.4 mL/g) between conditions. While the free fraction cannot be measured with this particular ligand, there was still a significant difference between the baseline and tryptophan depletion condition when we use V3?, (VT ROI/VT CER)-1, which is independent of the free fraction (1.79 ⫾ 0.18 vs. 2.10 ⫾ 0.15, p ⫽ 0.045). Conclusions These preliminary results suggest that the brainstem binding of [11C]McNeil 5652 is sensitive to endogenous serotonin, because tryptophan depletion is known to decrease intra-synaptic serotonin release and we find that it increases [11C]McNeil 56522 binding. The reason for the regionally selectivity of this effect to the midbrain remains to be elucidated. This paradigm, if replicated and reliable, could be used in humans to study not only transporter binding, but also the intra-synaptic levels of serotonin. NIMH MH62185.

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STRIATAL ALPHA4BETA2 NICOTINIC ACETYLCHOLINE RECEPTORS IN THE MPTP MODEL OF PARKINSON’S DESEASE: PET STUDY WITH 2-[18F]FLUORO-A-85380 Svetlana I. Chefer*, Alexey G. Mukhin*, Andrei 0. Koren‡, Alane S. Kimes*, Olga A. Pavlova*, John A. Matochik*, Varughese Kurian*, Edward F. Domino†, Andrew G. Horti*, Edythe D. London‡ *Brain Imaging Center, Intramural Research Program, NIDA, 5500 Nathan Shock Drive, Baltimore, MD 21224, USA †Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109 ‡University of California, Los Angeles, CA 90024 Introduction Postmortem assay of brain tissue from patients who died with Parkinson’s disease (PD) reveals abnormally low concentrations of nicotinic acetylcholine receptors (nAChRs) of the alpha4beta2 subtype in the nucleus caudate (NC) and putamen (Put). Up to now it was not possible to investigate in vivo the role of these receptors in pathogenesis of PD because suitable radioligands for in vivo imaging of nAChRs have not been available. 2-[18F]Fluoro-3-[(S)-azetidinylmethoxy]pyridine (2-[18F]Fluoro-A-85380), novel nicotinic agonist, has been synthesized (Horti et al, 1998; Dolle et al, 1998) and evaluated in our laboratory (Horti et al,1998; Chefer et al,1999, 2000) and by others (Valette et al,1999; Dolle et al,1999; Ding et al,2000) as a potential radioligand for in vivo imaging of the alpha4beta2-subtype of brain nAChRs by PET. 2-Fluoro-A-85380 has high affinity and selectivity for the alpha4beta2 subtype of nAChR (Kd 46 pM) and low toxicity compared with epibatidine analogues. Preliminary results of ongoing PET studies in human subjects at National Institute on Drug Abuse under a Phase 1 of IND application indicate that this tracer can be used for imaging of nAChRs in human brain (Kimes et al, 2002). The present study aimed to evaluate the ability of 2-[18F]Fluoro-A-85380 to measure the alterations of nAChRs in a primate model of PD in vivo. Methods Four hemiparkinsonian Macaca nemestrina each received a single unilateral 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intracarotid injection (Emborg and Domino,1998) 10-12 years before the PET scans in the current study. Unilateral striatal dopamine lesions persisted in these animals as demonstrated by PET studies with [11C]DTZB, which binds to vesicular monoamine transporter (Kilbourn et al, 2000). For the current study, two PET scans, control and blocking study with cytisine (1 mg/kg), a partial nAChRs agonist, were performed on each animal under Saffan anesthesia on two separate days after bolus injection of 22.2 - 62.9 MBq/kg (0.6 1.7 mCi/kg) of 2-[18F]Fluoro-A-85380. Data from blocking study were used to evaluate the non-displaceable accumulation of the radiotracer in the NC, Put and cerebellum (Cer). For placement of regions of interests (ROIs), PET images were coregistered with structural magnetic resonance imaging scans of each animal’s brain. Quantitative analysis of 2-[18F]Fluoro-A85380 binding in vivo were performed using the Logan method with the arterial plasma input function corrected for metabolites. Results The average values of specific binding volume distribution (VDsb) were significantly lower in the NC and Put on the lesioned side (by 24% compared with values on the contralateral side). These data are consistent with the results of postmortem assays of striatal nAChRs in PD patients. The values of non-displaceable VD measured in NC and Put did not differ either ipsilateral or contralateral to the side of the MPTP injection. The total VD in the Cer was lower that the nondisplaceable VD value in the striatum. Therefore, use of the Cer as a reference region for quantitative characterization of 2-[18F]Fluoro-A-85380 binding to nAChRs yields values that underestimate VDsb in the striatum and overestimate the percent decline of specific binding on the leisioned side. Conclusions 2-[18F]Fluoro-A-85380 can be used to measure specific binding of nAChRs even in brain regions with moderate nAChRs density (e.g., striatum). Quantitative analysis of specific binding of 2-[18F]Fluoro-A-85380 to nAChRs in vivo indicates that the density of nAChRs declined in the striatum on the side ipsilateral to MPTP injection. These findings support the idea that nAChRs are located on presynaptic dopaminergic terminals and suggests that the loss of dopaminergic terminals in PD procudes a concurrent reduction in the density of nAChRs. We conclude that 2-[18F]Fluoro-A-85380 may be useful for quantitative characterization of nAChRs in patients with PD and other neuropathologies.

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Human Imaging of Nicotinic Acetylcholine Receptors in Vivo: 5IA-SPECT Studies Koichi Ishizu*†, M. Mamede*, T. Mukai*, Y. Iida†, H. Fukuyama‡, H. Saji†, J. Konishi* *Dept. of Nuclear Medicine and Diagnostic Radiology, Graduate School of Medicine, Kyoto University †Dept. of Patho-Functional Bioanalysis, Graduate School of Pharmceutical Sciences, Kyoto University ‡Dept. of Brain Pathophysiology, Graduate School of Medicine, Kyoto University Introduction The nicotinic acetylcholine receptors (nAChR) are widely distributed in mammalian organisms, appearing in the central and peripherial nervous systems. They have been known to take part in various neurophysiological functions, as well as linked to neurodegenerative disease, such as Alzheimer disease. Neuronal nAChRs differ significantly according their multiple subtypes, however it appears that the majority of high affinity nAChRs in the brain comprise the alpha4-beta2 subtype. Therefore, the development of high affinity radioligands suitable for in vivo imaging of nAChRs has been of major interest. Demonstrate the in vivo labeling of high-affinity nicotinic cholinergic receptors (nAChR) in human using I-123 (S)-5-iodo-3-(2azetidinylmethoxy) pyridine, I-123 5-iodo-A-85380 (5IA) a specific radioligand for the alpha4-beta2 subtype. Methods This study was carried out with eight health volunteers (1 female and 7 males), with age range from 20 to 71 years old (45.3 ⫹/21.4). None of them had past history of tobacco use. They received a one-minute intravenous injection of approximately 150 MBq of 5IA at constant rate with an infusion pump. The images were acquired with a three-headed rotating gamma camera system (Picker, Prism 3000) equipped with low-energy, high resolution, fanbeam collimators. Data acquisition was performed in 64 x 64 matrices over 90 minutes (1min/frame) resulting 90 images. Simultaneously, twenty-four arterial blood samples were drawn and the metabolite correction by TLC-plate was done for each subject. Six circular regions of interest were placed for each different brain region on transaxial SPECT images, and a time activity curve (TAC) was generated for each ROI. In this study, a graphical analysis described by Logan and a two-compartment nonlinear fitting method were used for ROI based kinetic analysis to calculate distribution volume (DV) of 5IA. Results Peak plasma activity occurred between 60 and 90 seconds after the injection start of 5IA, decreasing rapidly to 7% to 10% of the peak at 5 minutes after the injection. Analysis of unmetabolized compound demonstrated high parent fraction of the 5IA in the plasma at 10 seconds (97.2% ⫹/- 3.4%), reducing to around 30% at 60 minutes. The cerebral cortex, cerebellum, thalamus, basal ganglia, white matter, brain stem were well visualized in the static image from 20 to 40 min. after injection. The TAC data demonstrated that the peak of thalamus, brain stem and white matter occurred around 50 minutes and around 30 minutes in the other cortices. The DV (ml/ml) (mean ⫹/- SD) using 90 minutes scan were obtained from the ROI based kinetic analyses as follows: 1) Logan Plot analysis: frontal cortex (14.2 ⫹/- 2.0), occipital cortex (12.5 ⫹/- 1.5), cerebellum (16.5 ⫹/- 2.8), thalamus (22.4 ⫹/5.2), basal ganglia (15.6 ⫹/- 2.4), brain stem (20.1 ⫹/- 4.0), and white matter (15.1 ⫹/- 2.82); and 2) two-compartment model analysis: frontal cortex (14.8 ⫹/- 2.2), occipital cortex (13.1 ⫹/- 1.6), cerebellum (17.4 ⫹/- 3.0), thalamus (26.1 ⫹/- 7.4), basal ganglia (17.1 ⫹/- 2.7), brain stem (23.0 ⫹/- 4.8), and white matter (16.6 ⫹/- 2.96). The correlations between DV values from different analyses were 97.6% using 90 minutes scan. Conclusion The 5IA demonstrated larger distribution of nAChR in thalamus and brain stem than that in the other regions. Logan Plot and 2-compartment model analyses showed similar DV values of 5IA.

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Comparison of two methods for quantification of NMDA receptor binding with [ 123I]-CNS 1261: Venous sampling and equilibrium analysis versus arterial sampling and kinetic modelling Rodrigo A Bressan*, Kjell Erlandsson†, Rachel S Mulligan†, Roger N Gunn‡, Vincent J Cunningham§, Jonathan Owens ¶, Ian D Cullum†, Peter J Ell†, Lyn S Pilowsky*† *Institute of Psychiatry, King’s College London, De Crespigny Park, Denmark Hill, London SE5 8AF, UK †Institute of Nuclear Medicine, University College London, London, UK ‡McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, Montreal, Canada §Imaging Research Solutions Ltd, Hammersmith Hospital, London, UK ¶

West of Scotland Radionuclide Dispensary, Western Infirmary, Glasgow, UK

Introduction [ 123I]-CNS 1261, a selective SPET ligand for the phencyclidine intrachannel site, was used to study the distribution of NMDA receptors in healthy volunteers. The aim was to establish an appropriate protocol for clinical studies of psychiatric disorders. Since NMDA receptors are ubiquitously distributed in the brain it is not possible to use a reference tissue model for this tracer, and receptor binding quantification must be done relative to the plasma concentration. With bolus studies, the arterial input function is needed. We developed a bolus/infusion protocol using venous blood samples for quantification. The results were compared with those from a previous study using a bolus protocol and arterial sampling [1]. Methods SPET scans were performed in healthy volunteers using a Prism-3000XP scanner (Marconi/Philips). 6 subjects were scanned for 6 h with a bolus plus constant infusion (B/I) protocol. Venous samples were taken throughout the scan. In one subject, the full arterial input function was also sampled. The total volume of distribution (V T) parameter was used for quantification of receptor binding in a set of brain regions. V T values were determined by equilibrium analysis, as the ratio of the tissue and venous plasma activity concentration, corrected for metabolites, arterial to venous ratio and the slope of the plasma curve. The results were compared with those obtained in the previous bolus study using kinetic modelling. Direct comparison between equilibrium analysis and compartmental modelling was also performed for the subject with full arterial sampling. Results There was a good correlation between the V T values obtained by equilibrium analysis and compartmental modelling in the same subject (slope⫽1.01, R 2⫽0.96, left graph below). There was also a good correlation between the mean V T values in the group of volunteers scanned with the B/I protocol (n⫽6) and those scanned with the bolus protocol (n⫽7) (slope⫽1.00, R 2⫽0.95, right graph below). Conclusion We developed a B/I protocol for [ 123I]-CNS 1261, which is advantageous for clinical studies as it eliminates the need for arterial sampling. The data is encouraging for future work with this ligand, and clinical studies investigating the hypoglutamatergic hypothesis of schizophrenia are underway. Reference [1] Erlandsson K, et al. (2001) J. Nucl. Med., 42:221P

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

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[C-11]N-(2-CHLORO-5-THIOMETHYLPHENYL)-N’-(3-METHOXYPHENYL)-N’-METHYLGUANIDINE ([C-11]GMOM) AS A POTENTIAL PET RADIOTRACER FOR THE PCP/NMDA RECEPTOR: IN VIVO EVALUATION IN RATS AND PET IMAGING IN BABOONS Rikki N. Waterhouse*, Filip Dumont*, Abida Sultana*, Mark Sliftein*, Yasuhiko Sudo*, Norman Simpson†, Marc Laruelle* *Department of Psychiatry and †Department of Radiology, Columbia University, New York, NY USA Objective The N-methyl-D-aspartate (NMDA) ion channel, a major site of action for glutamate, is known to play a central role in many biological functions including neuroprotection, neurodegeneration, long-term potentiation, memory, and cognition. Alterations in normal NMDA channel composition, densities or function have been implicated in the pathophysiology of several neurological and neuropsychiatric disorders such as Parkinson’s Disease, Huntington’s Chorea, schizophrenia, alcoholism and stroke. To provide a potential tracer for imaging the NMDA/PCP receptor, we synthesized and evaluated in vivo the novel radioligand [C-11]GMOM (Ki (PCP site) ⫽ 5.2 ⫾ 0.3 nM; log P ⫽ 2.34). Methods [C-11]GMOM was prepared by heating the phenol precursor with [C-11]methyl iodide at 65 °C in DMF for 5 minutes under basic conditions. The brain distribution and specific binding of [C-11]GMOM was examined through distribution studies in male rats (dose: 6 – 8 MBq, n ⫽ 4-7) and through PET scanning in baboons (dose: 50-80 MBq, scan time: 90 min, 3D mode; ECAT EXACT HR⫹). For PET studies, arterial blood samples were collected, and HPLC analysis was used to determine the formation of plasma metabolites. Regional total distribution volumes (VT) were derived by kinetic analysis using a 3-compartment model, and by Logan graphical analysis. Results [C-11]GMOM was synthesized in yields of 8.4 ⫾ 3.2% (EOS), with specific activities of 1.25 ⫾ 0.25 Ci/␮mol (EOS) and a radiochemical purity of 96.7 ⫾ 1.5% (n ⫽ 5). The brain uptake of [C-11]GMOM in rats was high and peaked by 10 minutes post-injection with 1.30 ⫾ 0.23, 1.28 ⫾ 0.21, 0.96 ⫾ 0.12, 0.72 ⫾ 0.07, 0.75 ⫾ 0.9 and 0.11 ⫾ 0.03 %ID/g in the frontal cortex, parietal cortex, hippocampus, cerebellum, medulla and blood, respectively. The radioactivity cleared from the brain fairly rapidly; regional %ID/g values were decrease by 50-67% a uniform regional distribution was obtained by 60 minutes post-injection. Blocking studies included pre-injection of GMOM or MK801 (2 mg/kg iv) 5 minutes prior to tracer administration, which caused a moderate and uniform decrease (20-38%, p ⬍ 0.05) in all regional %ID/g values, and a slight but non-significant increase in blood activity. No significant changes in regional ratios were found between the block and control animals. It is worthwhile to note that the NMDA receptor is expressed in all regions examined. Blood to regional tissue ratios were also decreased by 17-50% and was highly significant for most regions. However, these data obtained in rats should be considered with the knowledge that potential changes in blood flow and metabolite profiles were not determined. PET imaging in isoflurane-anesthetized baboons with high specific activity [C-11]GMOM (n ⫽ 3) provided fairly uniform VT values (12.8-17.1 ml/g) highest in striatum, thalamus and cingulate cortex and lowest in the parietal and occipital cortexes, and midbrain. Plasma free fraction of [C-11]GMOM averaged 15 ⫾ 1%, and the amount of intact [C-11]GMOM in the plasma was 81, 53, 36, 25, 14, and 8 percent at 2, 4, 12, 20, 40 and 90 minutes, respectivley. Only polar metabolites were noted over the course of the study. Blocking experiments were carried out by pre-administration of MK801 [i.v.; 0.5 mg/kg (n ⫽ 1) or 1.0 mg/kg (n ⫽ 1)] 10 minutes prior to tracer injection. Regional VTs and regional VT ratios were not reduced as a result of MK801 pre-treatment, indicating a lack of saturable binding. However, we note that the affinity of GMOM for the baboon PCP/NMDA receptor and any effect of isofurane (proposed to deactivate NMDA channels) on in vivo [C-11]GMOM binding have yet to be established. Conclusions [C-11]GMOM was successfully synthesized in good yield and high specific activity. In vivo evaluations indicate that [C-11]GMOM is likely not an optimum tracer due to a low degree of measured saturable binding, suggesting that a higher affinity ligand is likely required. Funding for this work was provided for by grants from the National Institutes of Health (NIAAA IP50AA-12870-01 and NIMH MH59342-01).

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

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NMDA Antagonism Impairs Working Memory Performance via Modulation of Dopamine in the Prefrontal Cortex: PET Study in Conscious Monkeys Hideo Tsukada, Mitsuru Suzuki, Shingo Nishiyama, Norihiro Harada, Takeharu Kakiuchi Central Research Laboratory, Hamamatsu Photonics K.K., 5000 Hirakuchi, Hamakita, Shizuoka 434-8601, JAPAN Introduction The N-methyl-D-aspartate (NMDA) receptor is known to interact with the central dopaminergic system in the striatum and nucleus accumbens (1, 2). The dopamine innervation of the prefrontal cortex (PFC) has also to be considered to be highly responsive to psychomotor stimulants. The role of the antagonistic effect of the NMDA receptor has been of interest because the hypofunction of glutamatergic neurotransmission in the PFC, which might be related to the dopaminergic dysfunction, has been hypothesized in schizophrenia. In the present study, we evaluated the effects of MK801, a non-competitive NMDA receptor antagonist, on the prefrontal dopamine D1 receptors (D1R) as measured by [11C]NNC112 (3), as well as on the working memory task performance (WMTP) in conscious monkeys. In addition, the relation between age-related D1R in the PFC and impairment of WMP was also demonstrated. Methods The extrastriatal neocortical D1R binding in the brains of young (6.2⫹/-1.5 years old) and aged (20.2⫹/-2.6 years old) conscious behaving monkeys (Macaca mulatta) was assayed with [11C]NNC112 using a high-resolution animal PET (HAMAMATSU SHR-7700) (4). PET scans were performed for 91 min, and time-activity curves of radioactivity in the cerebellum, used as an input function, and each ROI were fitted to two-compartment model using the least-square fitting method to estimate the kinetic parameters (K1 and k2°f), and distribution volume (DV) in each ROI was calculated as the ratio of K1/k2°f. The special WMTP of monkeys was evaluated in combination of two oculomotor tasks; a visually-guided saccade (VGS) task and an oculomotor delayed response (ODR) task. The levels of glutamate in the PFC were assessed by microdialysis method. MK801 was intravenously administered as doses of 0.03, 0.1 and 0.3 mg/kg. Results and Conclusion: Administration of MK801 at doses of 0.03, 0.1 and 0.3 mg/kg 30 min before [11C]NNC112 injection dose-dependently decreased the DV of [11C]NNC112 binding to D1R in the extrastriatal regions, especially in the PFC. MK801 impaired the special WMTP as assayed by ODR task in a dose-dependent manner without any deficiency of VGS task performance. Glutamatergic neurotransmission in the PFC as measured with microdialysis was decreased by MK801 administration. In aged monkeys, the special WMTP demonstrated significant lower level than that of young animals, showing the decreased DV of [11C]NNC112 binding to the prefrontal D1R compared to young. These results demonstrated that the changes in the dopaminergic neuronal system in the PFC might contribute to the cognitive impairment observed in schizophrenic patients as well as aged peoples. References (1) (2) (3) (4)

Tsukada, H. et al., Synapse, 37:95-103 (2000) Tsukada, H. et al., Synapse, 42:273-280 (2001) Halldin, C. et al., J. Nucl. Med., 39: 2061-2068 (1998) Watanabe, M., et al., IEEE Trans. Nucl. Sci., 44:1277-1282 (1997)

Acknowledgement The Authors are grateful to Professor Christer Halldin for his advices on [11C]NNC112 synthesis. This work was supported in part by Grant-in-Aid for Creative Scientific Research of Japan Society for the Promotion of Science.

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Imaging benzodiazepine receptors in the rat brain using [11C]flumazenil and microPET TD Fryer*, MC Cleij†, FI Aigbirhio*†, JS Beech‡, O Barret*, TA Carpenter*, DK Menon‡, JC Clark*, J-C Baron† *Wolfson Brain Imaging Centre, University of Cambridge †Department of Neurology, University of Cambridge ‡Department of Anaesthetics, University of Cambridge Introduction In this work a commercially available small-animal PET scanner (microPET P4, Concorde Microsystems Inc) has been used to image the central benzodiazepine receptors in the rat brain using [ 11C]flumazenil. The ultimate aim is to use this technique in an in vivo stroke model to assess neuronal loss. The work presented here concerns preliminary assessment of microPET through imaging of controls and assessment of the specific binding level from displacement studies. Methods Using a modified captive solvent method [1], [ 11C]flumazenil was prepared from [ 11C]iodomethane at very high specific activities, up to 15 Ci/mmol at EOS. At the time of injection the specific activity was 5-6 Ci/mmol. For each study the molar dose of stable flumazenil co-injected was kept constant at ⬃0.3 nmol by adjusting the injected activity, which varied between 65-72 MBq. To reveal the specific binding of [ 11C]flumazenil, in two subjects between 80-100mg of stable flumazenil was injected after 9 minutes. The brain was centred in the field of view of the scanner (78mm axial x 200mm diam) to maximise sensitivity and resolution (2mm), and in all cases the image frames were 10x0.5 ⫹ 5x1 ⫹ 15x2 ⫹ 7x5minutes. Transmission scan data was also acquired using a rotating 68Ge point source. The images were reconstructed using 3D filtered backprojection, with corrections applied for normalisation, randoms, dead time and decay. The data will be subsequently re-reconstructed with attenuation and scatter correction once these algorithms have been implemented. A region of interest encompassing the cortical grey matter was drawn on each subject using Analyze. Results The orthogonal views through the control brain 5-20 minutes post-injection (Figure 1) reveal delineation of binding in grey matter, white matter and the cerebellum. The ROI curves (Figure 2) for the displacement experiments show a high level of specific binding (77%, 87%).

Figure 1

Conclusions Preliminary images using high specific activity [ 11C]flumazenil and microPET reveal a high level of specific binding in the rat brain. The expected delineation of binding in grey matter, white matter and the cerebellum is visualised. Work is Figure 2. ROI values (normalised to the peak) for a control and ongoing to extend these qualitative results to quantitative a displacement scan ones through the implementation of additional data corrections (attenuation, scatter, and sensitivity) and input function determination through blood sampling. It is then intended that the technique will be used to examine in vivo neuronal loss in stroke. Reference [1] MC Cleij et al, submitted to Society of Nuclear Medicine meeting, Los Angeles 2002

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[11C]Ro15 4513 PET predominantly labels the ␣5-containing GABA-benzodiazepine receptor in vivo Anne Lingford-Hughes*, Sue Hume†, Adrian Feeney*, Ella Hirani†, Safiye Osman†, Vin Cunningham†, Vic Pike‡, David Brooks§, David Nutt* *Psychopharmacology Unit, University of Bristol, University Walk, Bristol, BS8 1TD, UK. †Imaging Research Solutions Ltd (formerly MRC Cyclotron Unit), Hammersmith Hospital, Du Cane Rd., London, W12 0NN, UK ‡Molecular Imaging Branch, National Institute of Mental Health, Building 1, B3-10, 1 Center Drive, Bethesda, MD 20892-0135, USA (current) §MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Rd., London, W12 0NN, UK Introduction The ␣5-containing subtype of the GABA-benzodiazepine receptor (GABA-BDZR) is highly expressed in limbic structures, including the hippocampus. This receptor subtype is proposed to play an important role in memory. [ 11C]Ro15 4513 has been developed as a PET radioligand, but its use has not been widespread. We have characterized this PET radioligand in man and rat and compared its distribution to [ 11C]flumazenil, which predominantly labels the ␣1-containing GABA-BDZR. Methods Human: Healthy male subjects (3) underwent a [ 11C]Ro15 4513 PET scan (370 MBq) on an ECAT EXACT3D HR⫹⫹ PET scanner. A group of 6 healthy male subjects underwent a [ 11C]flumazenil PET scan (370 MBq) using an ECAT-953B PET scanner in 3D mode. 20 dynamic frames (1 x 30, 4 x 15, 4 x 60, 2 x 150, 2 x 300, 7 x 600 s) of data were acquired over 90 min. During both PET scans, blood samples were taken for HPLC analysis to derive a metabolite-corrected plasma input curve. Spectral analysis was used to generate a volume of distribution (VD) map. A region of interest map was generated to measure radioligand uptake in particular brain areas. Rat. Experiments were performed with [ 11C] Ro15 4513 (⬃10 MBq) or [ 3H]Ro15 4513 (⬃0.3 MBq) in rats, using either a dedicated small animal PET scanner or post-mortem dissection respectively. A. Rat imaging. A dual-HIDAC scanner was used. [ 11C]Flumazenil or [ 11C]Ro15 4513 was given i.v. to isoflurane anaesthetised rats who were scanned for 60 min. A measure of specific binding was generated using the VOI:medulla ratio during the period 20 to 60 min. B. Blocking experiments: benzodiazepine ligands of known subunit selectivity were used as blocking agents and given i.v. 10 min before radiolabelled Ro15 4513 injection : flunitrazepam (5 mg/kg), Ro15 4513 (2 mg/kg), zolpidem (5 mg/kg), L655, 708 (2 mg/kg) or RY80 (2 or 5 mg/kg). The animals were killed at 60 min. The radioactivity content of brain tissues was measured. The medulla/pons was used as a reference region since there were low counts. Results Human. [ 11C]Ro15 4513 uptake was particularly high in limbic regions (Vds: medial temporal cortex 8.17 ⫹ 0.55, anterior cingulate cortex 8.94 ⫹ 1.23, ventral striatum 5.20 ⫹ 1.31) compared to that of [ 11C]flumazenil (Vds: medial temporal cortex 6.00 ⫹ 0.74, anterior cingulate cortex 7.98 ⫹ 0.90, ventral striatum 3.46 ⫹ 0.86). In the cerebral cortex there was an antero-posterior gradient of decreasing levels of [ 11C]Ro15 4513 Vds (frontal : 6.27 ⫹ 0.78; occipital : 5.46 ⫹ 0.32); the reverse was seen with [ 11C]flumazenil (frontal : 6.42 ⫹ 1.25; occipital : 8.61 ⫹ 0.81). In addition a higher [ 11C]Ro15 4513 Vd was seen in the ventral striatum (5.2 ⫹ 1.3) compared with the dorsal striatum (3.96 ⫹ 0.63). A lower [ 11C]Ro15 4513 Vd was seen in the cerebellum compared to [ 11C]flumazenil (2.65 ⫹ 0.21 vs 4.97 ⫹ 0.61). Rat. A. Rat Imaging. The highest levels of [ 11C]Ro15 4513 uptake were seen in the hippocampus (Vd: 2.5 ⫹ 0.2) and frontal/anterior cingulate cortex (Vd: 2.2 ⫹ 0.4), with negligible/low uptake seen in the cerebellum, occipital cortex or striatum (Vds: 1 ⫹ 0.1, 1.5 ⫹ 0.5, 1.5 ⫹ 0.2 respectively). [ 11C]Flumazenil uptake was greater throughout through the brain (Vds: hippocampus: 3.4 ⫹ 0.3, frontal/anterior cingulate cortex: 3.9 ⫹ 0.5, cerebellum: 2.0 ⫹ 0.3, occipital cortex: 2.7 ⫹ 0.6, striatum: 2.4 ⫹ 0.3). However, relative uptake of [ 11C]Ro15 4513 in the hippocampus (2.5 ⫹ 0.2) compared to that in the frontal cortex (Vd: 2.2 ⫹ 0.4) is higher than is seen with [ 11C]flumazenil (Vd: 3.4 ⫹ 0.3 vs 2.2 ⫹ 0.5). B. Blocking Experiments. Pretreatment with benzodiazepines having high affinity for the ␣5 subtype significantly (p⬍0.01) reduced uptake of [ 11C]/[ 3H]Ro15 4513 (flunitrazepam, RY80, L655,708, Ro15 4513), unlike the ␣1 selective, zolpidem. In the hippocampus, these compounds resulted in the following Vds: flunitrazepam 3.1 ⫹ 0.8, RY80 2.3 ⫹ 0.3 L655,708 2.4 ⫹ 0.3, Ro15 4513 1.9 ⫹ 0.4, and zolpidem 18.0 ⫹ 4.1 1 (c.f. control level: 16.9 ⫹ 3.7). Conclusions We have shown that brain uptake of [ 11C]Ro15 4513 in rat and in man has a primarily limbic distribution consistent with labelling of the GABA-BDZR containing the ␣5 subunit. The displacement studies support this since only benzodiazepines with high affinity for the ␣5-containing subtype reduced [ 11C]Ro15 4513 uptake to non-specific levels. This PET radioligand will allow us to explore in vivo the role of this particular GABA-BDZR subtype in man. Acknowledgement This work was funded by an MRC Programme grant.

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Decreased 11C-flumazenil cortical binding after subcortical stroke in baboons with temporary middle cerebral artery occlusion: a marker of selective neuronal loss? Cyrille Giffard*†‡§, Alan R. Young*‡, Florence Me´ zenge*‡, Brigitte Landeau*‡, Annick Brocquehaye*‡, Louisa Barre´ †, Jean Claude Baron* ¶ *INSERM U320 †CEA LRV10 ‡CYCERON §Universite´ de Caen, France ¶

Dept of Neurology, University of Cambridge, UK

Introduction Selective neuronal loss (SNL) after ischaemic stroke in humans is a controversial concept. Although some authors reported incomplete infarction in large areas remote from the actual infarct (pannecrosis), others performing detailed post mortem studies found it to be a very rare occurrence at best. This is an important issue to resolve, however, as it has implications in terms of mechanism of neurological deficit and recovery after stroke, especially in patients in whom severely ischaemic cortex (penumbra) is saved from pannecrosis by early therapeutic thrombolysis. In several SPECT studies, Iomazenil, a radioligand of the central benzodiazepine receptor (cBZR), has been used as an in vivo marker of SNL in humans, and reduced cortical tracer uptake has been reported in patients with subcortical stroke. However, there has been no histological validation of this so far, and reduced ligand binding may arguably reflect mechanisms other than SNL. One suitable way to address this issue would be to use an animal model mimicking the human situation, that is, including temporary middle cerebral artery occlusion (MCAo) and detailed histology in the chronic stage. To this end, we have performed 11C-Flumazenil PET studies in a stroke model in the baboon. Methods under appropriate anaesthesia, 5 young adult male baboons (12-18 kg) underwent a right temporary MCAo, according to the transorbital approach, with reperfusion carried out at 18 hrs. Following orbital reconstruction and closure, the animals were allowed to recover and to survive into the chronic stage. Around MCAo ⫹ 25 days, a 11C-flumazenil (FLU) PET study was performed using the ECAT Exact HR⫹ device under a previously published anaesthesia protocol (Sette et al, Stroke, 1993). Following transmission measurements, an IV bolus of FLU (5.2 ⫾1 mCi; SRA⫽ 271 ⫾ 71 mCi/mmol) was administered; PET data acquisition was performed from 20 to 60 min after injection. Around MCAO ⫹ 30 days, and immediately before euthanasia, a T1-weighted volume MRI was obtained and co-registered to the PET data set using AIR and MPITOOL娀 software. Using MRI coronal planes, a reference ROI encompassing the entire cortical mantle of the convexity was drawn on the non-occluded hemisphere, and mirrored onto the occluded side. An ROI delineating the infarct (which was essentially restricted to the striato-capsular area in these animals) was also drawn onto the MRI sets. Using an in-house software for voxel-based analysis, we calculated the mean FLU uptake (M) in the entire reference ROI, and mapped the voxels in the occluded hemisphere with FLU uptake below (M – 2SDs), termed voxels-of-interest (VOIs). To constrain our analysis to non-infarcted tissue, all VOIs located within 3mm from the border of the infarct ROI were excluded. Results VOIs in non-infarcted cortical areas were present in each baboon. They represented 9.8 ⫾ 2.3 % (range: 7.3-12.4%) of the voxel sample contained in the reference ROI, i.e., greater than would be expected by chance (i.e., 5%), even after exclusion of infarcted and peri-infarct VOIs. The average decrease in FLU uptake relative to the reference ROI was 37%. Regarding topography, the VOIs tended to cluster in the parietal cortex, i.e, remarkably caudal and posterior to the MRI-delineated infarct. Comments Our findings obtained using an objective, voxel-based method, indicate that decreases in FLU specific binding are constantly observed in cortical areas remote from the infarct in the chronic stage after temporary MCAo in the baboon. This is a novel observation, although its interpretation is speculative. These VOIs may represent widespread SNL in the cortical zones transiently affected by the ischaemic insult. The results of the presently ongoing detailed histological analysis of the cortical zones with low FLU binding so identified, will be the core of our future investigations. Reference Sette et al, Stroke, 24 (1993) 2046-2057

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Receptor binding quantification with high affinity ligands: A simulation study Kjell Erlandsson*, Vincent J Cunningham†, Peter J Ell*, Lyn S Pilowsky*‡ *Institute of Nuclear Medicine, University College London, Middlesex Hospital, Mortimer Street, London W1T 3AA, UK †Imaging Research Solutions Ltd, Hammersmith Hospital, London, UK ‡Institute of Psychiatry, KCL, London, UK Introduction Using the high affinity SPET tracer [ 123I]epidepride, Pilowsky et al. [1] found evidence of limbic selectivity for the atypical antipsychotic drug clozapine. This finding does not agree with the results obtained by Farde et al. [2] using the PET tracer [ 11C]FLB 457, a close analogue to epidepride. Based on simulation experiments, Olsson and Farde [3] suggest that the difference could be due to methodological errors (in particular underestimation of occupancy in striatum). However, their simulations only cover the time period for a 11C PET scan (60 min), and their results are therefor not relevant for an 123I SPET scan, which can be considerably longer (up to 6 h or more). Methods Tissue time activity curves were simulated based on the traditional 2-tissue compartment (2-TC) model, with a variable k 3; k 3⫽k on(B max-C B(t)/SA). The parameters values used (K 1, k 2, k on, k off and SA) were the ones corresponding to the tracer [ 11C]FLB 457 [3]. Data were simulated for up to 5 h p.i. with different values of B max, ranging from 0.1 to 40 nM. The data for the first 60 min were analysed using 3 compartmental models: the 2-TC model, and the full and the simplified reference tissue models (2-TCR and 1-TCR). Binding potential values (BP⫽k 3/k 4) were calculated with each method. An alternative quantitative measure, the binding index (BI), was also calculated: BI⫽C T/C R-1, where C T and C R are the activity concentrations in the target and reference tissue, respectively. BI was calculated for different times p.i.; 60, 120, 180, 240 and 300 min. Occupancy values were determined, for the case of a true receptor occupancy of 50%, as O⫽(1-B 50/B 0)⫻100%, where B x is the binding value obtained with x% of receptors blocked. Results As shown in the left graph below, all 3 compartmental models gave occupancy values very close to the true value over the whole range of B max (which was numerically equal to the true BP value, in this case). Large errors were obtained in the BI-based occupancy values calculated at 60 min p.i., as also shown in [3]. However, the errors were significantly smaller at later time-points (right graph). Discussion Although BI values can be used for receptor binding quantification with ligands of lower affinity, it can result in large errors with high-affinity ligands such as epidepride or FLB 457. Our results indicate that, when using BI values for comparing drug occupancy in striatum (BPⱁ20) and in temporal cortex (BPⱁ1), the data obtained around 4 h p.i. should be used. Pilowsky et al. [1] used the 3-4 h data to calculate D 2/D 3 occupancy by antipsychotic drugs, which, according to the present simulation, should not result in serious errors. Conclusions Based on our results we can draw the following conclusions regarding receptor binding quantification with high affinity ligands such as epidepride or FLB 457: 1) If data acquisition is only possible up to 60 min p.i., it is necessary to use kinetic modelling. 2) If BI values are used, data should be obtained much at a much later time than 60 min p.i. References [1] Pilowsky LS, et al., Lancet, 350:490-1, 1997. [2] Farde L, et al., Psychopharmacology, 133:396-404, 1997. [3] Olsson H and Farde L, Neuroimage 14:936-45, 2001.

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Is the peripheral benzodiazepine receptor ligand PK11195 a substrate for P-glycoprotein? Hassan Rahmoune, Andrew Lockhart, Jan Passchier, Antony Gee, Ron A. Leslie, John Brown GlaxoSmithKline, Clinical Pharmacology and Experimental Medicine Unit, Addenbrookes Centre for Clinical Investigation, Addenbrookes Hospital, Cambridge, CB2 2GG, UK The peripheral benzodiazepine receptor ligand PK11195, when labelled with carbon-11, has been used as an in vivo marker of inflammatory activity in human brain with PET. In the absence of pathology the ligand is characterised by a relatively low uptake in the brain, followed by a rapid washout. This is somewhat surprising in light of this compounds apparent lipophilicity. It is known that non-polar compounds can have a propensity to be a substrate for P-glycoprotein (P-gp), an ATP-driven efflux pump with an affinity for a wide variety of lipophilic substrates. In light of the observed kinetics in the brain, PK11195 was assayed for P-pg efflux activity. A drug accumulation assay was performed using the P-gp expressing human neuroblastoma cell line (SK-N-F1). The assay measures the intracellular accumulation of known P-gp substrates in the absence or presence of cyclosporin A (CsA, 10␮M), an established P-gp inhibitor. There was no difference in the accumulation of [3H]PK11195 in the presence or absence of CsA as opposed to [3H]vinblastine which was used as positive control and showed a 5-fold higher uptake in the presence of CsA. These findings suggest that PK11195 is not a P-gp substrate and that its lack of binding in the brain is due to other factors. At present, we cannot rule out the possibility that the compound is a substrate for other drug transporters.

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Mapping of Sigma 1 Receptors by 11C-SA4503-Distribution and Aging Effect in Normal Human Brain Kenji Ishii, Yuichi Kimura, Kazunori Kawamura, Keiichi Oda, Toru Sasaki, Kiichi Ishiwata Positron Medical Center, Tokyo Metropolitan Institute of Gerontology Background: Postmortem human brain studies have suggested that the sigma receptors are involved in a variety of neurological and psychiatric disorders. However, the physiological role of sigma receptors in normal human brain has not been clarified. We have developed and evaluated 11C-labeled 1-(3,4-dimethoxyphenethyl)-4-(3-phenylprophyl)piperazine ( 11C-SA4503) as a promising ligand for PET study for imaging central nervous system (CNS) sigma 1 receptors (1,2,3). Previous animal studies revealed a significant age related increase of sigma 1 receptors in rat and monkey brains by the in vitro membrane assay with 3H-SA4503 and by PET with 11 C-SA4503, respectively (submitted). The purpose of this study is to examine the normal distribution and its aging effect of sigma 1 receptors in human brain using 11C-SA4503 and PET. Methods We studied fifteen healthy volunteers (ages 20-71). All the subjects had physical and neurological examinations and MRI study prior to the PET study. A 500 MBq of 11C-SA4503 was administered intravenously and a dynamic PET scan was performed for 60 to 120 minutes after the injection. Serial arterial blood samples were obtained, and the plasma radioactivity and the amount of non-metabolised ligand were measured for an input function. The K1 and binding potential (BP) were estimated based on the 3-compartment 4-parameter model in 12 brain regions. The aging effect was evaluated by comparing two groups, young (n ⫽ 4, ages 20-22) and aged (n ⫽ 6, ages 63-71). Results The cerebellum, hippocampus, lateral temporal cortex, and anterior cingulated gyrus showed relatively higher BP values among the 12 brain regions investigated. There was no significant correlation between regional K1 and BP values. The K1 was significantly lowered in the cerebellum and anterior cingulated corgex in the aged group. Some brain regions had a trend to increase the BP as aging without statistical significant (Table 1) as observed in previous animal studies. K1 and BP of 11C-SA4503 in the Young and Aged Groups, Mean (SD)

Cerebellum Hippocampus Temporal ANT cingulate Prefrontal Parietal

K1-young

K1-old

BP-young

BP-old

0.46 (0.05) 0.36 (0.04) 0.41 (0.03) 0.45 (0.03) 0.42 (0.02) 0.44 (0.03)

0.41 (0.03)* 0.36 (0.04) 0.38 (0.05) 0.41 (0.03)* 0.40 (0.04) 0.42 (0.05)

10.41 (2.90) 10.46 (2.94) 8.13 (1.66) 9.86 (4.24) 6.97 (1.38) 7.23 (1.53)

12.41 (3.79) 11.47 (4.77) 10.91 (5.34) 9.15 (5.03) 8.88 (4.51) 9.12 (3.38)

* Significantly different (P ⬍ 0.05) between young and old. Conclusions The sigma 1 receptors might have a unique characteristics that the BP increases in the CNS as aging. The mapping of sigma 1 receptors with 11C-SA4503 will provide us a different kind of functional information from regional cerebral blood flow and metabolism, and is expected to be useful for evaluating phathophysiological mechanism of various CNS disorders. References 1. Kawamura K, et al. Nucl Med Biol 27:255-261, 2000. 2. Kawamura K, et al. Ann Nuc Med 14:285-292, 2000. 3. Ishiwata K, et al. Synapse 40: 235-237, 2001.

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The Effect of Acute DA Depletion on in vivo [3H]Rolipram Binding in Rodent Brain Ningning Guo*†, Dah-Ren Hwang, Zhihong Zhu, Marc Laruelle Department of Psychiatry, Columbia University Previous studies has demonstrated that pharmacologically induced acute dopamine (DA) depletion produced either no change ([11C]NNC112) or a paradoxical decrease ([3H]SCH23390) in D1 radioligand binding in the brain [1]. A recent report indicated that DA enhancer amphetamine facilitated the activity of the cAMP-specific enzyme phosphodiesterase-IV (PDE-IV), as measured by the binding of [11C]rolipram [2]. To further evaluate the impact of DA depletion on the activity of D1 receptor-coupled cAMP messenger system, in vivo binding of [3H]rolipram was examined on DA-depleted animals. Male Sprague-Dawley rats were pretreated (n⫽ 10/group) with a combination of reserpine (5 mg/kg i.p., 24 h before experiment) and AMPT (400 mg/kg i.p., 4 and 1h before experiment), a combination known to induce complete (⬎95%) depletion of striatal DA [3]. Time course of the accumulation of [3H]rolipram in the brain was performed in untreated rats to determine the time point when the highest binding ratio of tissue/brainstem would be obtained. Depleted and control rats were injected through a lateral tail vein with 100 ml of [3H]rolipram (4 mCi/rat). The normal and the treated rats were decapitated and the brain regions including striatum (ST), prefrontal cortex (PFC), hippocampus (Hipp), thalamus (Thal), cerebellum (Ceb) and brainstem (Bstem) were dissected. The brain samples were processed and counted by a scintillation counter. The brainstem was taken as the reference region [4]. The differences between controls and the treated animals in the ratios of tissue/Bstem were analyzed by a Student’s two-tailed t-test. For in vivo [3H]rolipram binding, the ratio of tissue/Bstem reached the highest at 60 min post the tracer injection. Although the ID % of [3H]rolipram increased in all the brain regions examined in the drug treated animals, no changes in the ratios of tissue/Bstem were tested between the control and the DA depleted rats (PFC/Bstem: control, 2.16 ⫾ 0.47, treated, 2.03 ⫾ 0.32; ST/Bstem: control, 1.97 ⫾ 0.32, treated, 2.07 ⫾ 0.17; Hipp/Bstem: control, 1.65 ⫾0.49, treated, 1.60 ⫾0.06; Thal/Bstem: control, 1.83 ⫾ 0.68, treated, 2.17 ⫾ 0.15; Ceb/Bstem: control, 1.43 ⫾0.16, treated, 1.79 ⫾ 0.92). Since these data only reflect the in vivo binding of [3H]rolipram at one time point, a full time frame scan of this radioligand might produce different binding data at certain time point. At the given time (60 min), the present data showed that acute DA depletion did not significantly change the in vivo binding of [3H]rolipram in the brain, suggesting that the drug treatment had no effect on the D1 receptor-coupled second messenger system at the level of PDE-IV activity. References [1] [2] [3] [4]

Guo, N. et al, Neuroimage, 2000, 11(6): S4. Tsukada, H. et al, Synapse, 2001, 42: 258-265. Guo et al., Neurosci Abstr, 1999, 25: 952. Pe´ rez-Torres et al, J. Chem. Neuroanatomy, 2000, 20: 349-374.

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Characterisation of the kinetics of both the R(-) and the S(ⴙ) enantiomers of [ 11C]Rolipram in the Papio anubis brain Julian C Matthews*, Christine A Parker*, Stephen T Knibb*, Jan Passchier*, Michel Bottlaender†, Fre´ de´ ric Dolle†, Jean-Robert Deverre†, John Brown*, Antony D Gee* *GlaxoSmithKline Pharmaceuticals, Box 128, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2GG, United Kingdom †CEA-Service Hospitalier Frederic Joliot, DSV/DRM, 4 Place Ge´ ne´ ral Leclerc, 91406 Orsay, France [ 11C]Rolipram is a radiotracer that binds to the enzyme phosphodiesterase-4 (PDE4). PDE4 catabolises the second messenger cAMP. The expression and activity of this enzyme is indirectly regulated by cAMP and hence [ 11C]Rolipram may be a potential tool for probing the agonism of cAMP coupled receptors and alterations of this major second messenger system with neurological and psychological disorders. [ 11C]Rolipram exists as two enantiomers with the S(⫹) form having a substantially lower affinity for PDE4 than the R(-) enantiomer and hence the S(⫹) form may be useful in measuring non-specific binding. However, a recent publication has questioned the utility of the S(⫹) enantiomer for this purpose (1). The aim of this study is to characterise the kinetics of the two enantiomers to examine similarities and differences. Two baboons (Papio anubis) were each scanned on two occasions, once with R(-)-[ 11C]Rolipram and once with S(⫹)11 [ C]Rolipram, with data collected for 90 minutes in 3D mode, with the camera position over the brain. During the acquisition, frequent arterial samples were taken for the determination of the radioactivity in arterial blood plasma and the contribution of unmetabolised [ 11C]Rolipram to this signal using radio-HPLC. For anatomical localisation, each baboon had a T1-weighted MRI scan of the brain. For each animal the two dynamic PET data sets and the MRI data set were registered into the same space using rigid body transformations and were consequently resliced into isotropic images. Ten regions of interest were defined on the MRI, each of which was delineated for both left and right hemispheres. Representative time activity curves for each region were then generated. From the blood data an input function of the unmetabolised [ 11C]Rolipram concentration in arterial blood plasma was calculated. Three models were then fitted to the data for each region of interest: (A) a spectral analysis model (2); (B) a two tissue compartmental model; (C) and a single tissue compartmental model. For both animals the percentage of the total radioactivity in blood plasma due to unmetabolised [ 11C]Rolipram was higher for the S(⫹) enantiomer, suggesting than there may be differences in the metabolism of the two enantiomers. However the differences of approximately 10% at 55 minutes were less than the differences between the two animals (25% and 40% for the R(-) enantiomer). Both the spectral analysis model and the two tissue compartmental model produced good fits to the data but the single tissue compartmental model fits were poor for both enantiomers. The apparent delivery (K 1) was similar for both enantiomers (figure 1a) but apparently twice as large for Baboon B than Baboon A. In contrast, the volumes of distribution were clearly different for the two enantiomers with 2-3 times higher values for the R(-) enantiomer (figure 1b). The only clear difference in the kinetics of R(-)-[ 11C]Rolipram and S(⫹)-[ 11C]Rolipram was the higher retention in brain tissue with the R(-) enantiomer. For both enantiomers the two tissue compartmental model adequately describes the tissue kinetics. However, a single tissue compartmental model failed to fit the S(⫹) enantiomer data which may be due to the small amount of specific binding observed by Lourenco et al (1). Additionally, there may be differences in the metabolism of the two enantiomers in Baboon. Further work is in progress to establish whether the S(⫹) enantiomer has utility in measuring the non-specific binding component of [ 11C]Rolipram in man. 1. Lourenco CM, Houle S, Wilson AA, DaSilva JN. Characterization of R-[ 11C]rolipram for PET imaging of phosphodieterase-4: in vivo binding, metabolism, and dosimetry studies in rats. Nuclear Medicine & Biology 2001; 28(4):347-358. 2. Cunningham VJ, Jones T. Spectral analysis of dynamic PET studies. J Cereb Blood Flow Metab 1993; 13:15-23.

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Evidence to support the use of S(ⴙ)-[11C]Rolipram in estimating the nonspecific binding component of R(-)-[11C]Rolipram to PDE4 in vivo using PET Antony Gee*, Christine Parker*, Julian Matthews*, Jan Passchier*, Donald Smith†, Dirk Bender†, Stephen Knibb*, Albert Gjedde†, John Brown* *GlaxoSmithKline ACCI Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2GG, UK †PET-Centre, Aarhus University Hospital, Norrebrogade 44, DK-800 Aarhus C,Denmark R-(-)-[11C]Rolipram ((-)-ROLI) has been proposed as a PET radioligand for probing in vivo phosphodiesterase 4 (PDE4) function in vivo(1,2). The indirect modulation of PDE4 by second messenger cAMP provides a potential opportunity to probe the function of this major second messenger cascade in vivo using PET. A possible caveat to the use of this tracer is the absence of a reference region in the brain, devoid of PDE4, to enable the estimation of the nonspecific binding component of the ligand. However, it was proposed that the stereoselectivity of rolipram enantiomers might provide a means of circumventing this problem (1). The S(⫹)enantiomer of rolipram ((⫹)-ROLI) exhibits 10 - 20 fold lower affinity than (-)-ROLI for PDE4 in vitro (1,3). PET imaging of the 11C-labelled enantiomers in the living pig brain led to the observation that the less active S(⫹)-rolipram enantiomer, labelled with 11C, may be useful in estimating the free and non-specifically bound component of R-(-)-[11C]Rolipram in vivo. However, a recent report suggested that the less active (S) enantiomer may have a significant specific binding component in the rat brain which could preclude the use of the (S)-enantiomer for this purpose (1). This aim of this study was to further examine the hypothesis that the S-(⫹)- enantiomer can be used to estimate the specific binding component of R(-)-[11C]rolipram in vivo using PET. Five [11C]rolipram tracer injections were sequentially administered to an aneaesthetised pig (40 Kg), before and after an intravenous injection of 0.5 mg/kg racemic rolipram in the following order: (⫹)-ROLI and (-)-ROLI at baseline followed by (⫹)-ROLI, (-)-ROLI and (⫹)-ROLI post 0.5 mg/kg racemic rolipram (rac-ROLI). The racemic rolipram was given approximately 5, 90, and 180 minutes prior to the last three scans. Emission scans, arterial plasma time-activity curves (TAC’s) and metabolite analysis were performed for 60 mins following each tracer injection. Regional tissue TAC’s were generated from the reconstucted data to include the following brain regions: frontal cortex, occipital cortex, temporal cortex, thalamus and cerebellum. Arterial plasma input functions were generated by correcting total plasma radioactivity concentrations for percentage unchanged radioligand. Tissue TAC’s were corrected for blood volume using a fixed blood volume of 4%, fitted to a two tissue compartment model and regional distribution volumes calculated from the estimated rate constants. Baseline (-)-ROLI showed heterogeneous uptake and retention of the ligand in the brain, as reported previously (1,2), with higher concentrations seen in grey matter compared to white matter. Post 0.5 mg/kg, (-)ROLI was homogeneously distributed and washout was rapid from all brain regions. All (⫹)-ROLI scans showed homogeneous distribution with rapid washout from tissue. An apparent increase in the rate of (⫹)ROLI metabolism was observed post racemic rolipram treatment. Fig 1 shows that the estimated regional distribution volumes for (⫹)-ROLI are similar at baseline compared with the (⫹)-ROLI scans performed at approximately 5 and 180 min post 0.5 mg/kg racemic rolipram, suggesting that there is little saturable binding for this enantiomer. Compared with (⫹)-ROLI, the (-) enantiomer shows distribution volumes which are substantially higher pre displacement but only slightly higher post displacement with rac-ROLI. This is probably due to (-)-ROLI binding to PDE4, which may not be completely blocked with 0.5 mg/Kg rac-ROLI. Discrepancies with a study reporting a specific binding component of (⫹)-ROLI in rat brain (1) could possibly be explained by a species difference. Alternatively, the observed increase in rate of metabolism of (⫹)-ROLI post 0.5 mg/kg of racemic rolipram, would lead to an absolute decrease in tissue (⫹)-ROLI concentration, which, if not accounted for, could be misinterpreted as displaced specific binding of (⫹)-ROLI to PDE4. In conclusion, this data supports the hypothesis that (⫹)-ROLI can be used to estimate the non-specific binding component of (-)-ROLI at PDE4 in vivo using PET. Reference List (1) Lourenco CM, Houle S, Wilson AA, DaSilva JN. Characterization of R-[11C]rolipram for PET imaging of phosphodieterase-4: in vitro binding, metabolism, and dosimetry studies in rats. Nuclear Medicine & Biology 2001; 28(4):347-358. (2) Gee A, Smith D, Ostergaard L, Bender D, Poulsen PH, Simonsen C et al. Mapping Second Messenger Activations in the Brain. NeuroImage 1998; 7(4):A38. (3) Schneider H, Schmischen R, nBrezinski M, Seidler J. Stereospecific binding of the antidepressant rolipram to brain protein structures. Eur. J. Pharm 1986; 127:105-115

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Age-related decline of dopamine transporters in F344/N rat striatum revealed by PET and [ 11C]␤-CFT Kentaro Hatano*, Mitsuru Suzuki†, Mikako Ogawa*, Yasuhiro Kawasumi*, Hideo Tsukada†, Kengo Ito* *National Institute for Longevity Sciences, Obu, Japan †Central Research Laboratory, Hamamatsu Photonics K.K., Hamakita, Japan High resolution PET scanners for animal studies have opened novel research areas besides the preclinical investigation of radiopharmaceuticals. The direct comparison of the in vitro and in vivo dopamine receptor binding property in aged rats indicated the possibility of this methodology to demonstrate the compensatory mechanism which could only be investigated in living animals (1). In the present study, we examined this age effect on dopamine transporters using [ 11C]␤-CFT as a radioligand. The tracer was prepared and purified according to the literature (2). Data acquisition procedure by animal PET camera (SHR-2000, Hamamatsu Photonics, Japan) and their analysis using cerebellar uptake as indirect input function were identical to those we reported for dopamine receptor measurement (1). The binding Dopamine Transporter Binding in F344/N Rats potential (BP) values of 18 and 24 months old rats Age (month) 6 12 18 24 were smaller than the es- BP (mean ⫾ SD) 2.32 ⫾ 0.07 2.12 ⫾ 0.27 1.80 ⫾ 0.39* 1.66 ⫾ 0.15* timates of younger animals. Although an insig* Significant decrease from 6M (P ⬍ 0.05). nificant decline of BP was observed between 6 and 12 months, the effect was likely to initiate at the age of 12 months as D1 or D2 dopamine receptor. This evidence support the parallel reduction of presynaptic and postsynaptic dopamine neuron marker argued on elderly humans (3). 1 Suzuki, M, Hatano, K, Sakiyama, Y, et al., (2001) Age-related changes of dopamine D1-like and D2-like receptor binding in the F344/N rat striatum revealed by positron emission tomography and in vitro receptor autoradiography. Synapse 41: 285-293. 2 Någren, K, Halldin, C, Mu¨ ller, L, et al., (1995) Comparison of [ 11C]methyl triflate and [ 11C]methyl iodide in the synthesis of PET radioligand such as [ 11C]␤-CIT and [ 11C]␤-CFT. Nucl. Med. Biol. 22: 965-970. Kawamura, K, Ishiwata, K, Futatsubashi, M, et al.,(2000) Efficient HPLC separation of [ 11C]␤-CFT or [ 11C]␤-CIT from an N-desmethyl precursor on semipreparative reversed phase ODS column. Appl. Radiat. Isot. 52: 225-228. 3 Volkow, ND, Wang, GJ, Fowler, JS, et al., (1998) Parallel loss of presynaptic and postsynaptic dopamine markers in normal aging. Ann. Neurol. 44: 143-147.

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Ultra-high resolution single photon emission tomography imaging of dopamine transporters in the mouse brain Paul Acton*†, Seok-Rye Choi, Karl Plossl, Hank Kung University of Pennsylvania Objective Functional imaging of small animals, such as mice and rats, using ultra-high resolution PET and SPECT is becoming a valuable tool for studying animal models of human disease. While several studies have shown the utility of PET imaging in small animals, few have used SPECT in real research applications. In this study we aim to demonstrate the feasibility of ultra-high resolution SPECT in quantitative studies of dopamine transporters (DAT) in the mouse brain. Previous SPECT studies of [ 99mTc]TRODAT-1 binding to DAT have shown the tracer is capable of quantitative measurement of DAT concentration, and also that the reference region model provides accurate results without the need for blood sampling in baboons and humans. We now extend this work into mice, to show that ultra-high resolution pinhole SPECT is capable of accurate, repeatable and quantitative measures of cerebral binding sites. Methods Four healthy CD-1 male mice were anesthetized with isoflurane, injected with 704 ⫾ 154 MBq [ 99mTc]TRODAT-1, and scanned using an ultra-high resolution SPECT system equipped with pinhole collimators (spatial resolution 0.83 mm at 3 cm radius of rotation). Each mouse had two studies, separated by a maximum of 2 weeks, to provide an indication of test-retest reliability. Reference tissue kinetic modeling analysis of the time-activity data in the striatum and cerebellum was used to quantitate the availability of DAT. A simple equilibrium ratio of striatum-to-cerebellum provided another measure of DAT binding. The SPECT imaging results were compared against ex-vivo biodistribution data from the striatum and cerebellum. Results SPECT images of [ 99mTc]TRODAT-1 uptake in the four mice showed the expected brain uptake and regional concentration in the striatum. The mean DVR for all four mice using the reference tissue model was 2.17 ⫾ 0.34, which is compared with the equilibrium model (DVR ⫽ 2.03 ⫾ 0.38) and the ex-vivo data (DVR ⫽ 2.32 ⫾ 0.20). Neither repeated measures ANOVA, nor post-hoc t-tests revealed any statistically significant differences between the values of DVR (p ⬎ 0.05). Regression analysis of DVR calculated using the various techniques showed strong correlations between the reference tissue model and the equilibrium ratio (R 2 ⫽ 0.86, p ⬍ 0.001). Similarly, correlation between the ex-vivo data and the reference tissue model and the simple ratio technique were both excellent (R 2 ⫽ 0.92, p ⫽ 0.04, and R 2 ⫽ 0.999, p ⬍ 0.001 respectively). Test-retest results for both the reference tissue model and ratio technique were both excellent (2.63 ⫾ 1.67 % and 6.64 ⫾ 3.86 %, respectively). Conclusion This study has demonstrated clearly that ultra-high resolution SPECT of small animals is capable of accurate, repeatable and quantitative measures of DAT binding, and should open up the possibility of further studies of cerebral binding sites in mice using pinhole SPECT.

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Prolonged neuroleptic exposure differentially alters the endogenous regulation of dopamine (DA): Serial 11C-raclopride PET studies of GABAergic and glutamatergic modulation of DA in primates treated with clozapine or haloperidol Wynne K. Schiffer*†, Doug A. Marsteller*†, Madina Gerasimov†, David Alexoff†, Jean Logan†, Jonathan Brodie†, Stephen L. Dewey*† *SUNY Stony Brook/Dept. Neurobiology & Behavior †Brookhaven Natl Lab/Chemistry Dept Introduction: The notion that DA D 2 blockade accounts for the antipsychotic effects of both haloperidol, which has a high affinity for D 2 receptors, and clozapine, with a much lower affinity for D 2 receptors, appears inconsistent with the superior clinical efficacy of clozapine. It is also possible that alterations in D 2 availability represent a productive interaction between dynamic, upstream events in related systems. Under this latter hypothesis, we used PET to explore the neurochemical plasticity associated with haloperidol versus clozapine in the absence of an ongoing disease process. Methods: Nine adult baboons were treated with vehicle (VEH), haloperidol (HAL) or clozapine (CLO) for total period of 6 wks. Following 1 wk treatment and 1 wk washout (1), animals received a baseline 11C-raclopride ( 11C-rac) PET scan followed by a challenge with either the DA depleting agent, alpha methyl para-tyrosine (AMPT), the GABA agonist, gamma-vinyl GABA (GVG), or the glutamate antagonist, phencyclidine (PCP), and a second 11C-rac scan. Animals returned to their respective treatment regimens (VEH, HAL or CLO) for 5 wks and were scanned again under an identical protocol as the first. To assess residual effects, animals were scanned again 9 wks following cessation. Basal DA concentrations and D 2 receptor density were estimated from changes in the distribution volume ratio (DVR) of striatal to cerebellar 11C-rac binding following AMPT treatment (Fig 1, circles). Results: Acute: After 1 wk HAL or CLO, AMPT increased 11C-rac by 11 and 26% respectively, with no change in D 2 density. Estimations of synaptic DA decreased with HAL and increased with CLO. GVG, which alone increased 11C-rac binding 15.1%, failed to modulate DA after one week of HAL but produced a normal effect in CLO animals (Fig 1, triangles). Alone, PCP decreased 11C-rac binding by 29 ⫾ 6.5%. After 1 week of HAL or CLO, PCP decreased 11C-rac binding by 28.1 and 11.4%, respectively. Chronic: AMPT increased 11C-rac binding more in HAL animals relative to CLO animals after 5 wks treatment, which coincided with a measured increase in D 2 density. HAL diminished the ability of GVG to inhibit DA, but did not affect PCP-induced changes in DA. Chronic CLO inhibited PCP-induced stimulation of DA (Fig 1, squares) without producing a marked effect on D 2 density, consistent with estimations of diminished synaptic DA in CLO animals challenged with AMPT. Residual: Nine wks after cessation of HAL or CLO treatment, AMPT induced changes in DA were restored to pretreatment levels (Fig 1, circles). However, HAL-induced alterations in GABAergic modulation of DA were maintained, with a 2.1% decrease in 11C-rac binding (Fig 1, triangles). There remained no significant effect of HAL on PCP-induced DA release. These PET studies suggest that the effects of prolonged antipsychotic exposure on DA synthesis and D 2 receptor number may not be confined to the initial site of action, but may also restore functional homeostasis to related systems. 1. M. Smith et al., Biological Psychiatry 23, 653-63(1988)

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Methylphenidate-Evoked Dopamine Release in Brain of Adolescents With Premature Birth: Correlation With Attentional Deficit Pedro Rosa*, Hans C. Lou*, Paul Cumming*, Ole Pryds*, Dean F. Wong†, Albert Gjedde* *Center of Functionally Integrative Neuroscience, Aarhus University, Aarhus, Denmark †Dept Radiology, Johns Hopkins University Medical Institutions RATIONALE Patients with attention deficit/hyperactivity disorder (ADHD) present with a higher dopamine transporter density compared to normal volunteers, suggesting a reduced dopaminergic tonus in this population. We hypothesized that the severity of attention deficit in adolescents who suffered birth trauma correlates with changes of dopaminergic neurotransmission. To test this hypothesis, we investigated a series of eight subjects (mean age 14.2⫹/-2.4 yrs) with documented birth trauma and/or low birth weight. METHODS Patients had two [11C]raclopride injections on the same day, the first scan in a baseline condition at 12:00 AM, and the second, 90 minutes later, 30 minutes after an oral dose of methylphenidate (0.3 mg/kg, p.o.). The relative binding of [11C]raclopride to dopamine receptors was measured in the striatum. Reproducibility of [11C]raclopride baseline scans was assessed in four adolescents. Behavioral measurements particularly impulsivity and inattention were measured with the TOVA psychological battery. RESULTS The methylphenidate challenge elicited a reduction of 7.8% (p⬍0.02) in the [11C]raclopride ratio in adolescents with premature birth. We found positive correlation between commission errors and the increased dopamine release assumed to be evoked by the methylphenidate (right side r ⫽-0.80, p⫽0.02; left side, r⫽-0.97, p⫽0.002). A similar trend was also observed in the scores for omission errors (right side, r⫽-0.72, p⫽0.06; left side, r⫽-0.59, p⫽0.16). However, no correlations were observed between S/C ratios of [11C]raclopride baseline conditions and neuropsychological measurements. COMMENT These preliminary results suggest that the extent of attention deficit in adolescents with birth trauma is associated with abnormal regulation of dopamine neurotransmission.

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A protocol to measure stress induced dopamine release using [ 11C] raclopride Mitul Mehta*, Andrew Montgomery*, Peter Bloomfield†, Terence Spinks†, Paul Grasby* *MRC Cyclotron Unit, Hammersmith Hospital, London, UK †Imaging Research Solutions Limited, Hammersmith Hospital, London, UK Stress responsivity is thought to be important in the exacerbation of a number of psychiatric disorders. However, much of the literature to date appears to focus on the psychosocial rather than the biological. The aims of the present study were to develop an experimental protocol to examine possible relationships between behavioural and biological measures of acute stress induction in healthy human volunteers. Behavioural measures included subjective ratings and performance on a stressful mental arithmetic task. For the stress induction task subjects counted backwards serially in 7s and 13s at a rate calibrated to allow them to perform the task, but with difficulty. The control task involved counting backwards at the same rate, but in 1s. The primary biological measure acquired was changes in binding potential of [ 11C] raclopride - an index of dopamine release. Six healthy subjects (M:F, 5:1) were scanned with positron emission tomography (PET) using bolus plus infusion [ 11C]raclopride (K bol 105 minutes)(Watabe et al. 2000). This method obviates potential confounding effects of task-induced blood flow changes. Subjects performed the control task from 0-49min and the stress task from 50min until the end of the scan. The CTI/Siemens 966 PET scanner was used to acquire the image data (Spinks et al. 2000). Regions of interest were defined as ventral (VS) and dorsal striatum (DS) using a template which had been spatially transformed into individual space. Binding potential (BP) was calculated as the ratio of (striatal counts-cerebellar counts)/cerebellar counts in two sampling periods: 38-50 minutes and 58-100 minutes. Head movement was minimised using a moulded headholder, firm sponge and a head strap. For all six subjects head position was closely monitored using laser crosshairs, but for the latter three a motion detection system was also introduced. Prior to the PET scan all subjects attended on a separate day for a “dummy” scan procedure in order to familiarize them with the study procedures and putatively minimise confounding novelty-induced stress responses. The figure shows individual subjective ratings of stress (on a scale of 1-10) during the non-stress condition, peak stress during the stress-induction task and post-scan rating. The data describe a significant subjective increase in stress during the difficult arithmetic [ANOVA, F(2,10)⫽27.4, P⬍0.01]. During the stress task subjects showed a decrease in tracer binding potential in both the dorsal and ventral [ 11C] Raclopride Binding Potentials striatum (see table). One-tailed, one-sample t-test on the Non-stress Stress % Change percentage “displacement” was significant only for the dorsal striatum [t(5)⫽-2.05, P⫽0.048]. Dorsal striatum 2.06 (0.52) 1.86 (0.56) ⫺9.94 (11.85) Preliminary results from the movement detection sysVentral striatum 2.37 (0.32) 2.32 (0.42) ⫺4.37 (10.02) tem indicate that even the most carefully monitored subjects show small but significant motion which may Note. Mean (standard deviation). bias the displacement effect presented. We have demonstrated reliable stress responses in subjects performing difficult mental arithmetic in a PET scanner environment. However, changes in head movement may be difficult to distinguish, in some cases, from genuine tracer “displacement”. Accurate and continuous measures of head position allowing realignment of emission data at the stage of image reconstruction are being introduced to help minimise confounding effects of head movement on tracer binding measurements. References Spinks et al. (2000) Physical characteristics of the ECAT EXACT3D positron tomograph. Phys Med Biol 45: 2601-2618. Watabe et al. (2000) Measurement of dopamine release with continuous infusion of [ 11C] raclopride: optimization and signal-tonoise considerations. J Nucl Med 41:522-530.

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Electrical Peripheral Sensory Stimulation Accelerates Dopamine Release in the Basal Ganglia Manabu Inoue*‡, Hidefumi Yoshida*‡, Chisako Oyanagi*‡, Yukinori Katsumi*‡, Takahiro Mukai†‡, Koichi Ishizu†‡, Kazuo Hashikawa*‡, Hidenao Fukuyama*‡ *Human Brain Research Center †Nuclear Medicine and Diagnostic Imaging ‡Kyoto University Graduate School of Medicine Introduction There are many reports that the functions of dopaminergic neurons in the midbrain are related to motor system, psychosis and reward. But the relationship between mesencephalic dopaminergic neurons and sensory system has been remained to be unclear. Clinically sensory trick in dystonia and kinesie paradoxale in Parkinson°fs disease have been regarded as the somatosensorydopamine system integration. Electrophysiologically sensory stimulation activated or inhibited the activity of dopaminergic neurons (1) (2). The purpose of this study is to demonstrate the relationship between mesencephalic dopaminergic neurons and sensory system using animal PET and C11-raclopride. Material and Methods Five cats were anesthetized sufficiently with halothane. The physiological parameters were monitored during experiments. PET with raclopride was used to evaluate dopamine release in the basal ganglia during the electrical stimulation at the paw of the right forelimb. PET scan data consisting of 31 slices were obtained using SHR 7700 (Hamamatsu Photonics K.K., Hamakita, Japan). Just after intravenous administration of 9 mCi of C11-raclopride, dynamic scans were obtained over a period of 60min. Parametric images of Binding Potential (BP) were calculated with the simplified reference region model proposed by Gunn et al (3). The imaging of the PET data was coregistrated with MRI. Regions of Interest ( ROI ) in the bilateral striatum and nucleus accumbens were determined on MRI, the cat brain atlas and coregistrated PET images. The value of BP data in these ROI was compared with t-test. Result The values of physiological parameters were within normal limits in this group. The physiological parameters were stable during PET scan. The BP data in the contralateral nucleus accumbens to the stimulation side was smaller than that in the ipsilateral nucleus accumbens (P⬍0.033) and caudate (P⬍0.058). These findings indicate that dopamine was released on the contralateral side to the stimulation of the peripheral nerves. The representative case is shown in the figure. Discussion This study showed that mesencephalic dopamine neurons were related with sensory input, but the anatomical connection between mesencephalic dopamine nuclei and sensory pathway has not been clear. One possible connection is that between nuclei intralaminares thalami and pars compacta of substantia nigra through striatum because the innervation of intralaminares thalami coupled with somatosensory pathways projects to striatum (4). Another possibility is the connection between the ventral tegmental area and tractus spinothalamicus through griseum centrale mesencephali, because griseum centrale mesencephali is innervated from tractus spinothalamicus (5). We provided the physiological evidence to prove the functional interaction between somatosensory input and dopaminergic system. References (1) Wolfram S., Ranulfo R.; Responses of Nigrostriatal Dopamine Neurons to High-Intensity Somatosensory Stimulation in the Anesthetized Monkey. J Neurophysiol 57: 201-17,1987 (2) Barasi S.; Responses of Substantia Nigra Neurons to Noxious Stimulation. Brain Res 171:121-30,1979 (3) Roger N. G., Adriaan A., Vincent J. C. et al; Parametric Imaging of Ligand-Receptor Binding in PET Using a Simplified Reference Region Model. NeuroImage 6:279-87,1997 (4) Matsumoto N., Minamimoto T., Kimura M. et al; Neurons in the Thalamic CM-Pf Complex Supply Striatal Neurons With Information About Behaviorally Significant Sensory Events. J Neurophysiol 85:960-76, 2001 (5) Harmann PA., Carlton SM., Willis WD.; Collaterals of spinothalamic tract cells to the periaqueductal gray: a fluorescent double-labeling study in the rat. Brain Res :441:87-97, 1988

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Can dopamine D2-receptor upregulation by antipsychotics be avoided by a different regimen of occupancy kinetics? A longitudinal [11C]raclopride PET study in cats Nathalie Ginovart, Lisa Richardson, Sylvain Houle, Shitij Kapur PET Centre, Centre for Addiction and Mental Health and University of Toronto, Toronto, Canada The antipsychotic effects of neuroleptics such as haloperidol are thought to be mediated through the blockade of dopamine D2-receptors. Despite their benefits for the treatment of schizophrenia, these drugs also induce side-effects such as tardive dyskinesia (TD). It is widely believed that long-term blockade of D2-receptors achieved by chronic treatment with neuroleptics leads to D2-receptor upregulation, and that this upregulation is a necessary condition for the occurrence of TD. However, little is known about the parameters of D2-receptor blockade (duration and percentage of blockade) that lead to upregulation. In this study, we hypothesized that only continuously high (ⱖ 80% % throughout the day) and not transiently high (ⱖ 80% for a few hours per day) D2-receptor blockade results in receptor upregulation. To test this hypothesis, we followed and compared the time-course of changes in D2-receptor density and affinity in 2 groups of cats (n ⫽ 3 per group) receiving either chronic daily injections (0.05 mg/kg/day; s.c) of haloperidol or constant infusion (0.25 mg/kg/day; s.c) of the drug through osmotic minipumps for 4 weeks. D2-receptor density (Bmax) and apparent affinity (Kd) were determined in vivo, using Positron Emission Tomography (PET), from the Scatchard analysis of [11C]raclopride experiments performed at baseline and at up to 3 weeks following cessation of injections or removal of the pumps. In each cat, PET experiments were performed after 1 week of treatment to determine D2-receptor occupancy. Occupancy measures showed that bolus injections of haloperidol at 0.05 mg/kg/day gave rise to a transiently high D2-receptor occupancy (82 ⫾ 4 % at 4 hours and 36 ⫾ 8 % at 24 hours post-injection) while a continuous infusion of the drug at 0.25 mg/kg/day led to a continuously high 86 ⫾ 4 % D2-receptor occupancy. Additional occupancy measures were performed at 3 weeks of treatment in one cat in each group and revealed D2-receptor occupancy levels similar to those obtained at 1 week (80 % at 4 hours and 29 % at 24 hours post-injection; 87 % for continuous infusion). A high test-retest reliability of Bmax (3.8 ⫾ 2.2%; range: -7% to -0.4%; n ⫽ 6) and Kd (6.7 ⫾ 4.2%; range: -11.1% to -0.2%; n ⫽ 6) values was observed at baseline conditions. Our results showed that haloperidol given by continuous infusion led to an increase in D2-receptor density that was maximal at 1 week withdrawal (40 ⫾ 4%; n ⫽ 3) and still detectable at 2 weeks withdrawal (26 ⫾ 3%; n ⫽ 2). D2-receptor densities were within the range of baseline values at 3 weeks withdrawal. Marginal increases in Kd values (range 8.4% to 24.2%) were observed during withdrawal, a result probably reflecting remaining small amounts of haloperidol in brain. In contrast, haloperidol given by daily bolus injection did not induce significant changes in D2-receptor densities (range: -10.1% to 5.0%) and affinity (-10.5% to 5.5%). Preliminary analysis of behavioral data showed a marked decrease in spontaneous locomotor activity (by 50 to 70%) in both groups of treated cats as compared to baseline conditions during the first week of chronic haloperidol administration. Spontaneous locomotor activity progressively increased during the following weeks of treatment and appeared to reach baseline activity values at the end of treatment or during the early withdrawal period. To our knowledge, this study constitute the first systematic examination of the parameters of D2-occupancy that lead to upregulation in clinicallly relevant doses and occupancies and suggests that intermittent high peaks of D2-occupancies are less likely to cause D2-receptor upregulation than continuous high levels of occupancies. These data could be used to develop principles for more safe and tolerable clinical dosing schedule, and for tailoring development of antipsychotic drugs with more favorable brain pharmacokinetics which could avoid upregulation. Support provided by the National Alliance for Research on Schizophrenia and Depression.

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18F-Fallypride Displacement in the Non-human Primate Brain by NicotineInduced and Amphetamine-Induced Dopamine Release J Mukherjee†, B Christian*, B Shi*, TK Narayanan*, J Mantil* *Kettering Medical Center †University of California, Irvine Objective Our goal in this study was to evaluate feasibility of using PET to measure dopamine release in extrastriatal regions (particularly the thalamus). Release of dopamine was induced either by amphetamine or nicotine which are well-known to release dopamine in several regions of the brain. These studies were carried out using 18F-fallypride which has been shown to be susceptible to competition by dopamine (Mukherjee et al., Synapse 27: 1-13, 1997) and is able to clearly visualize the thalamus in rhesus monkeys (Christian et al., Synapse 38: 71-79, 2000). Methods PET scans were carried out on a Siemens HR⫹ high resolution PET scanner and two rhesus monkeys. Scans extended for a total duration of 2.5 to 3 hrs after injecting 2 to 2.5 mCi of 18F-fallypride at specific activity of approx. 2800 Ci/mmol. Drug challenges were either carried out prior (-15 mins) to administering 18F-fallypride or after (⫹ 45 mins) administering 18F-fallypride. Doses of amphetamine ranged from 0.4 mg/kg to 0.8 mg/kg (free base) or nicotine 0.02 mg/kg (free base), administered intravenously. PET data provided time-activity curves and in all cases the cerebellum was used as a reference region. The effect on 18F-fallypride binding was evaluated as change in distribution volume (DVR) for the pre-drug experiments or as thal-cer/cer ratios (Bound/Free; B/F) for the post-drug challenge, and were compared to the control experiment. Results All regions of the brain containing dopamine receptors demonstrated a decrease in the binding of 18F-fallypride upon drug challenge. The pre-amphetamine studies allowed the quantitative evaluation of the reduction in 18F-fallypride binding using the DVR approach. This gives a measure of receptor occupancy by released dopamine. Thalamus, amygdala and cortical regions exhibited significant amounts of measureable dopamine release. In the competition experiment, displacement of the radioligand is clearly seen and corresponds to the time of injection of amphetamine and nicotine. This is indicative of the ability of dopamine to compete with 18F-fallypride in brain regions with lower concentrations of D2/D3 receptors. For the pre-drug challenge, the thalamus exhibited a decrease of approx. 17% (range 16 to 20%), amygdala decreased by 26% (range 24 to 27%), ventral striatum decreased by 15% (range 12 to 19%), pituitary decreased by 12% (range 9 to 13%) and cortical areas showed approx. 10-15% decreases. The DVR method of analysis accounts for possible changes in blood flow but is sensitive to noise in the low receptor density regions. Cortical regions, for example will have to be carefully evaluated. For the post-drug challenge, 18F-fallypride in the thalamus of control experiment provided an average B/F ratio (between 85 and 155 min) of 4.0. In the case of amphetamine, the B/F ratio was found to be 1.6, indicating a 61% reduction in 18F-fallypride binding. Nicotine provided a B/F ratio of 3.6, suggesting a 10% decrease in 18F-fallypride binding. Further dose-response studies will need to be carried out in order to assess the sensitivity of 18F-fallypride to nicotine using pre-injections of nicotine and analysis using the DVR approach. Conclusions We have demonstrated that binding of 18F-fallypride in extrastriatal regions, such as thalamus, amygdala, ventral striatum and other regions of the monkey brain is sensitive to drug-induced changes in endogenous dopamine. Our findings suggest that both nicotine and amphetamine are able to induce a reduction in the specific binding of 18F-fallypride in the thalamus. A moderate dose of nicotine induced a significantly smaller change in 18F-fallypride binding than amphetamine and may be indicative of the amount of dopamine released.

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Displaceability of [ 11C]FLB-457 from extra-striatal D 2 receptors Andrew Montgomery*, Marie-Claude Asselin†, Lars Farde‡, Paul Grasby* *MRC Clinical Sciences Centre, Hammersmith Hospital, DuCane Rd., London, UK †IRSL, Hammersmith Hospital, DuCane Rd., London, UK ‡Psychiatry Division, Karolinska Institute, Stockholm, Sweden Background [ 11C]FLB-457 has been developed as a high affinity (K i 18pmole) D 2 PET radioligand which may be used to measure receptor binding in extra-striatal regions of the brain. It is not currently known if [ 11C]FLB-457 binding is susceptible to changes in dopamine concentration in extra-striatal regions in a manner analogous to other D 2 radiotracers such as [ 11C]raclopride in the striatum. Two studies in primates have assessed the potential to “displace” [ 11C]FLB-457 from its binding site using an amphetamine challenge. Chou et al reported reductions in region of interest to cerebellar ratios of between -2 and -16% in three cynomolgus monkeys (Chou et al 2000), and Okauchi et al found mean reductions of BP of -3 to -6.5% in frontal cortex and thalamus respectively in three rhesus monkeys (Okauchi et al 2001). Here we report preliminary data examining the displaceability of [ 11C]FLB-457 from extra-striatal sites using methylphenidate 40 mg orally in man. Methylphenidate robustly reduces binding of [ 11C]raclopride when administered orally (Volkow et al 2001). Methods Three healthy male volunteers (age range 26-34) were scanned on two occasions on the Siemans ECAT 966 PET scanner. One hour before the start of the PET scan they were given either placebo or 40mg methylphenidate in a balanced-order, double-blind design. [ 11C]FLB-457 (mean injected dose 3.1␮g, 301MBq) was administered as a bolus over 1 minute. For this initial analysis scans were modelled using the simplified reference tissue model with the cerebellum as reference tissue. Regions of interest in thalamus, lateral frontal cortex, medial frontal cortex and temporal cortex were defined on individual MR scans which had been co-registered into PET space. Results Binding potentials in the two conditions placebo (P) and methylphenidate (M) for the three subjects are shown below. Differences between the two scans fell within previously reported test-retest variability for this method of analysis of [ 11C]FLB-457 PET data (Vilkman et al 2000). There was no difference in injected dose of FLB-457, activity injected or specific radioactivity at the time of injection. Conclusion In this group of three subjects there was no reduction in BP after methylphenidate in the regions examined. Any changes which may be present are likely to be small and within the test-retest range for the radiotracer. Further pharmacological challenges and higher doses of methylphenidate will be required to comprehensively test the displaceability of [ 11C]FLB-457. References Chou YH, Halldin C, Farde L (2000): Effect of amphetamine on extrastriatal D2 dopamine receptor binding in the primate brain: a PET study. Synapse 38:138-43. Okauchi T, Suhara T, Maeda J, Kawabe K, Obayashi S, Suzuki K (2001): Effect of endogenous dopamine on endogenous dopamine on extrastriatal [(11)C]FLB 457 binding measured by PET. Synapse 41:87-95. Vilkman H, Kajander J, Nagren K, Oikonen V, Syvalahti E, Hietala J (2000): Measurement of extrastriatal D2-like receptor binding with [11C] FLB-457 - test-retest analysis. Eur J Nucl Med 27:1666-1673. Volkow ND, Wang G, Fowler JS, et al (2001): Therapeutic doses of oral methylphenidate significantly increase extracellular dopamine in the human brain. J Neurosci 21:RC121.

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Occupancy of dopamine D2 receptors in the mouse brain measured using ultra-high resolution single photon emission tomography Paul Acton, Mei-Ping Kung, Catherine Hou, Karl Plossl, Cindy Keeney, Hank Kung University of Pennsylvania Objective Functional imaging of small animals, such as mice and rats, using ultra-high resolution PET and SPECT should be a valuable tool in studies of drug occupancy of cerebral binding sites. Previously, we have demonstrated that small animal SPECT is capable of quantitative measurement of dopamine transporter binding sites in the mouse brain. In this study we aim to demonstrate the feasibility of using ultra-high resolution SPECT to measure the occupancy of dopamine D2 receptors by a competing drug, using the dopamine D2 receptor-specific radioligand [ 123I]IBF. Methods Preparation of [ 123I]IBF, a highly selective dopamine D2 receptor radioligand, was achieved by an iododestannylation of the corresponding tin derivative, and purified by a simple C4-cartridge filtration method. Normal male mice (CD-1) were jugular vein-cannulated and a bolus-infusion protocol was used to deliver 360 MBq [ 123I]IBF into the mouse (bolus-to-infusion ratio 1.8:1). The mice were scanned using an ultra-high resolution triple-headed SPECT system equipped with pinhole collimators (focal length 24 cm, spatial resolution 0.83 mm at 3cm radius of rotation using 0.5 mm pinholes). After sustained equilibrium had been achieved, 1 mg/kg raclopride, a potent dopamine D2 receptor antagonist, was injected through the tail vein and the tracer was allowed to regain equilibrium. A simple equilibrium ratio of striatum-to-cerebellum provided a measure of D2 receptor binding both before and after injection of raclopride. Results The radioligand, [ 123I]IBF, exhibited high uptake in the striatum, with much lower uptake in the background regions, such as the cerebellum, which contained minimal concentrations of dopamine D2 receptors. Sustained equilibrium was achieved after 60-90 min. Following raclopride administration, the system returned to equilibrium after approximately 30 min, with lower specific binding in the striatum. The counts in the cerebellum were unaffected by the raclopride. The mean equilibrium ratio of striatum-to-cerebellum before administration of raclopride was 2.27 ⫾ 0.25, which dropped to 1.40 ⫾ 0.16 after raclopride injection (t-test, p ⬍ 0.001). This implied a mean occupancy of dopamine D2 receptors by a dose of 1 mg/kg raclopride of 68 ⫾ 11 %. Conclusion This study has demonstrated clearly that ultra-high resolution SPECT of small animals is capable of measuring displacement and occupancy of dopamine D2 receptors by competing ligands. This opens up exciting possibilities for the measurement of drug binding to receptors in occupancy and behavioral mouse studies.

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Investigation of dopamine D2/D3 receptors in the monkey brain: a PET study with two radioligands of different selectivity profiles Judit Sovago, Balazs Gulyas, Christer Halldin, Lars Farde Department of Clinical Neuroscience, Psychiatry Section, Karolinska Institute, S-171 76 Stockholm, Sweden Introduction Discrimination of dopamine D2 and D3 receptors in the brain with in vivo imaging techniques requires highly selective ligands with sufficiently high affinity. Raclopride is a ligand with high affinity for D2 dopamine receptor family (D2, D3 receptors). RGH-1576 is a novel selective D3 receptor ligand, with high affinity for D3, and markedly lower affinity for D2 receptors (Ki is approximately one magnitude higher for D2, and one magnitude lower for D3 receptors than respective values for raclopride). Objective Visualization of D2 and D3 receptor binding separately in the monkey brain using positron emission tomography with two different selective D2/D3 radioligands: [11C]-labelled raclopride and RGH-1756. Methods Dynamic PET measurements were made in two cynomolgous monkeys under baseline and pre-treatment conditions. In one monkey [11C]raclopride was the administered radioligand (dose: 30 and 41 MBq, respectively), and in the pre-treatment condition 0.5 mg/kg RGH-1756 was given 20 min prior to radioligand administration. In the other monkey [11C]RGH-1756 was the administered radioligand (dose: 22 and 23 MBq, respectively), and in the pre-treatment condition 1 mg/kg raclopride was given 10 min prior to radioligand administration. The radioligand time-activity-curves and global and regional brain uptake values were determined in anatomically defined volumes-of-interest (VOI’s), using a computerised monkey brain atlas system. Regional binding potential (BP) values were calculated according to Logan’s method. Results [11C]raclopride readily entered the brain, and reached a maximal uptake (5.10 % of the total injected activity) 90 seconds after injection. The highest uptake was observed in the nucleus accumbens, the nucleus caudatus and the putamen. Pretreatment with RGH-1756 markedly suppressed BP values of [11C]raclopride in the above brain structures (nucleus accumbens: to 46%, the nucleus caudatus: to 62 % and the putamen: to 58 %). At the same time, the global and regional radioactivity values were elevated as compared to the baseline values. The uptake of [11C]RGH-1756 in brain was rapid and 1.17 % of the totally injected radioactivity was in the brain 90 seconds after drug administration. Regional activity was highest in the nucleus accumbens, followed by the nucleus caudatus, putamen and amygdala. Pretreatment with raclopride resulted in slightly elevated regional and global radioactivity values, but had no evident effect on BP values. Conclusion The large decreases in raclopride BP values in the ventral striatum due to pre-treatment with RGH-1756 indicate that RGH-1756 in pharmacological doses enters the brain and specifically binds to brain structures rich in D2/D3 receptors. Even though the affinity of RGH-1756 is a hundred times higher to D3 receptors than to D2 receptors, at the present dose binding to D2 receptors is as considerable as to D3 receptors. The relatively low BP values of [11C]RGH-1756 in D3-receptor rich brain structures such as nucleus accumbens (BP ⫽ 0.282) and putamen (BP ⫽ 0.287) indicate low specific binding. Pre-treatment with RGH-1756 suppressed BP values of [11C]raclopride, and it was more effective in the nucleus accumbens than in other ventral striatal structures. This finding indicates that RGH-1756 can indeed be more selective for D3 receptors than to D2 receptors in in vivo conditions, as well. The elevated regional and global uptake values in both experiments in the pre-treatment condition can be attributed to either metabolic interaction or mutual inhibition of extra-brain binding.

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Selection of Reference Regions in the Analysis of D2 Receptor Binding Data with [11C]Raclopride: Cerebellum versus Occipital Cortex Y. Ma, G. Smith, V. Dhawan, T. Chaly, B. Pollock, D. Eidelberg Center for Neurosciences, North Shore-Long Island Jewish Research Institute and NYU School of Medicine, Manhasset, NY 11030. Introduction PET studies of neuroreceptor systems are usually performed to estimate binding parameters in the brain relative to a reference tissue (1-3). This method is appealing because of its simplicity and elimination of invasive blood sampling. Both specific-to-nonspecific ratio and kinetic measures such as distribution volume ratio (DVR) can be computed using a reference region of interest (ROI) in cerebellum or occipital cortex, assumed to have negligible specific binding sites. However there have been insufficient formal comparisons on the selection of these reference regions. In the present paper we evaluate this issue in typical clinical D 2 receptor binding studies with [ 11C]raclopride (RAC) and PET. Methods 3D dynamic RAC scans were acquired on a GE Advance PET system from 11 normal subjects before and after intravenous administration of a selective serotonin reuptake inhibitor citalopram (40 mg infused over 60 min). PET images were separately realigned within each time-series and between the two treatment conditions. Each image was integrated over 4 striatal slices and time-activity curves (TAC) were generated with a set of striatal and occipital ROI template. Cerebellum TAC was also extracted by summing three adjacent slices. DVR was computed over the linear ranges of 20-60 min using both cerebellum and occipital input function. RAC data from five non-manifesting DYT1 carriers were also analyzed this way. Maps of D 2 binding were finally created and normalized into Talairach space for SPM analysis. Results DVR CB in caudate and putamen were significantly higher by 17 % than DVR OCC in both baseline and treatment conditions (p ⬍ 0.00001). Both parameters increased similarly in the putamen relative to the caudate. Additionally DVR CB were highly correlated with DVR OCC in the combined baseline and treatment group (R 2 ⫽ 0.4; p ⬍ 0.002). Both values decreased comparably following acute treatment with citalopram, indicating postsynaptic D 2 receptor occupancy. The same behaviors were seen when comparing RAC binding in asymptomatic DYT1 carriers with normal controls. These observations were confirmed by corresponding SPM analyses. Conclusion These data show a significant bias in D 2 binding parameters calculated using the cerebellum and occipital reference tissues. However this bias has no significant effect on the sensitivity of both indices for detecting altered binding in abnormal functional state or following pharmacological challenge. The occipital method is more convenient in clinical RAC studies focusing specifically on serial images covering the striatal sections of the brain. 1. Lammertsma, A.A., Bench, C.J., Hume, S.P. et al. J. Blood Flow Metab., 16: 42-52 (1996) 2. Logan, J., Fowler, J.S., Volkow, N.D. et al. J. Cereb. Blood Flow Metab., 16: 834-840 (1996) 3. Sossi, V., Holden, J.E., Chan, G. et al. J. Cereb. Blood Flow Metab., 20: 653-660 (2000)

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Evaluation of [123I] IBZM Pinhole-SPECT and MRI co-registration as a useful method for the in vivo detection of striatal dopamine D2 receptor distribution in small animals Christoph Scherfler*, Sonja Scholz*, Eveline Donnemiller†, Michael Schocke‡, Clemens Decristoforo†, Werner Poewe*, Gregor Wenning* *Department of Neurology, University Hospital, Innsbruck †Department of Nuclear Medicine, University Hospital, Innsbruck ‡Department of Magnetic Resonance Imaging and Spectroscopy, University Hospital, Innsbruck This study was performed in order to assess the feasibility of in vivo imaging of the post synaptic dopamine 2 receptor (D2R) function in healthy rats, using single photon emission computer tomography (SPECT) and magnetic resonance imaging (MRI). To evaluate the optimal injection dose of the D2Rligand [ 123Iodine] iodobenzamide ([ 123I]IBZM) and to determine the adequate acquisition period of [ 123I]-IBZM uptake 15 male Wistar rats were divided into three groups receiving 44.4MBq, 37MBq and 29.6MBq of [ 123I]IBZM. SPECT acquisitions were performed over a time period of 280min with a rotating single-head gamma camera (Siemens, Orbiter) and a pinhole collimator equipped with a 2.0mm aperture insert. Additionally, five animals were scanned from time 0 to 150min following the injection of 44.4MBq [ 123I]IBZM and subsequently the [ 123I]IBZM uptake was measured in the dissected striatum, parietal cortex, hippocampus, occipital lobe, cerebellum, lacrimal glands and thyroid using a gamma counter and correlated with the SPECT measurements. Transaxial, sagital and coronal slices were reconstructed using a filtered backprojection algorithm (Nuclear diagnostics, Sweden). MRI was performed by modifying a whole body 1.5 Tesla MRI scanner (Siemens) equipped with a flex loop. The MRI protocol included a T2 weighted spine echo sequence (TR, 3000ms; TE, 96ms; matrix 256 x 256 pixels, slice thickness 2mm, FoV 65mm; acquisition time ⬃13min). Fully automated SPECT-MRI co-registration was achieved applying a co-registration algorithm which is based on mutual image information and implemented into the software package SPM99. Measurements of counts per voxel in the striatum (ST) and cerebellum (CE) were higher in animals receiving 44.4MBq [ 123I]IBZM compared to those receiving 37MBq and 29.6MBq [ 123I]IBZM 70min after tracer application (animal group receiving 44.4MBq [ 123I]IBZM: ST, 127.1 ⫾21.9; CE, 52.5 ⫾10.7; animal group receiving 37MBq [ 123I]IBZM: ST, 83.2 ⫾21.4; CE, 32.6 ⫾6.3; animal group receiving 29.6MBq [ 123I]IBZM: ST, 104.4 ⫾11.4; CE, 42.1 ⫾11.5). The ratio of activity in the striatum to cerebellum peaked between 70min and 150min following tracer application in all three animal groups (animal group receiving 44.4MBq [ 123I]IBZM: ST/CB, 2.4 ⫾0.4; animal group receiving 37MBq [ 123I]IBZM: ST/CB, 2.4 ⫾0.5; animal group receiving 29.6MBq [ 123I]IBZM: ST/CB, 2.3 ⫾0.5). Linear regression analysis showed that the [ 123I]IBZM uptake measured with SPECT and gamma counting in various regions as mentioned above was highly correlated (r 2⫽0.8; p⬍0.001). Pinhole SPECT and co-registered MRI studies revealed simultaneous high-resolution imaging and reasonable count statistics allowing semi-quantitative measurements of [ 123I]IBZM uptake in the rat basal ganglia within 70min to 150min following tracer application of 44.4MBq [ 123I]IBZM. Our system will be applicable for the in-vivo monitoring of the effects of therapeutic interventions in animal models of postsynaptic D2R degeneration.

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

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Characterization of 18F fallypride: in vivo affinity, in vitro temperature sensitivity and in vivo vulnerability to amphetamine challenge Mark Slifstein*†, Raj Narendran*†, Peter Talbot*†, Yasuhiko Sudo*†, Ningning Guo*†, Dah-Ren Hwang*†, Yiyun Huang*†, Elizabeth Hackett*†, Marc Laruelle*† *Columbia University †New York State Psychiatric Institute Introduction 18

F fallypride is a new PET radioligand enabling visualization of D 2 receptor density in both striatal and extrastriatal areas. The goal of this study was to quantitatively characterize several properties of this radioligand. The in vivo equilibrium dissociation constant (K D) was measured in a series of PET experiments in baboon. The sensitivity of in vitro K D measurement to temperature was studied in displacement experiments in rat striatal homogenate tissue. Finally, the vulnerability of 18F fallypride binding to acute fluctuations in endogenous synaptic dopamine concentration following amphetamine challenge was investigated in baboons using PET. Methods Affinity Studies: One adult male baboon was studied 8 times with 18F fallypride using PET (ECAT EXACT HR⫹ in 3D mode) for 180 min following bolus injections with mass dose ranging from 5 to 1600 ng/kg. K D estimates were derived in striatum (STR), thalamus (THA) and hippocampus (HIP) using 3 methods. Peak equilibrium analysis was performed by Scatchard plot (SP, method 1) and by fitting to the Michaelis Menten equation (MM, method 2). Kinetic analysis was also performed in the 4 high mass experiments and K D was derived using nonlinear kinetic modeling with arterial input function (NL, method 3). Methods 1 and 2 provided one K D value per region; method 3 provided one value per region per high mass experiment. In vitro displacement: The potency of fallypride to displace 3H raclopride from rat striatal homogenate membrane was measured at 37°C (n ⫽2) and 25°C (n⫽3). Amphetamine Challenge: Single bolus (SB) and bolus plus constant infusion (B/I) PET experiments were performed. In SB experiments, one baboon was scanned for 180 min under control conditions (n ⫽ 4) and following amphetamine (1 mg/kg, n ⫽ 3). The outcome measure was the equilibrium specific to nonspecific partition coefficient (V⬙3), derived by kinetic analysis (2 tissue compartments) using an arterial input function and the cerebellum as a region of reference. Regions of interest were STR, THA and HIP. In B/I experiments, two baboons (A and B) were studied for 420 (A) and 480 minutes (B). Preliminary experiments defined the optimal bolus to infusion ratio required to reach equilibrium in striatal, extrastriatal and reference regions by 200 min. Amphetamine was injected at 260 min (0.5mg/kg). Regional V⬙3 was measured as ROI activity divided by cerebellum activity minus 1, averaged over a period before (200-260 min) and after amphetamine (360-420 (A) and 420-480 minutes (B)). Results Affinity: All NL estimates of K D fell within the range of 0.10 to 0.14 nM. K D estimates by MM and SP were similar to NL in STR (0.13 and 0.19 nM) but higher in THA (0.32 and 0.34 nM) and HIP (0.66 and 0.86 nM). Theoretical analysis suggests that small regional differences in nonspecific binding could account for higher K D estimates by equilibrium methods in regions of low receptor density. In vitro displacement: In vitro K D estimates in rat striatum were highly temperature dependent (1.1 ⫾ 0.5 nM at 37°C and 0.07 ⫾ 0.05 nM at 25°C). Amphetamine challenge: In SB studies, amphetamine (1 mg/kg) induced significant decrease in 18F fallypride V⬙3 of 47% in STR (p ⬍ 0.001, two tailed t test), 20% in THA (p ⫽ 0.016) and 32% in HIP (p ⫽ 0.022). In B/I studies, amphetamine (0.5 mg/kg) decreased 18F fallypride V⬙3 by 24, 24 and 30% (baboon A) and 12, 22 and 18% (baboon B) in in STR, THA and HIP, respectively. Conclusion The most robust in vivo K D estimates were derived by NL (0.10 to 0.14 nM) and were intermediate between in vitro values measured at room temperature (0.07 nM) and physiological temperature (1.1 nM). Our estimate of the in vivo affinity of 18F fallypride is lower than previously reported in vitro in transfected cells at room temperature (0.03 nM). This lower affinity is consistent with the observation of significant wash out from the striatum and reduced binding in the challenge studies. Following amphetamine, both scanning/ analytical strategies showed a reduction in 18F fallypride V⬙3. This agreement suggests that the phenomenon is not an artifactual effect of nonspecific changes in flow or metabolism. These results imply that 18F fallypride could be used to study DA synaptic function in extratriatal areas. Such a method will be very important to characterize the mesocortical DA system in neuropsychiatric conditions.

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

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Rostrocaudal Distribution of D1 and D2 Dopamine Receptors in Striatum With [11C]NNC-112 and [11C]Raclopride Pedro Rosa*†, Paul Cumming*†, Ole Munk†, Albert Gjedde*† *Center of Functionally Integrative Neuroscience, Aarhus University, Aarhus, Denmark †PET Center, Aarhus University Hospitals, Aarhus, Denmark RATIONALE The topology of neurotransmitter systems in the brain is important to neurotransmission. Dopamine (DA) is present in most parts of the brain, but particularly in neostriatum, frontal cortex, and limbic structures. The D1 and D2 dopamine receptors are the most abundant dopamine receptors in the mammalian brain, with lower densities and heterogeneous distribution in the cortex than in the neostriatum. The aim of this work is to investigate the segregation of DA receptor densities in the neostriatum with parametric imaging of [11C]NNC-112 and [11C]raclopride radioligand mapping of the distribution of D1 and D2 receptors, respectively. METHODS The Danish National Committee on Animal Research Ethics approved this protocol. In brief, 3 healthy male Gottingen minipigs had [11C]raclopride (500 MBq) and [11C]NNC-112 (500 MBq) scans as 60-minute dynamic studies performed in the same day. The same animals were scanned with the two radioligands four and eight months later. For each study, anesthesia was induced with ketamine and midazolam and maintained with fluothane (1-2%). The arterial radioligand concentration curves were calculated as the metabolite-corrected arterial input functions. The dynamic records were resampled in a standard stereotactic space. MRI probabilistic volumes of interest using 50% confidence thresholds (Watanabe et al., 2001) were applied to the dynamic emission sequences in order to extract time-radioactivity curves in the striatum and cerebellum. Unidirectional blood-brain clearance (K1, ml g-1 min-1) and equilibrium distribution volume (Vd, ml g-1) of tracers in cerebellum and striatum were calculated by fitting a one-compartment model to the brain time-radioactivity curves using nonlinear regression. Several other methods for calculation of binding parameters using a cerebellum reference input function were tested. Parametric images of the binding potentials were generated using the linearized reference region slope method (“Logan”). RESULTS The results of different models are summarized in Table 1. Results of the segregation of D1 and D2 receptors are summarized in Figure 1. COMMENT These preliminary results corroborate previous studies in humans and animals suggesting a rostrocaudal gradient in the density of D1 and D2 receptors in the striatum. These results are limited by small sample size, partial volume effects and short dynamic acquisition for [11C]NNC.

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Multiple ligand concentration receptor assays (MLCRA): Sequential vs nonsequential measurements of density and affinity of dopamine D2 receptors with [11C] raclopride. Application to amphetamine effects Doris J. Doudet*, James E. Holden† *Dept. Medicine/Neurology, Univ. British Columbia, Vancouver, BC, Canada †Dept. Medical Physics, Univ. Wisconsin, Madison, WI, USA In in vivo MLCRA PET studies, a scan after administration of high specific activity (SA) raclopride (RAC) is followed, later in the day by 1 or 2 further RAC scans with progressively lowered SA. The data are obtained sequentially throughout the course of one day, and the outcome of the second or third study may be influenced by the potential effect of circadian changes in endogenous transmitter release and/or the prior sequential administration of pharmacological doses of the antagonist or a challenge agent. We compared the data obtained within a one day, routine in vivo MLCRA study with that of a MLCRA study in which each scan was acquired on separate days, at the same time of the day. In order to evaluate the effects of varying delays between an acute pharmacological manipulation and the high and low SA scans, we applied these 2 methods to the study of the effects of amphetamine on the density Bmax and apparent affinity Kdapp of D2 receptors. Six rhesus monkeys were scanned at baseline using a routine one day MLCRA (3 RAC injections 2-3 hrs apart with high, mid and low (respectively, ⬎1000; 25-35 and 6-10Ci/mmole) SA). The same animals also received 3 RAC scans at the same SA on 3 separate mornings at least 2 weeks apart. Two of these animals and 4 others were scanned using the routine in vivo MLCRA after amphetamine administration (2mg/kg i.p.20 min prior to the first, high SA RAC scan) and received a further low SA scan (6-10 Ci/mmole) on a separate day, 20 min after administration of amphetamine. RAC was administered as a bolus (50%) ⫹ infusion (50%) to achieve equilibrium. Bound/free (B/F) ligand ratios were estimated at each SA as the total distribution volume ratio derived from tissue-input Logan analysis minus one. Bound mass B was estimated as B/F times the time-averaged cerebellar radioactivity concentration and divided by the SA at the time of injection. B/F vs B plots were fitted to yield the “true” binding potential BP, density Bmax and the apparent affinity Kdapp. At baseline, there was no significant difference in the density (25⫾3.57 vs 23.99⫾3.22 pmol/ml), apparent affinity (12.69⫾1.97 vs 12.55⫾1.14 pmol/ml) and BP (2.1⫾0.47 vs 2.03⫾0.44) measured with the non-sequential vs sequential methods respectively. The test-retest reproducibility for each parameter of interest between the 2 acquisitions was similar to the test-retest reproducibility reported for the BP of single RAC studies (below 8%). After amphetamine, sequential MLCRA revealed a significant decrease in BP (35%) and a very significant increase in Kdapp (90%). There was also a surprising 18% increase in Bmax, however non significant in this small sample. Analysis of the non-sequential high and low SA scans obtained on different days but both 20 min after amphetamine injection revealed similar data: 34% decrease in BP, 82% increase in Kdapp and 20% increase in Bmax compared to baseline. There was no significant difference between the density and affinity of D2 receptors obtained with the sequential and non-sequential methods after amphetamine. The test-retest reproducibility for the density and affinity after amphetamine was about 16-17% but was only 5-6% for the BP. In conclusion, our data suggest that neither physiologic baseline variations in endogenous synaptic DA throughout the day nor previous administrations of RAC inducing less than 60% occupancy of D2 receptors significantly affect the measurement of density and affinity of D2 receptors. As expected, acute administration of amphetamine increase the apparent Kdapp of the receptor and decrease the BP. Two findings in this study deserve further considerations and replication in a larger population and confirmation using a different drug manipulation regimen: 1) the good agreement between the MLCRA study performed over a 6 hrs period after a single acute amphetamine injection with that of the MLCRA study performed 20 min after acute amphetamine and 2) the 20% increase in Bmax after amphetamine with both the sequential and non-sequential methods.

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Effects of catecholamine depletion on D2 receptor binding, mood, attentiveness and personality in humans Nicolaas P.L.G. Verhoeff*†‡§, Bruce K. Christensen‡§, R. Michael Bagby§ ¶, Doug Hussey†, Maggie Lee㥋, George Papatheodorou‡, Lili Kopala**, Qing Rui**, Robert B. Zipursky‡§, Shitij Kapur†‡§ *Kunin-Lunenfeld Applied Research Unit, Baycrest Centre for Geriatric Care, Toronto, Ontario, Canada †PET Centre, Centre for Addiction and Mental Health, Toronto, Ontario, Canada ‡Schizophrenia and Continuing Care Division, Centre for Addiction and Mental Health, Toronto, Ontario, Canada §University of Toronto, Toronto, Ontario, Canada ¶

Section on Personality and Psychopathology, Centre for Addiction and Mental Health, Toronto, Ontario, Canada 㛳Psychopharmacology Research Laboratory, Centre for Addiction and Mental Health, Toronto, Ontario, Canada **Department of Psychiatry, Dalhousie University, Halifax, Nova Scotia, Canada

The effect of catecholamine depletion, achieved by per-oral administration of 4.5-5.25 g ␣-methyl-para-tyrosine (AMPT) over 25-29 h, was studied on measures of dopamine (DA) release, mood, attention, and personality. Neostriatal DA levels in vivo were estimated by comparing the neostriatal DA D 2 receptor binding potential (D 2RBP) before and after catecholamine depletion using positron emission tomography (PET) and the radiotracer [ 11C]raclopride. Twelve healthy subjects completed the protocol. The AMPT treatment increased D 2RBP significantly from 3.16 ⫾ 0.32 to 3.61 ⫾ 0.27 and decreased plasma levels of the DA metabolite homovanillic acid by 66 ⫾ 14% and levels of the norepinephrine metabolite 3-methoxy-4-hydroxyphenethyleneglycol by 64 ⫾ 11%. Catecholamine depletion resulted in decreases in happiness, talkativeness, feeling high, vigor, attentiveness, and openness to feelings, and in increases in eye blink rate, drowsiness, sleepiness, sedation and persistence. These changes were not correlated with D 2RBP changes as measured with [ 11C]raclopride PET. Supported by NARSAD, OMHF, and CIHR/MRC Canada.

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Lorazepam Modulates the Thalamus as the Generator of Cortical EEG Alpha Rhythm Gerhard Gru¨ nder*, Mathias Schreckenberger†, Matthias Lochmann†, Christian Lange-Asschenfeldt*, Thomas Siessmeier†, Christoph Hiemke*, Klaus Mann*, Peter Bartenstein† *Department of Psychiatry, University of Mainz, Mainz, Germany †Department of Nuclear Medicine, University of Mainz, Mainz, Germany Introduction Electroencephalographic (EEG) alpha power is believed to reflect regional brain activity. Based on animal studies, which demonstrated significant relations between thalamic and cortical rhythmic activity, the thalamus has long been implicated in the generation of cortical EEG alpha rhythm. While findings from human studies are conflicting, the exact nature of the relationship between cortical electrical and thalamic metabolic activity remains to be clarified. In order to elucidate the functional circuitry between the cortex and the thalamus in humans, we studied regional cerebral glucose metabolism (rCGM) with [18F]fluorodeoxyglucose (18-FDG) PET, while simultaneously recording the EEG. This paradigm was applied both before and after a pharmacological challenge with lorazepam, which was chosen because benzodiazepines are known to be potent suppressors of cortical EEG alpha activity. Subjects and Methods Ten healthy male volunteers (age 24-30 years) were examined in a randomized single-blind study undergoing two 18-FDG PET scans (180 ⫾ 20 MBq) with simultaneous EEG recording. Studies were performed at least one week apart. Placebo (sodium chloride solution) or lorazepam (30␮g/kg body weight), respectively, was injected intravenously 15 min prior to tracer injection. EEG recording began at time of injection. Blood was withdrawn at various time-points for determination of lorazepam plasma levels as well as prolactin and cortisol concentrations. EEG power spectra were computed by means of Fourier analysis. The 18-FDG PET data were analyzed using SPM99: after image realignment, the scans were stereotactically normalized and proportionally scaled to a mean global cerebral activity. In order to investigate the neuro-metabolic effects of lorazepam parametric t-maps were computed and the correlation between rCGM and EEG alpha power was calculated on a voxel-by-voxel basis for placebo and lorazepam. Results The comparison lorazepam vs. placebo revealed a reduced rCGM in the bilateral thalamus as well as in the occipital cortex (p⬍0.001). Lorazepam administration significantly reduced EEG alpha power (p⬍0.01). In the baseline condition, the correlation analysis between EEG activity and rCGM yielded a positive correlation between the magnitude in EEG alpha power and the metabolic activity in bilateral thalamus as well as in the parieto-occipital cortex (p⬍0.001). In the lorazepam condition, the (reduced) EEG alpha power was significantly correlated with rCGM in the thalamus only (p⬍0.005). Conclusion These results support the view that the cortical EEG alpha rhythm is closely related to metabolic activity in the thalamus. The benzodiazepine lorazepam reduces both EEG alpha power as well as thalamic metabolic activity. However, it seems unlikely that lorazepam exerts these effects directly on a thalamic level. The thalamus is distinguished from other brain regions by its high amount of benzodiazepine receptors with an alpha4 subunit, which is almost exclusively restricted to this brain structure. Interestingly, lorazepam like other classical benzodiazepines does not bind to alpha4-containing benzodiazepine receptors with significant affinity. Therefore, it seems more likely that lorazepam-induced suppression of cortical input to the thalamus leads to reduction in its’ metabolic activity, which in turn may result in reduced cortical alpha power. The reduction of input from the ascending reticular activating system (ARAS) to the thalamus seems to be a less likely explanation, because the number of benzodiazepine receptors in brain stem and midbrain structures is comparably low. Pharmacological challenge studies with compounds interacting with the cortico-striato-thalamo-cortical circuits involved in this regulation are needed to further clarify the situation.

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Detection of Neuromodulatory Changes in Specific Neurotansmitter Systems: Experimental Design and Strategy Nathaniel Alpert*†‡, Rajendra Badgaiyan*†‡, Alan Fischman*†‡ *Division of Nuclear Mediciene †Massachusetts General Hospital ‡Havard Medical School Introduction During the past decade, using brain activation paradigms, it has become possible to study theories of cognition with 15O-H2O/PET and fMRI. Such studies detect the loci of flow-related changes elicited by cognitive tasks; however, they cannot provide information about the underlying neural mechanisms. Cognitive activation tasks also increase the synaptic level of endogenous neurotransmitters and this effect can be used to alter the kinetics of receptor ligands. Previous work by Koepp et al (Nature 393, 1998) has demonstrated the basic feasibility of the concept; however, their techniques are not fully optimized, requiring two scan sessions and a cohort of subjects. A new experimental and analytic approach is proposed and analyzed. Methods The new experimental design requires one tracer injection during a control state and serial PET scans over a 90 min interval. After tracer is bound to the receptor system, a cognitive task is used during the rest of the study to increase the synaptic neurotransmitter level, which, in turn, increases the apparent dissociation rate of the radioligand for the receptor. For example, dopaminergic neurons show variable response following sustained stimulation, but measurements and estimates suggests that dopamine levels rise 50-100%. Data analysis relies on an extension of the simplified reference region model (SRRM), which is mathematically equivalent but can also model the alterations of receptor kinetics induced by cognitive tasks. It is assumed that cognitive activation causes a step increase in endogenous neurotransmitter level. The operational equation includes a parameter, ␥, that accounts for changes in dissociation rate. Because the new model is linear in its parameters, weighted least squares estimation can be done on a voxel-by-voxel basis. The null hypothesis, that ␥ ⫽0, can be tested as a t-score using the theory of random fields, ala SPM. Simulation, used to explore the feasibility of observing the effect of activation on 11C-raclopride binding, was based on the following assumptions: 1. Raclopride will be given as a bolus with specific activity greater than 1000 ␮Ci/mmol. 2. Following the discussions of Fisher et al and Morris et al (HBM 3, 1995), we took K D⫽100 nM and B’max⫽56 pmol/ml for dopamine. We were particularly interested if it might be possible to detect changes in ligand dissociation in a single individual. Noiseless PET curves were simulated using the standard receptor model, noise were added to form ensembles of PET data. The resulting curves were fit with our extension of the SRRM. The simulations optimized the task initiation time, examined the confounding effect of changes in rCBF and tested the effects of several plausible synaptic alterations in dopamine level (including the effect of statistical noise). Results Simulation suggests that initiating the task 25-30 min. post injection makes the results insensitive to rCBF changes. The figure displays some important findings. The inset diagram shows simulated concentration vs. time (PET) curves for a control measurement (solid line) and varying increases (%ACTIVATION) in endogenous dopamine (dashed lines) initiated 30 min. after injection. The bars, presented in groups of three, represent simulated z-scores. The different bar-hatchings indicate 20, 25 and 30% increases in endogenous dopamine level. The abscissa indicates increasing noise level in the PET curves. A systematic decline in z score is seen for decreasing activation and increasing noise level. It is likely that some degree of spatial averaging will be necessary for hypothesis testing. Conclusions The SRRM has been extended to include the effects of endogenous neurotransmitter and cognitive activation. The results of simulation for the dopamine system suggest it may be possible to detect the loci of dopaminergic neurons involved in specific cognitive tasks. Furthermore, loci detection may be feasible in individual subjects during a single scan session.

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Dual Logan Plot Analysis for PET Studies with Pharmacological Challenge Robert Koeppe*†‡ University of Michigan, Department of Radiology, Division of Nuclear Medicine This study evaluates an analysis method for quantifying pharmacologically or behaviorally induced change in radiotracer binding using data from a single PET scan. Such challenge studies have been performed either using two separate injections, one during baseline and one during challenge conditions, or using a single injection of a rapidly equilibrating radiotracer with the challenge given during the session. While single-scan approaches have advantages of savings in time, effort, and subject cooperation, they require a radiotracer having rapid kinetics. Reported here are results from analysis of single-scan PET data using a dual-Logan plot approach, where a baseline measure is obtained from “early” data (⬃0-40min) and a “challenge” measure is obtained from later data. Logan analysis has the advantage that true equilibrium does not need to be achieved to estimate tracer binding. While the time to reach equilibrium may be sufficiently short for some radiotracers such as [11C]flumazenil (FMZ), more slowly equilibrating tracers such as [11C]carfentanil (CFN) require 30-40min before binding measures can be initiated. Following intervention, another 20-30min is required to re-establish equilibrium. For Logan analysis, data as early as 5-10min after injection may be used while following the challenge, again only 5-10min of equilibration is needed. This timesaving is particularly important for [11C]-labeled tracers where the 20min half-life limits acquisition to ⬃100min. PET studies were performed using [11C]FMZ (n⫽16) and [11C]CFN (n⫽7 completed-to-date; 16 planned). For [11C]FMZ, 8 subjects received baseline-only studies (no intervention) while 8 received challenge studies. Half had single scans (15-18 mCi) and half had two scans (7-10 mCi per scan) separated by 2hrs. Baseline-only studies were performed to examine reproducibility of binding measures under conditions of no expected change. In challenge studies, subjects received 0.005 mg/kg cold flumazenil as a bolus, followed by a continuous infusion of 0.0001 mg/kg/min for the duration of the study, a dose designed to block ⬃50% of the specific binding sites. For single-injection studies, the challenge was initiated 40min post-injection, while for the double studies it was begun just prior to the second scan. For all studies, radiotracer was administered as a partial bolus followed by continuous infusion in order to achieve equilibrium as rapidly as possible. PET data was acquired for 70min for double studies and 100min for single studies. A similarly designed set of studies using [11C]CFN, a more slowly equilibration tracer, is partially complete. Intervention studies will use naloxone at a dose designed to block ⬃50% of the specific binding. Each subject’s data was analyzed using both equilibrium and Logan analyses. For double studies, equilibrium-binding measures were obtained from data 40-70min post-injection, while Logan analysis used data from 10-70min. For single studies, equilibriumbinding estimates were obtained from 30-40min post-injection for the initial measure and 70-100min for the second measure. For Logan analysis, data from 10-40min and 45-100min were used for initial and repeat binding measures, respectively. For the [11C]FMZ studies, equilibrium analysis did not differ significantly from Logan analysis for either single or dual studies, or for either first or second binding measures. The ratio of estimates (Logan/Equilibrium) for the 16 subjects (x2 measures each) averaged 99.7⫾3.5% and ranged from 93.2% to 108.1%. For baseline-only double studies, the percent change from scan 1 to scan 2 averaged 0.8⫾3.9% and -1.1⫾4.0% respectively for equilibrium and Logan analyses. For single-scan studies, the corresponding changes averaged 3.3⫾8.7% and 5.1⫾3.0%, demonstrating a slight positive bias in the repeat measure. The variability of the equilibrium approach was about double that of the Logan approach. For challenge studies using the rapidly equilibrating [11C]FMZ, again both equilibrium and Logan analysis yielded similar results. For double studies decreases in binding of 50.5⫾4.3% and 50.6⫾5.7% were measured for equilibrium and Logan analyses, respectively. For single studies, decreases in binding of 55.3⫾6.2% and 54.6⫾5.4% were measured for equilibrium and Logan analyses, respectively. Results from the baseline-only [11C]CFN, a much more slowly equilibrating tracer, show some differences between double and single studies. For double-scan studies, equilibrium and Logan approaches show similar results, while for single-scan studies, equilibrium analysis yielded binding measures that were considerably more variable both within (different regions) and across subjects than those obtained using dual-Logan analysis. Single-scan Logan results were slightly but not appreciably more variable than results from the double-scan studies. In conclusion, studies using [11C]FMZ show that dual-Logan plot analysis can provide multiple receptor measures from a single tracer injection, with as good precision and as little bias as double-injection studies. The studies proving utility for slowly equilibrating radiotracers, such as [11C]CFN, are underway, and appear promising.

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PET receptor assay with multiple ligand concentrations and true equilibrium: mathematical and neurochemical aspects James E. Holden*, Salma Jivan†, Thomas J. Ruth†, Doris J. Doudet‡ *Department of Medical Physics, University of Wisconsin Madison, WI, USA †TRIUMF, University of British Columbia, Vancouver, BC, Canada ‡Department of Medicine, Division of Neurology, University of British Columbia, Vancouver, BC, Canada The ligand-receptor binding potential determined by in vivo PET studies at high ligand specific radioactivity (SA) reflects both the receptor density and ligand-receptor affinity. This ambiguity has been resolved by various methods based on the administration of multiple unlabeled ligand concentrations. We have implemented and refined an approach to multiple ligand concentration receptor assay (MLCRA) that combines maximum simplicity and a minimum of assumptions and model dependence that nonetheless reliably distinguishes density from affinity effects. The approach uses administration by bolus followed by infusion (Laruelle et al., 1994; Carson et al., 1997) to obtain a true equilibrium between bound ligand and the other components of the ligand concentration, and does not require measurements of ligand in blood plasma. The increase in the binding potential of raclopride to D2-type receptors in the striata of nonhuman primates lesioned with MPTP (Falardeau et al., 1988; Doudet et al., 2000) provided an example application of our approach, and allowed particular aspects to be investigated in detail. Data from 14 rhesus monkeys (Macaca mulatta) were used: 8 were normal and 6 had right unilateral MPTP-induced lesions of the nigrostriatal pathway. These latter displayed symptoms of parkinsonism. 11C-raclopride was administered by a bolus-plusinfusion protocol designed to give constant time-courses in both specifically binding and reference regions of the brain. Studies were performed with either three or four progressively increasing ligand concentrations. All the animals had at least 3 raclopride scans: high SA (⬎1000 Ci/mmol), mid-low SA (25-40 Ci/mmol), and low SA (8-11 Ci/mmol). Five of the animals also had a fourth scan with a mid-high SA (70-100 Ci/mmol) and four had a fourth scan with an extra-low SA (3-6 Ci/mmol). The scans were separated by a minimum of 2 hours (between SA1 and SA2) and up to 3.5 hours (SA3-SA4). The equilibrium radioactivity concentrations in striatum and in cerebellum, divided by the SA at the time of injection, were assumed to represent (B⫹F) and F, respectively, where B is the equilibrium concentration of ligand that is specifically bound and F represents the sum of all ligand components that are not specifically bound. At equilibrium, these concentrations are related by B⫽B max F/(K dapp⫹F) (direct plot); the goal of MLCRA is to evaluate the total receptor density B max and the ligand-receptor affinity K dapp from the measured relationship between B and F. The superscript app (apparent) reflects the several differences between the apparent affinity determined in vivo and that determined under ideal conditions using in vitro receptor assay methods. We evaluated the properties of four different formulations of the B vs. F relationship for performing the parameter optimization, including the direct plot itself. Two were linearizations of the relationship, optimized by conventional least-squares optimization, and two were non-linear optimizations. Our results showed the benefits of having the equilibrium ratio B/F as one of the fitted data sets and the binding potential as one of the optimized parameters, as these are predicted to be equal in the limit of high SA. The small risk of inappropriate propagation of measurement error into parameter error is more than compensated for by the robustness of the fits and the decreased correlation between the fitted parameters. Analysis using the direct plot failed to find a significant difference in binding potential between the normal and lesioned groups, whereas the other methods yielded unpaired t-test p values of order 10 -4. Ligand administration by bolus-infusion and the use of the tissue-input Logan graphical method (Logan et al., 1996) allowed the ratio B/F to be evaluated from a maximum amount of the measured data with a minimum of prior assumptions. MLCRA requires the assumption that the optimized parameters are independent of SA. We investigated the possibility that high antagonist concentrations induced significant changes in synaptic dopamine concentrations, which would cause each equilibrium study to be characterized by a different K dapp value. The parameter K dapp can be estimated from (B,F) values at two SA values. For each animal, the data from high SA were combined in turn with the data from each of the other SA values to give either two or three two-point K dapp estimates. The results confirmed that K dapp for raclopride is independent of unlabeled ligand concentration, at least over the range of ligand concentrations used (maximum saturation about 65%.) Carson RE et al. (1997) J Cereb Blood Flow Metab17:437-447 Doudet DJ et al. (2000) Synapse 38:105-113 Falardeau P et al. (1988) Neurosci Lett 86:225-229 Laruelle M et al. (1994) J Cereb Blood Flow Metab 14:453-465 Logan J et al. (1996) J Cereb Blood Flow Metab 16:834-840

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Detailed multi-receptor mapping in the human brain Zsolt Cselenyi, Lars Farde, Hans Olsson, Christer Halldin, Balazs Gulyas Psychiatry Section, Department of Clinical Neuroscience, Karolinska Institute, S.171 76 Stockholm, Sweden Introduction Several brain neurotransmission systems are linked on neurodevelopmental and physiological levels. A fundamental question is whether the regional distribution of a single receptor as well as its co-distribution with other neurotransmission systems is genetically determined or influenced by stimuli during early life. To approach these questions novel techniques are required that allow combined correlational analysis of multiple receptor systems in the human brain. The objective of the present study was to create a conceptual framework and algorithm for quantitative and qualitative analysis of the spatial coexistence of markers for neuroreceptor systems in the human brain. Methods The dopamine and serotonin systems, two major and phylogenetically early transmission systems, were analysed in this study based on radioligand binding data in control human subjects. The radioligands were [11C]FLB457 for D2-like dopamine receptors and [11C]WAY100635 for the serotonin 5HT1A-receptor. Binding potential images were created from the raw input PET images using a wavelet aided parametric mapping method described previously. The multitude of results was explored in an n-dimensional “receptorspace” (where n is the number of neuroreceptor systems analysed) by means of a cluster analytic framework (Figure 1). Using the Karolinska Institute’s computerised human brain atlas software, the clusters were compared with the cytoarchitectural (Brodmann’s) areas of the average population’s. Results The proposed approach allowed identification of co-distribution patterns by classifying significant groupings of receptor density values. The cluster analytic exploration of individual binding maps was matched against that of the group mean binding maps. This allowed for identification of co-distribution patterns that may be stable and/or consistent within the population (Figure 2). Conclusions The results indicate that on the basis of multi-ligand mapping of central neuroreceptor systems the human neocortex can be characterized in terms of well-defined areas on a receptor co-localization level. These maps should be useful for detection of deviant patterns in patients with neuropsychiatric disorders.

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5-HT2C receptor activation detected in humans by fMRI Ian Anderson*†, Lynn Clark*†, Rebecca Elliott*†, Bhavna Kulkarni‡, Steve Williams*§, Bill Deakin*† *University of Manchester †Neuroscience and Psychiatry Unit ‡Rheumatic Diseases Centre, Hope Hospital, Salford §Imaging Science and Biomedical Engineering Functional brain imaging techniques using selective drug ‘probes’ offer the opportunity to investigate regional neuronal activation linked to receptor stimulation or inhibition. The validity of using fMRI for drug studies has been questioned; nevertheless there are examples in humans and animals where direct effects of drugs have been reported (pharmacoMRI) (1,2). Here we report the use of pharmacoMRI to identify brain regions involved in serotonergic neurotransmission. Eight healthy male volunteers (26-42 years) were given saline and the 5-HT2C agonist, m-chlorophenylpiperazine (mCPP), infusion (0.08mg/kg over 90 seconds) in a randomised, balanced order, single-blind design. Following an initial 8-minute baseline fMRI scan (S1) subjects received drug or saline given at the start of a second 8-minute scan (S2). Forty T2*weighted contiguous axial slices were acquired on a Phillips 1.5T Gyroscan machine using a single shot echo planar pulse sequence (TR ⫽ 5000 ms, isotropic in plane resolution 3.5 x 3.5 mm), with T1-weighted anatomical images acquired for co-registration. Data were analysed using SPM99 with a random effects model. The sum of subjective ratings during S2 were used as a regressor which was superimposed on a factorial design (S2-S1 under saline subtracted from S2–S1 under mCPP), to model direct drug effects. Plasma mCPP and prolactin and cortisol responses were correlated with activations due to mCPP. mCPP infusion increased BOLD signal in the hypothalamus, caudate, pallidum, amygdala and pyriform cortex, anterior cingulate gyrus and choroid plexus (p⬍0.001 uncorrected, Figure 1). Activation seen in the hypothalamus correlated significantly with the prolactin response (p⬍0.05, small volume correction based on a priori hypothesis, Figure 2). Correlations with prolactin were also seen in the L insula, L amygdala, L thalamus and anterior cingulate, for cortisol the ventral cingulate and L putamen, and for mCPP concentration bilateral orbitofrontal cortex (p⬍0.001, uncorrected). This study demonstrates regionally-specific changes in BOLD activation following the 5-HT2C agonist mCPP which largely corresponds to areas with a high density of 5-HT2C receptor binding in humans; choroid plexus, hippocampus, substantia nigra, basal ganglia, amygdala, hypothalamus and prefrontal cortex (3). Given mCPP’s neuroendocrine and temperature effects, the activation of the hypothalamus, and its correlation with subsequent prolactin secretion, is consistent with the result being of functional importance. Our findings suggest that pharmacoMRI is a potentially powerful tool for the examination of neurotransmitter function in normal subjects and patients with psychiatric disorders. References 1. Breiter, H.C., et al. Neuron 19, 591-611 (1997). 2. Stein, E.A., et al. Am.J.Psychiatry 155, 1009-1015 (1998). 3. Marazziti, D., et al. Eur.Neuropsychopharmacol. 10, 21-26 (1999). Acknowledgement This study was supported by a grant from the Central Manchester Healthcare Trust Research and Development Fund.

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Effect of OS-EM Reconstruction on Kinetic Modeling of [Carbonyl-11C] WAY-100635: implications for other neuroreceptor ligands M-J Be´ langer*†, Ramin V. Parsey*†, J. John Mann*† *Department of Psychiatry, Columbia University, NY, NY, USA †Division of Neuroscience, New York State Psychiatric Institute, NY, NY, USA Background Receptor imaging using 3D PET of the brain is an important tool in determining what are the mechanisms of psychiatric illnesses. The reconstruction algorithm: Ordered-Subset Expectation Maximization (OS-EM) (Hudson and Larkin, 1994), has emerged as a practical iterative alternative to filtered backprojection (FBP). EM algorithms have been found to improve image resolution (Liow et al, 1997). We investigate here the effect of choosing the OS-EM reconstruction algorithm versus FBP on kinetic modeling parameters of the serotonin 5-HT-1A receptor ligand: [Carbonyl-11C] WAY-100635 (Parsey et al., 2000). Methods We used seven dynamic studies from healthy volunteers acquired for 110 mins with the EXACT HR⫹ PET scanner. The 3D sinograms were preprocessed into 2D data using Fourier rebinning (FORE). Each of those studies were reconstructed by FBP or OS-EM (16 subsets and 6 iterations: necessary for DRN convergence) using ECAT 7.2. CTI software. The time-activity curves (TAC) were obtained for the: 1) cerebellum (CER) as an index of non-specific binding, 2) dorsal raphe nucleus (DRN) which represents an area of high specific binding surrounded by non-specific binding, 3) orbital prefontal cortex (OPFC) an area of moderate specific binding. The volume of distribution (VT) was calculated for each reconstruction using a three compartment unconstrained kinetic model and a measured arterial input function. Results The TACs of the CER calculated from OS-EM images, were found to level-off at late frames while the FBP TAC continued towards zero (see figure). This late frame bias was attributed to the non-negativity condition of the EM algorithm. As a result, the VT CER was found to be larger for OS-EM than for FBP (16⫾10%). For the DRN, the TAC at late frames was found to be smaller for OS-EM than for FBP. This compensatory reduction in the DRN’s reconstructed activity was presumably due to the positive bias of the surrounding tissue. Consequently, the DRN VT was found to be smaller for OS-EM than FBP (-14⫾7%). The OPFC TACs of the OS-EM were not found to differ from the FBP TAC. The visualization of the DRN was also examined for both OS-EM and FBP. The DRN region was drawn on OS-EM and FBP studies by four independent image analysts. The standard deviation of the VT from the drawn DRNs was smaller on images reconstructed by OS-EM compared with FBP. Conclusion We found that OS-EM introduced a positive bias in low count regions (ex. CER and indirectly, DRN at late frames). Similarly, neuroreceptor ligands that exhibit low non-specific binding can have the non-specific VT overestimated with OS-EM reconstructed studies and consequently, their specific binding potential can be underestimated. On the other hand, OS-EM improved the image sharpness required to draw the DRN in [Carbonyl-11C] WAY-100635 PET images. It would be beneficial to have an unbiased OS-EM implementation to use in neuroreceptor imaging. Supported by the PHS grants: MH62185 and MH40695.

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DEPICT: Data-driven Estimation of Parametric Images based on Compartmental Theory Roger Gunn*, Steve Gunn†, John Aston*‡, Federico Turkheimer§, Vincent Cunningham‡§ *Montreal Neurological Institute, McGill University, Montreal, Canada †Dept. of Electronics and Computer Science, University of Southampton, U.K. ‡Imperial College, London, U.K. §IRSL, London, U.K. Traditionally, quantification of PET data has usually required explicit specification of a compartmental model to describe the fate of the tracer in vivo. This work concerns the development of a general data driven bio-mathematical modelling technique for dynamic positron emission tomography (PET) data, which requires no a priori model specification. This transparent estimation method is based on a general compartmental description of the tracer and determines the appropriate model order from the data itself. The framework encompasses models using either a plasma or reference tissue input and the full range of tracer administration schemes. Here, the method is applied to neuroreceptor binding studies to produce parametric images of receptor function and model order. All linear compartmental systems, either using a plasma or reference tissue input, result in an impulse response function (IRF) which is composed of a sum of exponentials and a delta function (Gunn, 2001). The macro parameters of the system may be derived simply from the systems IRF. For example, for reversible radioligands with a plasma input, the total volume of distribution (VD) is equal to the integral of the IRF and for reversible radioligands with a reference tissue input, the binding potential (BP) is equal to the integral of the IRF minus one. Thus, it is possible to construct the parameter estimation problem in a basis function framework. The use of kinetic basis functions has the advantage that computer intensive convolutions may be performed initially before considering individual voxel time courses offering a great increase in speed over more traditional least squares optimization methods. For a suitable number of basis functions, which span the full kinetic range, this leads to an under-determined set of linear equations. This ill-posed problem requires the addition of constraints to impose a unique solution on the estimation process. The solution here is to use penalized least squares techniques, which have been popularized recently in the wavelet community, for the estimation of sparse representations from over-complete dictionaries (Chen, 1995). The introduction of a 1-norm regularizer or penalty function to the standard least squares metric constructs the problem, in terms of basis pursuit denoising. The addition of a 1-norm to the standard 2-norm metric allows for a uniquely determined parameter estimation process. With the introduction of slack variables basis pursuit de-noising can be written as a simple bound constrained quadratic program. Basis pursuit denoising requires the determination of the regularization parameter. Here, this is obtained by the method of leave one out cross-validation. Leave one out cross validation is a “resampling” method which allows us to minimize the generalisation error over the regularization parameter. Fits are performed in which each data point is omitted in turn, and the generalisation error is then estimated by summing up the prediction error for each omitted data point. This function is then minimized numerically over the regularization parameter. This is a transparent method because it will return information about the underlying compartmental structure. The number of non-zero coefficients returned corresponds to the model order which is related to the number of tissue compartments. For plasma input models the model order equals the number of tissue compartments (ignoring the blood volume component) and for reference tissue models the number of non-zero coefficients corresponds to the total number of tissue compartments in the reference and target tissues. The presented method allows for the estimation of parametric images or regional parameter values from dynamic PET data. The method requires no a priori description of the tracers fate in vivo, derives the model description from the data and returns information on the number of compartments (model order) present. This has application to a wide range of PET radiotracers but the emphasis here is with respect to the analysis of radioligands binding studies. References 1. Gunn R. N., Gunn S. R. and Cunningham V.J. Positron Emission Tomography compartmental models. J Cereb Blood Flow Metab. 2001;21(6):635-52 2. Chen S. PhD Thesis, Stanford University, U.S.A. 1995.

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MODEL AVERAGING USING AKAIKE WEIGHTS Federico Turkheimer, Rainer Hinz, Vincent Cunningham IRSL, Cyclotron Building, Hammersmith Hospital, London UK

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Amphetamine-induced dopamine release in patients with schizotypal personality disorders studied by SPECT and [123I]IBZM Marc Laruelle*, Lawrence Kegeles*, Yolanda Zea-Ponce*, Osama Mawlawi*, Diana Martinez*, Anissa Abi-Dargham*, Larry Siever† *Departments of Psychiatry and Radiology, Columbia University, NY †Department of Psychiatry, Mt Sinai School of Medicine, NY Objective Schizotypal personality disorder (SPD) is a schizophrenia-related personality disorder and family studies suggest that both conditions might be genetically related (1). Patients with SPD share with schizophrenia cognitive deficits and negative symptoms, but do not exhibit severe psychotic episodes. Previous imaging studies reported that amphetamine-induced dopamine (DA) release is elevated in untreated patients with schizophrenia, suggesting that this condition is associated with dysregulation of DA function (2). The goal of this study was to investigate if patients with SPD also exhibit this deficient regulation. Methods 13 unmedicated SPD patients (10M/3F, 36 ⫾ 12 y.o) and 13 matched healthy subjects (9M/4F, 34 ⫾ 9 y.o.) underwent SPECT scan during bolus plus constant infusion of the radiolabeled D2 receptor antagonist [123I]IBZM. Striatal V3⬙, calculated as striatal-cerebellar/cerebellar activity ratio, was measured under equilibrium condition, at baseline and following amphetamine administration (0.3 mg/kg) with the PRISM 3000. The decrease in [123I]IBZM V3⬙ induced by amphetamine was used as outcome measure. Results No significant differences were observed in baseline [123I]IBZM V3⬙ between SPD patients (0.77 ⫾ 0.11) and controls (0.72 ⫾ 0.08, p ⫽ 0.19). Amphetamine induced a larger decrease in V3⬙ in SDP patients (-12 ⫾ 5%) compared to controls (-7 ⫾ 5%, p ⫽ 0.03). Discussion The reduction in [123I]IBZM V3⬙ induced by amphetamine in patients with SDP was significantly larger compared to control subjects, suggesting that this condition is also associated with deficient control of DA activity. Amphetamine induced decrease in [123I]IBZM V3⬙ in SPD patients had a similar magnitude than that previously observed in remitted schizophrenic patients (-10 ⫾ 9%, n ⫽ 17) using a similar method. However, it was lower than that observed in patients with schizophrenia scanned during episodes of illness exacerbation (-24 ⫾ 13%, n ⫽ 17). This result suggests that the dysregulation of DA function revealed by the amphetamine challenge in schizophrenia spectrum disorders might have two components: a trait component, presumably of genetic origin and shared by remitted patients with schizophrenia and patients with SPD, and a state component, exhibited by patients with schizophrenia during psychotic episodes. 1.Siever LJ, Kalus OF, Keefe RS: The boundaries of schizophrenia. Psychiatr Clin North Am 1993; 16(2):217-44 2.Laruelle M, Abi-Dargham A, Gil R, Kegeles L, Innis R: Increased dopamine transmission in schizophrenia: relationship to illness phases. Biol Psychiatry 1999; 46(1):56-72

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Selective Upregulation of Prefrontal D1 Receptors in Schizophrenia Predicts Poor Working Memory Performance Anissa Abi-Dargham, Ning-Ning Guo, Ilise Lombardo, Osama Mawlawi, Roberto Gil, Dah-Ren Hwang, Yiyun Huang, Mohammed Ali, Diana Martinez, John Keilp, Ronald Van Heertum, Jack Gorman, Marc Laruelle Departments of Psychiatry and Radiology, Columbia University, NY Studies in nonhuman primates documented that appropriate stimulation of dopamine (DA) D1 receptors in the dorsolateral prefrontal cortex (DLPFC) is critical for working memory processing (1). Deficit in working memory is a core feature of schizophrenia, and it has been postulated that this impairment relates to a deficiency in mesocortical DA function. To test this hypothesis, we measured D1 receptors availability with PET and the selective D1 receptor antagonist [11C]NNC 112 in 16 patients with schizophrenia (7 drug naı¨ve and 9 drug free patients) and 16 matched healthy controls. [11C]NNC 112 binding potential (BP) was significantly increased in the DLPFC of patients with schizophrenia (1.63 ⫾ 0.39 mL g-1) compared to control subjects (1.27 ⫾ 0.44 mL g-1, p ⫽ 0.02). No significant differences in [11C]NNC 112 BP between patients and controls were observed in other regions examined. In patients, increased DLPFC [11C]NNC 112 BP was a strong predictor of poor performance at the n-back task, a test of working memory (r2 ⫽ 0.45, p ⫽ 0.008) involving the DLPFC. The result of this study contrasts with the result of a previous study (2) that reported a decrease in [11H]SCH 23390 binding in the frontal cortex in patient with schizophrenia. These conflicting results may simply relate to the heterogeneity of the illness. Alternatively, chronic DA depletion may have opposite effects on the binding of the two tracers due to differential regulation of the in vivo binding of the two D1 radioligands [11C]NNC 112 and [11H]SCH 23390 following chronic DA deficit. To test this hypothesis, in vivo binding of [11C]NNC 112 and [3H]SCH 23390 was evaluated in rats treated with either vehicle or reserpine (1 mg/kg, i.p.) daily for 14 days. Neurochemical analysis indicated that the drug treatment produced 98% reduction of DA. Compared to saline treated controls, rats with chronic DA depletion showed significant increases in [11C]NNC 112 in vivo binding in the PFC (p⬍ 0.002) and striatum (ST) (p⬍ 0.02). In contrast, [3H]SCH 23390 binding was unchanged in the PFC and a significantly decreased in the STR (p⬍ 0.03) in chronic DA-depleted rats. These studies suggest that the in vivo binding of these radiotracers is differentially affected by chronic DA depletion and might reconciliate the results of both clinical studies. In summary, studies in rodents documented that the in vivo binding of [11C]NNC 112, but not [11H]SCH 23390, is increased in the PFC following chronic DA depletion, suggesting that the increased D1 receptors availability observed in patients with schizophrenia might represent a compensatory (but ineffective) upregulation secondary to deficiency in mesocortical DA function. These findings confirm that alteration of DLPFC D1 receptor transmission is involved in working memory deficits presented by patients with schizophrenia. NIMH, NARSAD, and the Lieber Center for Schizophrenia Research. 1.Goldman-Rakic PS, Muly EC, 3rd, Williams GV: D(1) receptors in prefrontal cells and circuits. Brain Res Brain Res Rev 2000; 31(2-3):295-301 2.Okubo Y, Suhara T, Suzuki K, Kobayashi K, Inoue O, Terasaki O, Someya Y, Sassa T, Sudo Y, Matsushima E, Iyo M, Tateno Y, Toru M: Decreased prefrontal dopamine D1 receptors in schizophrenia revealed by PET. Nature 1997; 385(6617):634-6

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The antiparkinsonian effects of deep brain stimulation in the subthalamic nucleus are not mediated by an increase of tonic striatal dopamine receptor stimulation: evidence from serial [11C]raclopride-PET in parkinsonian humans Ruediger Hilker, K. Herholz, Alexander Schuster, Juergen Voges, Mehran Ghaemi, Volker Sturm, Klaus Wienhard, Wolf-Dieter Heiss Department of Neurology, University and Max-Planck Institute, Cologne, Germany Deep brain stimulation of the subthalamic nucleus (STN-DBS) has become an effective treatment option in advanced Parkinson’s disease (PD). Recent animal data indicate increased neuronal firing in dopaminergic neurons of the substantia nigra pars compacta and elevated extracellular levels of striatal dopamine metabolites under effective STN-DBS. In this study, we sought to determine changes of the striatal dopamine release in PD patients under effective STN-DBS. For this purpose, serial PET scans with the 11C-labeled reversible dopamine-D2/3-receptor-antagonist raclopride (RACLO) were performed in the stimulator-on- and stimulator-off-condition in six STN-DBS patients with advanced PD and levodopa-associated complications (2 males, 4 females, mean ⫾ 1 SD: age 61.8 ⫾ 4.9 yrs, disease duration: 15.3 ⫾ 4.4 yrs, HY stage 3.7 ⫾ 0.5). Differences of RACLO binding between conditions reflect intervention-induced changes in endogenous transmitter release, since striatal RACLO binding is inversely related to dopamine levels in the synaptic cleft. Two RACLO-PET-scans (ECAT Exact HR, Siemens-CTI, Knoxville, Tenn, USA) were undertaken in a 3D data acquisition mode on consecutive days in the stimulator-on- and stimulator-off-condition six months after surgery. After correction for decay and attenuation a 370 MBq 11C-raclopride bolus (specific activity 30-70 GBq/mmoL) was injected. PET scanning started after a 30-second background frame, recording a total of 18 serial time frames (3 x 1 ⫹ 4 x 2 ⫹ 3 x 3 ⫹ 2 x 5 ⫹ 6 x 10 minutes) over a total study period of 90 minutes. For identification of individual anatomical structures, high-resolution MRI was performed on a 1-Tesla Magnetom (Siemens AG, Erlangen, Germany) in a three-dimensional fast low-angle shot mode. The individual position of brain structures was determined by inspection of a summed RACLO image representing the activity collected 30-60 minutes after tracer injection. The PET images were exactly coregistered to the individual T1-weighted MRI-scan aligned to the AC-PC line according to a previously described procedure (MPI-Tool; Pietrzyk 1994). With the use of an interactive software package (3D-Tool; von Stockhausen 1998), the striatal volumes of interest (VOIs: caudate nucleus and putamen) were drawn by outlining the appropriate anatomical structures in transversal slices of the individual T1-weighted MRI. The entire cerebellum was defined as a reference region for measuring free and non-specifically bound RACLO. In each subject, the same VOI set was then automatically transferred to the PET scans. Decay-corrected time-radioactivity curves (TAC) were obtained from the dynamic PET images for each VOI. Specific RACLO binding values were computed using a graphical analysis method for assessing reversible radioligand-receptor-interactions (Logan 1996). This analysis approach employs the VOI-specific and cerebellar TAC to provide a linear regression slope that reflects the VOI-specific RACLO binding as a measure of the total radioligand distribution volume (DVR). Changes in striatal endogenous dopamine D2/D3 receptor binding were assessed as the percent difference in regional DVR between STN-off- and STN-on-conditions : DDVR ⫽ ([DVR STN-on – DVR STN-off] / DVR STN-off) x 100 %. STN-DBS proved to be a highly effective treatment for PD which significantly decreased the UPDRS III motor score in the medication-off-condition by nearly 57% (UPDRS III scores: STN off 39.2 ⫾ 7.9, STN on 16.7 ⫾ 5.5, P ⫽ 0.003 paired t test). After switching the stimulators on, the mean caudate und putaminal RACLO-DVR changed by – 4.7 ⫾ 10.4 % and 1.7 ⫾ 10.9 %, respectively. No significant RACLO-DVR differences between the two STN-DBS-conditions were found. The changes did not correlate to the patients’ improvement in the UPDRS motor scores. Though we observed a highly significant improvement of clinical scores in the STN-on-condition, no concomitant changes of striatal raclopride binding were found. Therefore, our data provide no evidence for a substantial increase of striatal dopamine receptor stimulation as a possible mechanism of delayed antiparkinsonian effects of STN-DBS. They rather suggest to consider long-term modifications of neurotransmitters apart from the dopaminergic system to explain the delayed effects of STN-DBS, e.g. changes of GABA and glutamate transmission within the striatum as well as the substantia nigra pars reticulata. Furthermore, the shift of activity within distinct thalamocortical projections that is believed to underly the aleviation of parkinsonian motor signs or the delayed occurence of levodopa-mimicking dyskinesias may occur with various and occasionally slow time courses. References Pietrzyk U et al. (1994) J Nucl Med 35: 2011-2018 Logan J et al.(1996) J Cereb Blood Flow Metab 16: 834-840 Von Stockhausen HM et al. In: Carson E, Daube-Witherspoon ME, Herscovitch P, editors. Quantification of functional brain imaging with positron emission tomography. San Diego: Academic Press, 1998: 139-42

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Neurobiology of personality traits: a [18F] FESP-PET study Rosa Maria Moresco*, Massimiliano Dieci†, Clara Gobbo‡, Cristina Messa*, Laura Galli‡, Giovanna Rizzo§, Andrea Panzacchi‡, Antonio Vita ¶, G. Invernizzi ¶, Ferruccio Fazio*‡§ *University of Milan-Bicocca †Ospedale di Melzo ‡Scientific Institute H San Raffaele §INB-CNR ¶

University of Milan

Neurotransmitter systems have been associated with aspects of personality traits. Using Positron Emission Tomography (PET) and [11C]raclopride, an association between striatal D2 dopamine receptors and emotional detachment has been reported (L. Farde et al., 1997; A Breier et al., 1998). In this study we investigated the relationship between “novelty seeking”, “reward dependence”, “harm avoidance”, and “persistence” evaluated by the Cloninger’s Temperament and Character Inventory Questionnaire and the in vivo binding of [18F]FESP in the brain of a group of young healthy volunteers. Eleveen healthy volunteers (8M, mean age 27⫹4, range 22-32 yrs.) were enrolled for the study and underwent a PET scan. A [F-18]FESP binding index (BI) to either striatal D2 or cortical 5HT2A receptors was measured between 90 and 120 minutes after injection using the cerebellum as a reference region. PET images were analyzed using both a manual (ROIs) and an automatic operator independent (SPM96) techniques. Spearman correlation coefficients were calculated among each of the four personality dimensions total score, age and the mean, right and left BI values and a multiple linear regression analysis, considering: each cortical region and age as independent variables and the personality dimension as the dependent variable. For these analyses, all data were transformed into rank values. Results The personality dimension “harm avoidance” showed a significant inverse correlation to [18F]FESP binding in the cerebral cortex, particularly in the frontal cortex (R square⫽ -0.709, p⫽0.0145) and left parietal cortex (R⫽-0.629), p⫽0.038) but not in the basal ganglia (r⫽-0.176, p⫽0.651). Similar results were obtained using SPM setting a p thresholds of 0.05. A significant direct correlation between cortical [18F]FESP binding and the trait “persistence” was also observed using both ROIs (R⫽0.712, p⫽0.04) or SPM analysis (p threshold lower than 0.001). No significant correlation of [18F]FESP binding were observed for the trait “novelty seeking” or “reward dependence” in any of cerebral area examined. Conclusion In the cerebral cortex, high values of [18F]FESP binding values are associated to an high tendency to avoid danger in otherwise normal subjects, indicating an involvement of serotoninergic system and in particular of 5HT2A receptors, in this trait of personality. In addition, the high correlation observed between the trait “persistence” and the in vivo binding of [18F]FESP to cortical 5HT2A receptors is in agreement with recent findings indicating an high association between that 5HT2A receptor gene and persistence. The results of our as well as previous studies on personality dimensions indicate not only the existence of a relationship between specific behavioural and neurobiological factors but more in general, represent important findings to sustained the concept that variability of PET data may be strongly explain by neurochemical aspects.

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Reduced GABAA Receptor/Benzodiazepine Binding Sites in Insular Cortex of Panic Disorder Patients Kirk Frey*, Oliver Cameron*, Grace Huang, Satoshi Minoshima†, Michael Kilbourn*, Thomas E. Nichols*, Robert Koeppe* *The University of Michigan †University of Washington Prior functional imaging studies suggest involvement of limbic cortex, including the hippocampal formation, during panic attacks. Because of the striking therapeutic effect of benzodiazepine agonists on symptoms of panic and anxiety, we hypothesized a potential role of altered GABA A receptor/benzodiazepine binding sites in the limbic system of panic disorder patients. In parallel with our data collection, 3 other research groups reported in vivo findings of benzodiazepine binding sites in panic and anxiety disorder patients. One group reported a generalized decrease in binding sites throughout the forebrain with additional focal reductions in the frontal lobe and insular cortices. The 2 other groups of investigators reported no significant abnormalities. In the present analysis we report findings from 11 patients with panic disorder (ages 22 - 45 years) and 22 age-similar (ages 19 - 44) normal controls. Panic disorder patients satisfied DSM-IVR criteria for panic disorder. Six had panic with agoraphobia and 4 had additional history of major depressive disorder. No subjects were taking medications that interact with the benzodiazepine binding site. After obtaining informed consent, subjects underwent benzodiazepine binding site imaging with the antagonist radioligand [ 11C]flumazenil (FMZ) and PET. Subjects were positioned supine in the gantry of a CTI-Siemens Exact 921 tomograph and an intravenous bolus/infusion injection of FMZ was administered simultaneously with the beginning of a 60-minute dynamic emission brain scan sequence. The tracer infusion protocol was calculated to result in constant arterial FMZ levels after approximately 15 min, and resulted in stable brain tracer levels after 30 min. FMZ binding was quantified by calculation of the brain distribution volume (DV) relative to metabolite-corrected arterial plasma between 30 and 60 minutes after initiation of tracer injection. Specific FMZ DV images were derived by expression of DV relative to the pons, a region where the majority of FMZ DV is non-saturable, representing free and non-specifically bound tracer distributions. There was no significant group difference in the FMZ DV of the pons relative to arterial plasma (Control: 1.09⫾0.12; Panic: 1.10⫾0.24; p⫽0.75). Voxel-wise specific FMZ DV images were co-registered with FMZ transport images obtained during the initial 5 minutes of scanning, and then were spatially transformed to a stereotaxic atlas space configuation. Mean images were formed for both subject groups and were compared on a voxel-wise basis for areas of difference. There was no significant global neocortical difference in FMZ DV, either expressed relative to arterial plasma (Control: 5.39⫾0.90, mean⫾SD; Panic: 5.17⫾0.90; p⫽0.52) or specifif DV relative to the pons (Control: 4.81⫾0.48; Panic: 4.76⫾0.60; p⫽0.78). However, there were significant peaks of reduced FMZ DV in the dorsal insular cortices bilaterally. In the left insula, specific FMZ DV measured 4.40⫾0.57 in controls and 3.44⫾0.83 in panic disorder (p⫽0.0033 uncorrected). In the right insula, specific FMZ DV was 3.81⫾0.66 in controls and 2.95⫾0.52 in panic subjects (p⫽0.0004). There were no other significant FMZ DV abnormalities in limbic corticies. Panic disorder subjects had mean binding values within 7% or closer to control mean FMZ DV elsewhere in the brain. Inspection of the FMZ transport parameter images did not reval a significant reduction in the insula of panic disorder subjects, and did not confirm the prior report of reduced right hippocampal cerebral blood flow. Our findings confirm the prior observation of a focal reduction of GABAA/benzodiazepine binding sites in the insular cortices of panic disorder patients. However, we fail to reproduce the main finding of the prior report indicating a global reduction of FMZ binding in panic disorder. Our present analysis also indicates the need for caution in voxel-based analyses of ligand binding. We initially explored these data with an algorithm that identifies cerebral cortical binding on hemispheric brain surfaces only; the insular cortex is not represented in this system, and accordingly, was not analyzed. The present findings suggest that panic attacks may arise from diminished inhibition in the insular cortex, a brain region associated with the highest representation of autonomic regulation. Our findings do not, however, specify the nature of the insular abnormality. There may be a generalized change in the insular cortical neurons and neuropil, or alternatively, there may be a specific effect on inhibtory synapses. There is further, no specification of the relationship between the insular findings and the evolution of panic dosorder. It remains open to speculation whether the insular reduction is the result of pathological stress and anxiety, or alternatively, whether the insular reduction is the neural substrate leading to panic attacks.

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Different reduction of cortical acetylcholine esterase and cortical glucose in Alzheimer’s disease measured by positron emission tomography S. Weisenbach*, K. Herholz†, G. Zu¨ ndorf†, Olaf Lenz†, R. Hilker†, B. Bauer*, W.-D. Heiss*† *Max-Planck-Institute for Neurological Research Cologne, Germany †Department of Neurology, Medical University of Cologne, Germany Introduction Significant cerebral cholinergic deficit is among the most consistent findings in Alzheimer’s disease (AD). Previous positron emission tomography (PET) studies demonstrated a general decrease of cortical acetylcholine esterase (AChE) activity (Kuhl et al., 1999; Herholz et al., 2000; Shinotoh et al., 2000). With parametric imaging, we compared the distribution of AChE reduction with the severity and distribution of glucose metabolism impairment, which is an indicator of local neuronal function. Methods AChE activity was measured in a group of healthy control subjects (5 female, 7 male, age 65.8 ⫾ 8.5, MMSE 28 or higher) and a group of patients with probable AD according to the NINCDS-ADRDA criteria (8 female, 2 male, age 67.3 ⫾ 7.2, MMSE 21 ⫾ 3.5, CDR 1.5 ⫾ 0.8, disease duration 4.5 ⫾ 5). Patients and controls did not use AChE inhibiting drugs. We used dynamic positron emission tomography (PET) on an ECAT EXACT scanner with i.v. injection of C-11 – labelled N – methyl – 4 – piperidinyl – acetate (MP4A). All images were aligned to each other and normalized spatially in stereotactic space. We calculated local k3 values by dynamic curve analysis with tracer accumulation in the putamen as a reference using recently developed and published methods (Herholz et al., 2001; Zu¨ ndorf et al., 2002). Cerebral glucose metabolism was measured with PET and F-18 – labelled deoxyglucose (FDG) in the same group of patients and another group of control subjects (6 female, 6 male, age 61.2 ⫾ 3.9, MMSE 28 or higher). Images were transferred into stereotactic space for generation of parametric (k3) images and statistical analysis. For both tracers, MP4A and FDG, patients were compared to controls using SPM99. The t-maps resulting from these comparisons were then used to compare extent and location of alterations of glucose metabolism with those of AChE activity. Results Significant impairment of glucose metabolism was present in the typically affected areas (posterior cingulate, temporo-parietal and frontal association cortex). Impairment of AChE activity was more widespread and was most severe in temporobasal cortex. In contrast to the severe impairment of glucose metabolism in the posterior cingulate cortex, little impairment of AChE activity was found there, whereas AChE activity was more severely impaired than glucose metabolism in temporal and parietal regions. Conclusion Our findings demonstrate that the regional distribution of cholinergic impairment does not coincide with that of glucose metabolism. Findings are consistent with the hypothesis that a severe cholinergic deficit is present mainly in mesial and basal temporal lobe structures already in mild dementia that may be responsible for severe memory disturbance. Disturbance of glucose metabolism in posterior cingulate cortex may either be a remote effect or a pathophysologically independent process. Reference List 1. Herholz K., Bauer B., Wienhard K., Kracht L., Mielke R., Lenz O., Strotmann T. and Heiss W.-D. 2000. Measurements of Regional Acetylcholine Esterase Activity with C-11-MP4A in Degenerative Dementia: Comparison with Blood Flow and Glucose Metabolism. Neuroimage 11 (Suppl.):S49 2. Herholz K., Lercher M., Wienhard K., Bauer B., Lenz O. and Heiss W.-D. 2001. PET measurement of cerebral acetylcholine esterase activity without blood sampling. Eur.J.Nucl.Med. 28:472-477 3. Kuhl D. E., Koeppe R. A., Minoshima S., Snyder S. E., Ficaro E. P., Foster N. L., Frey K. A. and Kilbourn M. R. 1999. In vivo mapping of cerebral acetylcholinesterase activity in aging and Alzheimer’s disease. Neurology 52:691-699 4. Shinotoh H., Namba H., Fukushi K., Nagatsuka S., Tanaka N., Aotsuka A., Ota T., Tanada S. and Irie T. 2000. Progressive loss of cortical acetylcholinesterase activity in association with cognitive decline in Alzheimer’s disease: a positron emission tomography study. Ann.Neurol. 48:194-200 5. Zu¨ ndorf G., Herholz K., Lercher M., Wienhard K., Bauer B., Weisenbach S. and Heiss W.-D. 2002 PET functional parametric images of acetylcholine esterase activity without blood sampling. In Brain imaging using PET (Senda, M., Kimura, Y., and Herscovitch, P. Eds.). Academic Press, San Diego, Ca.

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Attenuation of preferential occupancy of 5-HT1A autoreceptors by pindolol in depressed patients: effect of SSRIs or an endophenotype of the depressed state? Eugenii Rabiner*†, Zubin Bhagwagar‡, Roger Gunn§, Christopher Bench ¶, Philip Cowen‡, Paul Grasby† ¶ *Experimental Medicine Division, GlaxoSmithKline, Cambridge †MRC Cyclotron Unit, Hammersmith Hospital, London ‡Psychopharmacology Research Unit, University of Oxford, Oxford §McConnel Brain Imaging Centre, Montreal Neurological Institute, Montreal ¶

Division of Neuroscience and Psychological Medicine, Imperial College, London

Introduction The mixed ␤-adrenergic/5-HT 1A partial agonist pindolol binds preferentially to 5-HT 1A autoreceptors in the midbrain raphe nuclei (RN), compared to post-synaptic 5-HT 1A receptors. This finding has been demonstrated in healthy humans in vivo, human brain tissue in vitro 1,2, and rat in vivo 3. The reason for this preferential binding of pindolol to 5-HT 1A autoreceptors is unclear, however differences in the proportion of G-protein coupled receptors between the RN and other brain regions may be involved. In this study we investigated the binding of pindolol to 5-HT 1A receptors in a group of depressed patients treated with selective serotonin re-uptake inhibitors (SSRIs). Methods Seven healthy volunteers and 7 depressed patients on SSRIs treatment received 2 [ 11C]WAY-100635 PET scans in the ECAT 953B PET camera. The first PET scan was baseline (on SSRIs only for the depressed patients), while the second PET scan was conducted 2 hours following a single oral dose of 10mg of pindolol. The PET data were analysed using a simplified reference tissue model and binding potential values (BP⫽f2Bavail/Kd) were generated for autoreceptor and post-synaptic receptor regions. 5-HT 1A receptor occupancy for each region was estimated as Occupancy⫽(BPbaseline-BPpindolol)/BPbaseline. Preferential occupancy was calculated as the difference between autoreceptor and post-synaptic receptor occupancy. Results The preferential occupancy of patients with MDD (3⫾10%) was significantly lower than that of healthy volunteers (21⫾10%, t2,12⫽3.25, p⫽0.007). There was no difference in post-synaptic occupancy (14⫾10% versus 18⫾12%, t2,12⫽-0.71, p⫽0.489), but the depressed patients had a noticeable, though non-significant decrease in autoreceptor occupancy (35⫾16% versus 21⫾13%, t2,12⫽1.83, p⫽0.092). There appeared to be a negative correlation between the degree of preferential occupancy and the Hamilton Depression rating scale (HAM-D) in depressed patients (r⫽-0.61, p⫽0.197). This correlation is reinforced by an inclusion of 8 depressed patients on SSRIs, studied previously 4, who received sustained dose pindolol (7.5mg or 15mg daily for 1-2 weeks, r⫽0.702, p⫽0.005). Conclusion Our studies indicate that preferential occupancy is attenuated in depressed patients on SSRIs. The mechanism of this attenuation may be an effect of antidepressant treatment, or an effect of depression. The putative negative correlation of the degree of preferential occupancy with the severity of depression (as measured by the HAM-D) indicates that it may be an endophenotype of the depressed state. 1. Rabiner et al, Neuropsychopharmacol 23: 285-293 (2000) 2. Martinez et al, Neuropsychopharmacol 24: 209-229 (2001) 3. Hirani et al, Synapse 36: 330-341 (1999) 4. Rabiner et al, Am J Psychiatry 158: 2080-2082 (2001)

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A Variational Approach for Noise Removal of Parametric Images in Tracer Kinetic Modelling Wei-Hsun Liao*, Leilani Kashida‡, Luminita Vese†, Marvin Bergsneider§, Sung-Cheng Huang* *Department of Biomathematics, UCLA School of Medicine †Department of Mathematics, UCLA ‡Department of Computer Science, UCLA §Brain Injury Research Center, UCLA School of Medicine Introduction Variational approach in image processing is a rapidly evolving technique in the past decade. In this study we extend the noise removal technique proposed by Aubert and Vese [1]. The technique is closely related to the Total Variation noise removal proposed in [2]. We generalize the method by replacing the fidelity term with non-linear model fitting terms for generating parametric images from dynamic PET studies. Our results show that the method yeilds low-noise images in the parameter space while preserving sharp edges and the estimators are shown to be unbiased. Methods Let u(i) be the observed data at time i, and f(i) be the predicted value at the same time point given n model parameters (␪(1), ␪(2),. . .,␪(n)). We propose the following variational problem:

As in [1], the method tries to reconstruct the image by penalizing large gradients in the reconstructed image, which is now the parameter space. The ␣’s are the weights for the penalizing terms. A proper G function could smooth the image without blurring across edges. For faster convergence, initial values are given by pixelby-pixel model fitting. In discretizing the equation, we only use values at the current time step for the model fitting part and the nonlinear part of the divergence operator, whereas in the linear part of the divergence operator we use values at the next time step. The system is then solved by an iterative method. For more details please refer to [1][3]. Results We use a two-parameter (␪(1)⫽a and ␪(1)⫽b) one-compartment model to elucidate our method, in which the output f(i) can be written in the form:

Here * is the convolution operator. A 16 by 16 square is divided into 4 subsquares where 4 sets of parameters ((a1,b1), (a2,b2), (a3,b3) and

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(a4,b4)) are used (Table 1). Pixels belonging to the same subsquare share the same set of parameters. Figure 1 shows the simulated dynamic data. 15 time points (t⫽0.5, 1, 2, 3, 4, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23) are used for simulation and at each time point a Gaussian random noise with variance proportional to the mean is added to each pixel. The input curve is linear from t⫽0 to t⫽0.5, where the input is 0.0 and 0.4 respectively, and after t⫽0.5 the input is kept constant at 0.04. The input is chosen such that it has unit area and resembles the inputs in tracer kinetic studies. The PDE that governs the minimization of the functional could be written in the form:

The weights for the regularizing terms are determined by experiments. The smallest value that could visually suppress the noise is preferred. It is shown in Table 2 that we did not introduce large bias by applying our spatial smoothing (bias⬃2%) while significantly increase the SNR** of parameter images. In Table 3 we compare the standard deviation in each subsquare and it is shown that the SD is substantially decreased in all 4 subsquares. Figure 2 and Figure 3 show the parameter images in 2D and 3D Table 1 A1

A2

A3

A4

B1

B2

B3

B4

(1)

(2)

0.2

0.3

0.45

0.6

0.2

0.33

0.46

0.6

0.008

0.015

before and after applying our technique. One can see the reconstructed images preserve the edge pretty well while smoothing within each subsquare. Discussion In this study we propose a PDE formulation of parameter estimation on an image basis. The technique could be used as long as the model could

Table 2

Before After

BIAS A

BIAS B

SNR A

SNR B

⬃1% ⬃2%

⬃1% ⬃2%

⬃13 ⬃20

⬃11 ⬃19

Table 3

Before After

SD A1

SD B1

SD A2

SD B2

SD A3

SD B3

SD A4

SD B4

0.0167 0.0064

0.0212 0.0098

0.0250 0.0136

0.0330 0.0193

0.0362 0.0131

0.0424 0.0136

0.0460 0.0209

0.0495 0.0163

be formulated as a variational problem. The procedure we propose for solving the PDE is fast and stable and little modification is needed for other tracer models. ** The SNR of a reconstruced image is given by 10log[(variance of the reconstruced image)/(variance of the (reconstructed minus noise-free) image)] References [1] G. Aubert and L. Vese, A variational method in image recovery, SIAM J. NUMER. ANAL. vol. 34, No. 5, pp. 1948-1979 [2] L. Rudin, S. Osher, and E. Fatemi, Nonlinear total variation based noise removal algorithms, Phys. D, 60 (1992), pp. 259-268 [3] T. Chan and L. Vese, Active contours without edges, IEEE Transactions on Image Processing, vol. 10, (No. 2) (2000), pp. 266-77

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Strategies to Reduce the Bias in Estimating Reversible Neuroreceptor Parameters by Linear Regression Analysis Masanori Ichise, Richard E. Carson, Hiroshi Toyama, Robert B. Innis Molecular Imaging Branch and PET Department NIH Bldg 1 B3-10 One Center Drive, Bethesda MD 20892 USA Introduction In the presence of statistical noise in the PET data, the graphical analysis (GA) of Logan et al. (1) is known to underestimate the distribution volume (V) of reversible neuroreceptor binding (2, 3). The purpose of the present study was to evaluate alternative linear regression methods that are potentially less affected by bias and applicable to reversible radiotracers with three-compartment kinetics. Methods In GA, a noise-associated term, C ROI, in the denominator of the independent variable causes a biased least squares estimation. Therefore, one approach was total least squares (TLS) estimation that allows noise in the independent variable (4). Another approach was to rearrange the GA equation in multilinear form (MA1) to remove C ROI from the denominator of the independent variable. Finally, because both GA and MA1 are linear only beyond a time t*, another multilinear equation (MA2) without C ROI in the denominator of the independent variable was derived from a three-compartment model, which is linear from time zero and provides more time points for analysis.

where C ROI (T) ⫽ regional time radioactivity concentration, V ⫽ total distribution volume [K 1/k 2(1⫹k 3/k 4)], C P(T) ⫽ plasma parent tracer concentration, V P ⫽ plasma volume in brain region, b ⫽ constant; and K 1, k 2, k 3, and k 4 ⫽ three-compartment kinetic parameters. In addition to V, MA2 allows calculation of specific volume of distribution, V 3 ⫽ K 1k 3/k 2k 4, from the partial regression coefficients. These methods and unconstrained, nonlinear three-compartment kinetic analysis (KA) were tested for bias by data simulations of 5-HT 1A tracer, [ 18F]FCWAY (5, 6), and 5-HT 2A tracer [ 11C]MDL 100,907 (7); and then compared with examples from actual PET data of these tracers. Results and Discussion For simulations, the bias due to random noise in V (mean percent deviation of 1,000 runs from the true value) was smallest for MA1 followed by KA, MA2, TLS and GA, whereas the variability of bias (SD of 1,000 runs) was smallest with GA followed by TLS, MA1, KA and MA2 (Table). The noise bias and variability in V3 by MA2 showed a similar pattern to that for V by MA2. V values [mean ⫾ % BIAS (%SD) [noise]

GA

TLS

MA1

MA2

KA

FCWAY [15%] MDL [9%]

⫺14 (7) ⫺17 (7)

⫺8.0 (8) ⫺14 (8)

⫹0.6 (10) ⫹0.9 (13)

⫹7.6 (12) ⫹3.1 (19)

⫹2.6 (11) ⫹1.5 (11)

SD (mL/mL), n ⫽ 5] for actual PET studies for GA, TLS, MA1, MA2 and KA were 3.4 ⫾ 1.5, 3.6 ⫾ 1.5, 4.2 ⫾ 1.5, 4.5 ⫾ 1.6 and 4.4 ⫾ 1.7 for FCWAY(raphae, noise ⱁ 8%); and 21.2 ⫾ 6.0, 21.3 ⫾ 6.0, 21.2 ⫾ 6.1, 25.2 ⫾ 6.3 and 25.7 ⫾ 6.2 for MDL(thalamus, noise ⱁ 2 %), respectively. Thus, the bias for FCWAY was consistent with the simulation results. However, for MDL despite very low noise, GA, TLS and MA1 equally gave lower V values than did MA2 or KA. Further simulations showed that this was due to the real t* being late or beyond the MDL PET duration of 120 min. V3 values estimated by MA2 for actual MDL PET studies [mean ⫾ SD (mL/mL), two regions, basal ganglia and thalamus, n ⫽ 10] were 17.1 ⫾ 7.5 and correlated well with those by KA (17.7 ⫾ 7.9, r2 ⫽ 0.98), although physiological interpretation of V3 obtained by unconstrained fitting is unclear. TLS, MA1 and MA2 thus all appear to reduce the noise bias compared to GA. For tracers with early t*, MA1 appears the method of choice. However, for slower tracers, MA2 may provide the best balance between the bias and the computational ease suited for pixel-wise parameter estimation. References 1. Logan J, Fowler JS, Volkow ND, et al. J Cereb Blood Flow Metab 10:740-747 (1990). 2. Carson RE. Quantification of brain function. Elsevier Science Publishers pp 499-507 (1993). 3. Slifstein M, Laruelle M. J Nucl Med 41:2083-2088 (2000). 4. Van Huffel S, Vandewalle J. The total least squares problem. Society for Industrial Mathematics pp 1-300 (1991). 5. Carson RE, Spanaki Y, Ma MG, et al. Neuroimage 11:S45 (2000). 6. Carson RE, Lang L, Watabe H, et al. Nucl Med Biol 27:493-497 (2000). 7. Watabe H, Channing MA, Der MG, et al. J Cereb Blood Flow Metab 20:899-909 (2000).

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Reference Region Hyperplots of Brain Uptake of FDOPA and Raclopride Albert Gjedde, Dean F. Wong, Pedro Rosa Neto, Paul Cumming Center of Functionally Integrative Neuroscience, University of Aarhus, Aarhus, Denmark The multiple-time graphical analysis methods provide precise and reasonably accurate estimates of transient kinetic processes of brain uptake and metabolism. One great advantage of the methods is their ability to directly reveal key features of kinetic processing, which can be invisible in more conservative presentations of uptake data. The hyperplot extends the MTGA method by integrating the reference region and region of interest curves prior to the normalization. As in the conventional MTGA method, the new independent variable has unit of time, and the the new dependent variable is the ratio between the integrals of the regions of interest and the integral of the reference region curve. The definition of the independent variable is shown below (Left Equation), in which n is the radioactivity in the reference region as a function of time. The definition of the dependent variable is shown below (Center Equation), in which m is the radioactivity in the region of interest as function of time. The relationship between the dependent and independent variables is shown above (Right Equation), where R AUC is the ratio of the areas under the curves as functions of time, and ⌰ is the independent time-variable defined above. The parameters are R o, the ratio between the initial uptakes in region of interest and reference region, p B, the binding potential index, defined as the ratio between bound or trapped tracer and freely exchangeable tracer, and ␣, the rate of approach to steadystate. The analysis is particularly useful in the case of trapping of FDOPA in human brain. For FDOPA, the binding potential is the ratio between the index of the rate constant of FDOPA decarboxylation, k’ 3, and the rate constant of dopamine breakdown, k7, where k7 equals the rate constant of approach to steady-state, ␣. The relationships between real time and the transformed time variable ⌰ are shown in Figures 1 for FDOPA (Left Panel, 8 normal volunteers and one patient) and raclopride (Right Panel, 15 normal volunteers). The transformed time variable is lower than real time because of the shape of the reference region activity curve. The method was applied to eight normal volunteers and to patients with Parkinson’s Disease, of whom one is shown in Figure 2 (Left Panel). The average hyperplot of raclopride for the 15 normal volunteers is shown in Figure 2 (Center Panel): Figure 2 (Left Panel) shows the hyperplot curves of four regions in the brains of normal volunteers, the left and right putamina and the left and right caudates, as well as the combined putamina of one patient with Parkinson’s Disease (H&Y score 3-4). Figure 2 (Center Panel) shows the average hyperplot of raclopride in 15 normal volunteers. The fits of the equation to the data are shown in the table below:

In the patient, the FDOPA binding potential was reduced to one-quarter to one-third of the binding in normal volunteers, but the index of the fluoro-dopamine removal rate (k 7) estimated as alpha was elevated in the patient. The index of the FDOPA decarboxylation rate is the product of the estimates of ␣ and p B (corresponding to the initial slopes of the curves). It is apparent that the slopes are much less different than the estimates of p B and ␣. For raclopride, this method promises to reveal salient features of the uptake process by direct visual demonstration of the differences between patients and healthy controls. To test the raclopride hyperplot, we reanalyzed data reported in the literature by Schlaepfer et al (1997) for the effect of a street-dose of cocaine on raclopride binding, and by Koepp et al (1998) for the effect of playing a reward-generating videogame of virtual violence. In both cases, the raclopride binding potential declined as reported in the original publications but with greater precision, as shown in Figure 2 (Right Panel) (⫹SEM). The decline was three-fold greater for the videogamers than for the cocaine abusers. References Koepp MJ, Gunn RN, Lawrence AD, Cunningham VJ, Dagher A, Jones T, Brooks DJ, Bench CJ, Grasby PM. Evidence for striatal dopamine release during a video game. Nature. 1998 May 21;393(6682):266-8. Schlaepfer TE, Pearlson GD, Wong DF, Marenco S, Dannals RF. PET study of competition between intravenous cocaine and [11C]raclopride at dopamine receptors in human subjects. Am J Psychiatry. 1997 Sep;154(9):1209-13.

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Impact of Graphical Analysis Bias on Group Comparisons of Regional [carbonyl- 11C]WAY Binding Potential Measures Julie C. Price*, Lei Xu*, Sati Mazumdar*, Carolyn C. Meltzer*, Wayne C. Drevets†, Chester A. Mathis*, David E. Kelley*, Christopher M. Ryan*, Charles F. Reynolds, III* *Departments of Radiology, Biostatistics, Psychiatry, or Medicine, University of Pittsburgh, Pittsburgh, PA †National Institute of Mental Health, Bethesda, MD Introduction Previously, we described a pilot study that measured serotonin-1A (5-HT 1A) receptor binding in humans with type 2 diabetes (1). The results of the study supported our hypothesis of elevated 5-HT 1A receptor binding in type 2 diabetes that was largely based on animal studies that showed hyperglycemia-induced alterations in the serotonin system and structural and functional alterations in hippocampus. The pilot PET study indicated greater [carbonyl- 11C]WAY 100635 Logan graphical binding potential measures in the mesial temporal cortex (including hippocampus) of type 2 subjects, relative to medically healthy controls (p⬍0.05). The graphical analysis also indicated a greater binding potential in the brainstem raphe region but these results were weakened by low linear correlations and concerns of measurement bias in this small and noisy region (2). In the present work, the [carbonyl11 C]WAY 100635 pilot data set was re-analyzed using a bias-reduction method described by Logan (3) that applies generalized linear least squares smoothing (4) prior to graphical analysis. The goal of this work was to examine the impact of the graphical measurement bias on group comparisons of regional [carbonyl- 11C]WAY 100635 binding potential measures. Methods Medically healthy controls (5M, 1F: 54⫾17 years) and subjects with type 2 diabetes (NIDDM, non-insulin-dependent diabetes mellitus) (5M, 1F: 55⫾13 years) were studied. High specific activity [carbonyl- 11C]WAY 100635 PET data were acquired (ECAT HR⫹, ⬎1200 Ci/mmole, 13.6⫾3.0 mCi, metabolite-corrected arterial plasma) (1). Data were analyzed and compared over 60 min. Specific receptor binding was assessed for several regions-of-interest (ROIs): mesial temporal cortex (MTC, including hippocampus: HIP), brainstem raphe (RAP), lateral orbitofrontal (LOF) and occipital (OCC) cortices. The cerebellum (CER) was used as reference region. Generalized linear least squares (GLLS) smoothing (1-tissue compartment) was applied after segmentation of the PET time-activity curve into two (2-segment) or three (3-segment) parts. The 2-segment smoothing was applied to: 1) 0 to 18 min; 2) 8 min - end time (3). The 3-segment curve was comprised of an initial unsmoothed portion (0 min to time of plateau) and two overlapping smoothed segments: plateau to 18 min and 10 to 60 min. Overlapping data were averaged to generate a single curve. The smoothed [carbonyl- 11C]WAY 100635 data were analyzed using the Logan graphical approach to estimate distribution volume (DV), using 14-60 min of data. The binding potential (BP) measure was calculated ([DV ROI ⫼ DV CER] – 1) and used to assess 5-HT 1A receptor binding. Results The assessment of group differences in the regional GLLS-Logan BP measures provided results that were consistent with the original hypothesis of greater 5-HT 1A receptor binding for the type 2 subjects, as greater BP measures were indicated in MTC and HIP (p⬍0.05), although no group difference was indicated for the RAP BP. No group difference was evident in the CER DV, with or without GLLS smoothing. The 2-segment and 3-segment GLLS methods provided similar results with average absolute differences of ⬍4% for all data, except the control RAP BP (7%). Across the subject groups, the average absolute difference between the 2-segment GLLS Logan BP and conventional Logan BP ranged from 3-12% for MTC and HIP and 4-7% for LOF and OCC. The graphical bias had greatest impact on the RAP BP for which the average absolute difference in BP was more than 50% for controls and about 20% for diabetics (GLLS smoothing relative to no smooth). Conclusions The GLLS smoothing had greatest impact on the small and noisy RAP region. This comparison study showed that differences with marginal p-values may not retain statistical significance after accounting for bias in the graphical method or if group differences in the noise-related bias exist. However, strong differences that are evident, prior to smoothing, can prove to be robust even for data in small areas that may be susceptible to noise. by the Whitaker Foundation and NIH grants K01 MH01976, MH01410, MH59768, P30 MH52247, and NARSAD. 1. Price JC et al., J Nucl Med, 42(5):P18 (2001): Price JC et al., Brain Research, in press 2. Parsey RV et al., J Cereb Blood Flow Metab, 20:1111-1113 (2000) 3. Logan J et al., J Cereb Blood Flow Metab 21: 307-320 (2001) 4. Feng D et al., IEEE Trans Med Imag, 12:182-188 (1993)

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Likelihood Approach to Parameter Estimation in Logan Graphical Analysis R. Todd Ogden*†‡, Ramin V. Parsey*†, J. John Mann*† *Department of Neuroscience, New York State Psychiatric Institute †Department of Psychiatry, Columbia University ‡Department of Biostatistics, Columbia University Logan et al. (1990) offer an alternative to using kinetic modeling methods for quantifying neuroreceptor binding. This is based on estimating parameters in a linear relationship between two variables which result from transforming measured quantities. In particular, if C a(t) represents the actual concentration of the ligand in the plasma at time t and ROI(t) is the actual ligand concentration within a given brain region of interest at time t, then the relationship is given by for all values of t greater than some specified time point. The total volume of distribution V T for the region of interest under study is equal to font face⫽“symbol”b, the slope of the linear relationship. Thus, the inference problem consists of estimating font face⫽“symbol”b and font face⫽“symbol”g in (1) using only noisy measurements (taken from the PET camera) for the concentration in the ROI. (The plasma concentration function is typically fit by some parametric model, such as the sum of three exponentials, and fitted values are used in place of C a(t). This convention is followed here as well.) Because this general approach to estimating V T is model-free (in the sense that no choices have to be made between the various competing compartmental model candidates), the “graphical analysis” method has become popular in PET ligand studies. The usual approach for estimating the slope of the line with the graphical method involves applying ordinary least squares (OLS) regression techniques to the data. The primary advantages of this approach are that it is easy to implement, ubiquitous in statistical software, and that it runs without iterating. A drawback with this approach, as shown by Slifstein and Laruelle (2000), is that when using standard methods for fitting a line to scatterplot data, there is a substantial negative bias when estimating font face⫽“symbol”b, especially in regions with relatively high noise levels. This bias is a result of the assumptions of the simple linear regression model (namely, independent additive error only on the “Y” variable) not being satisfied in this application. With the C a(t) function considered as fixed, the error is additive in the ROI(t) component, which appears in the denominator of both the “X” and the “Y” terms in the linear relationship given in (1). To avoid the bias problem, the estimation needs to be approached from another angle. Let 0⫽t 0 ⬍ t 1 ⬍ . . . ⬍ t n be the time points of the PET images and let Z i * be the “true” concentration in the region under study at time t i, i.e., Z i * ⫽ ROI(t i). Also, let x i represent the integral of C a(t) up to time t i. Replacing the integral of ROI(t) by a trapezoidal approximation, the “true” relationship (1) can be rearranged to give a recursive relationship for the Z i * values in terms of parameters and x i values: where t k is the point at which linearity is achieved. The alternative approach to parameter estimation is to write the ROI data as Z i ⫽ Z i * ⫹ font face⫽“symbol”e i for i ⫽ k, k⫹1, . . . n. If the font face⫽“symbol”e i’s are thought to be independent Gaussian random variables with equal variances, then the maximum likelihood estimators of font face⫽“symbol”b and font face⫽“symbol”g are the minimizers of font face⫽“symbol”S (Z i - Z i *) 2. (This sum of squares may be replaced by a weighted version to account for heteroscedastic errors.) No general analytic solution can be written for the estimators but they may be computed using standard nonlinear optimization techniques. Parameter estimates obtained through the likelihood approach are known to be asymptotically unbiased, but sample sizes in these studies tend to be too small for the asymptotic results to apply. Through simulation, it is shown that for sample sizes as small as 20, for a variety of noise levels, the likelihood method is nearly unbiased. The likelihood approach is shown to improve on the mean squared error performance of the OLS estimation as well. This new estimation technique provides a model-free approach to estimation in neuroreceptor binding studies that is unbiased even for rather noisy regions. As it is not too computationally intensive, it may prove useful in estimation at the individual voxel level. References Logan, J., et al. (1990). Graphical analysis of reversible radioligand binding from time-activity measurements applied to N- 11C-methyl-(-)-cocaine PET studies in human studies. Journal of Cerebral Blood Flow Metabolism 10: 740-747 Slifstein, M. and Laruelle, M. (2000). Effects of Statistical Noise on Graphic Analysis of PET Neuroreceptor Studies. Journal of Nuclear Medicine 41: 2083-2088.

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Partial Volume Correction: A Recipe John Aston*†, Alexander Hammers*, Vincent Cunningham*‡, Roger Gunn† *Imperial College, London, UK †McGill University, Montreal, Canada ‡IRSL, London, UK Partial Volume Correction (PVC) is now a routine part of many analyses especially when there is the possibility of underlying physiological differences within the subjects under study [1]. It is well known that partial volume effects can cause large changes in quantified results [2]. Partial volume is now a well understood problem and there are many different types of analysis available for its solution. Here, a new automated procedure will be proposed, in order for PVC to be applied objectively and quickly. The procedure is modular in form, and whilst an algorithm for each module has been proposed, any suitable algorithm for that module could be used. The principles behind each module, however, will allow a full PVC analysis with little or no supplementary interaction from the user after the data has been gathered. The Method & Algorithms The algorithm is based on the assumption that the following initial data is available for each scan in the study; A PET image (dynamic or static); An adjunct Magnetic Resonance structural image; A regionally defined template. In the case of normal subjects, a regionally defined average template [3] can be used, reducing further the need for interaction with the process, whilst in the case of subjects with abnormal brains, individual templates may be required. The method then proceeds as follows: 1. Tissue Classification. The MRI is automatically segmented [4] to define separate regional classified images for GM, WM and CSF. 2. Regional Alignment. If an average regional template is used, then alignment with the subjects own MRI scan is necessary. Routines in SPM [5] can be combined successfully into an automated process to normalise the regional template to each individual scan (assuming the regional template has an associated average MRI). 3. Co-registration of the regional template, tissue classified and MRI images can be carried out using SPM through the mutual information routines [6] which allow accurate image registration. 4. PVC can then be undertaken. New routines allow PVC with different noise model specifications [7,8], and can include measured point spread functions if available. These routines also enable the information to be given in the format of region definition and classification images, thus reducing the need for specification of very large regionally classified data sets. These methods allow for rapid and reproducable estimates of PVC. This can be carried out in as little as 1 hour for a 128x128x31 static image (25 minutes for tissue classification, 10 mins for realignment and co-registration and 25 mins for Partial Volume Correction). The Assumptions The method is based upon the assumption of accuracy at each step. A failure of the routines to converge at any step will result in poor or indeed incomputable estimates of partial volume. There is also an implicit assumption in the algorithm that the probability distributions of tissue classifications are not indentical within any one region as this would lead to incomputable estimates. In practice though, this never occurs, as the tissue classifications are probablistic in nature and as such are a continuum of values between [0,1]. Conclusions The methods presented enable PVC to be routinely carried out as part of any study with the minimum of expertise required by the end user. The individual algorithms can be replaced if so desired but the overall method allows for rapid calculation of partial volume effects, facilitating the use of PVC as part of common practice. References [1] [2] [3] [4] [5] [6] [7] [8]

Labbe´ , C. et al, Alz Dis Assoc Dis, 10: 141-70 (1996) Rousset, O. et al, Synapse, 37: 81-9 (2000) Hammers, A. et al, Hum Brain Mapp, in press Lemieux, L. et al, Magn Reson Med, 42: 127-35 (1999) Ashburner, J. et al, Hum Brain Mapp, 7: 254-66 (1999) Studholme, C. et al, Med Phys, 24: 25-35 (1997) Aston, J. et al, Neuroimage 11:6 (3/3) S15 (2000) Aston, J. et al, JCBFM, submitted

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Image Space Variance from Wavelet Transforms John Aston*†, Federico Turkheimer‡, Vincent Cunningham*‡, Roger Gunn† *Imperial College, London, UK †McGill University, Montreal, Canada ‡IRSL, London UK Wavelets are now increasingly used in data analysis of PET studies [1,2,3]. Dynamic PET images are spatially transformed into the wavelet domain, where linear tracer kinetic models are used to derive kinetic parameters such as volume of distribution. These parameters are thresholded in the wavelet domain and then transformed back into image space. These methods utilise the entire four-dimensional data set, making use of temporal estimates of error to allow thresholding of the wavelet components in the spatial wavelet domain. However current methods only provide estimates of variance of the wavelet coefficients prior to thresholding and performing the inverse transform. Here a method for the estimation of the resulting error estimates of the parameters in image space is developed through the use of quasi-tensor algorithms. Theory Wavelet transforms and hence inverse transforms are linear transforms when regarded in one dimension. However, algorithms for multidimensional wavelet transforms make use of the multidimensional structure of the wavelet transform, in that it can be performed firstly in one dimension, then the second and then the third. However, this multidimensional structure is not linearly multidimensional (i.e. cannot be expressed as linear products in each dimension) due to the decimation step which is not the product of linear decimation steps. The wavelet transform, as opposed to the inverse transform, can be modified to be seen as a series of linear multidimensional transforms, one for each of the k levels of decimation. However the inverse transform cannot be written in a linear multidimensional way, as the inverse transforms of the linear multidimensional wavelet transforms are not well defined (some of the matrices of the multidimensional wavelet transform are singular). This is not a problem for calculation of the inverse transform, but variance components are related by the point-wise square transforms which require multidimensional linearity for rapid calculation. Therefore a quasi-tensor (quasi multidimensional linear) algorithm has been developed to allow point-wise estimation of variance components. It is not as rapid as the original wavelet transforms, but can be calculated as opposed to the full inverse transform in one dimension, which is prohibitively large. The k level inverse transform for pixel (ijm) is given by the following: n

(k) IW ijm ⫽ ( nIW i(1) o nIW j(1) o nIW m(1)) o

n⶿2

IW (1) o . . . o

n/(2 (k-1)

)IW (1)

where n is a vector of the number of pixels in each dimension and ‘o’ a convolution or tensor operation. The first term in the expansion is the only non multidimensional linear term, and can easily be reshaped to allow operation by the tensor algorithms comprising the rest of the terms. The variance transform of the wavelet variances to image variances is then given by (k) (k) V I(ijm) ⫽ ( nIW ijm V W) T ( nIW ijm V W)

if the wavelet variances are assumed to be independent, which is likely, based on the decorrelational properties of the wavelet transform. Simulations Simulations have been performed in two dimensions and shown to yield identical answers to the full one dimensional solution for the two dimensional case. It is not possible to verify this for the three dimensional case, as the one dimensional solution for the three dimensional case is too large to calculate, although the theory is independent of the number of dimensions. Conclusions The methods described above yield an image of the errors on the estimates obtained using wavelet modelling. This allows any analysis using error estimates that has previously been applied in image space to be converted into wavelet space, assuming a linear temporal model. References [1] Turkheimer, FE et al, JCBFM 19: 1189-208 (1999) [2] Turkheimer, FE et al, JCBFM 20: 879-93 (2000) [3] Turkheimer, FE et al, JCBFM 20: 1610-8 (2000)

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Evaluation of the Reliability in PET Kinetic Analysis for Dual Tracer Injection Yoko Ikoma*, Hinako Toyama†, Tetsuya Suhara†, Akihiko Uchiyama* *Waseda University †National Institute of Radiological Sciences INTRODUCTION A PET kinetic analysis of dual tracer injection using the 2-input compartment model is challenging. If the dynamic data of dual tracer can be acquired at the same time, different two functions will be measurable for a short time and in the same condition. In this study, we investigated the reliability of parameter estimates for various injection protocols and evaluated the possibility of kinetic analysis for dual tracer injection by means of a computer simulation. METHOD Decaying tissue time activity curves were simulated with 2-input compartment model (Fig. 1) for various input functions and injection intervals of two tracers. The noise depended on the collected total count was added to each time activity curve. The rate constants were estimated by nonlinear least square method, and the mean of absolute differences (MAD) between the true and estimated value was calculated for one thousand curves. RESULTS In a case of 18F-FDG and 11C-flumazenil (FMZ), parameters were estimated most reliably when FDG was injected 15 minutes later than FMZ injection (Fig. 2). In this injection interval, the reliability of parameter estimates did not depend on the ratio of administration dose of FDG and FMZ. Error of parameter estimates became larger as the noise increased. When the noise level at the last frame was 10%, the MAD of Ki, reflecting the uptake of FDG, was about 20%, that of distribution volume for FMZ was about 13%. CONCLUSION In the simulation study, the rate constants of FDG were able to be estimated simultaneously with that of FMZ by 2-input compartment model, and the possibility of kinetic analysis for dual tracer injection was shown. With this method, the radioactivity distribution images of two tracers are able to create by calculating parameters pixel by pixel.

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[11]C-diprenorphine PET in malformations of cortical development (MCD): voxel-based analysis and region-based partial volume effect correction Alexander Hammers*†, Matthias J. Koepp*†, John A.D. Aston*, Louis Lemieux†, Mark P. Richardson*†, David J. Brooks*, John S. Duncan† *MRC Clinical Sciences Centre and Division of Neuroscience, Faculty of Medicine, Imperial College, Hammersmith Hospital, DuCane Road, London †Department of Clinical and Experimental Epilepsy, Institute of Neurology, UCL, Queen Square, London, and National Society for Epilepsy MRI Unit, Chalfont St Peter Background Up to 15% of adult patients with medically refractory epilepsy referred for epilepsy surgery have malformations of cortical development (MCD). Surgery in these patients has a less favourable outcome than if there is a discrete lesion, probably due to the presence of abnormalities beyond those appreciated on standard MRI. [11C] diprenorphine (DPN) PET images all subtypes of opioid receptors. There is evidence for release of endogenous opioids during or after partial seizures. Subjects and common methods We studied 14 patients with partial epilepsy due to MCD. Four had subcortical forms of MCD: 2 laminar (band)heterotopia (LH), 1 subependymal nodular heterotopia (SNH) and one subcortical heterotopia (SH). Ten had cortical or mixed forms: 3 focal cortical dysplasias (FCD), 2 dysembryoplastic neuroepithelial tumours (DNT), 1 schizencephaly (Schiz), 1 perisylvian polymicrogyria (PMG) and 3 tuberous scleroses (TS). 21 healthy controls were studied for comparison. All had high resolution MRI and quantitative [11C]-DPN PET. We used spectral analysis with an arterial plasma metabolite-corrected input function to produce parametric images of DPN volume-of-distribution (Vd). Voxel-based analysis Statistical Parametric Mapping (SPM99, Wellcome Department of Cognitive Neurology, London) with explicit masking was used for the comparison of individual patients and controls. 13 of the 14 patients showed abnormal DPN-Vd. Within subgroups, findings tended to be similar. In the two LH patients, the bands were detected as an increase in DPN binding, and there were widespread cortical decreases. Similarly, the heterotopia in the patients with SNH and SH were detected as increases compared with the homologous regions in controls, but both had significant decreases of DPN-Vd in the vicinity of the lesion. The patient with Schiz had increases within the thickened cortex in the clefts but decreases in the surrounding areas. Of the three patients with FCD, only one had a decrease within the lesion; there were no increases, one normal study and one patient with remote decreases. Both patients with DNT showed increases in the lesion and contralaterally. The patient with PMG had widespread increases and decreases. The three patients with TS had no increases, and decreases were confined to only some of the multiple cortical tubers. Region-based analysis with partial volume correction (PVC) 13 controls and 10 patients were available for this preliminary analysis. We used a maximum probability map in stereotactic space (Hammers et al., this meeting) to automatically define 49 standard volumes of interest (VOI). The MCD and overlying/adjacent areas were individually defined on the patients’ MRIs, and these areas transferred to all controls to derive normal ranges for the area of MCD for each patient individually (Hammers et al. (2001), Brain 124:1555-1565). MRIs were segmented into grey matter (GM), white matter and CSF (Lemieux (2001), Proc SPIE 4322:159-169) and PVC performed using a new algorithm (Aston et al., this meeting). In the subcortical forms of MCD, the 2 patients with LH had largely normal values; one patient had a decrease of 30% in one region. The patient with SNH had normal DPN-Vd in the lesion and the overlying cortex but widespread remote cortical increases (14 regions, ⫹14 to ⫹93%). The patient with SH showed decreased DPN binding in 1/2 lesions (-42%) and ipsilateral increases in 4 VOIs in the vicinity of the lesion (⫹42 to ⫹116%). In the cortical forms of MCD, of the 3 patients with FCD, 1/3 had decreased DPN-Vd in the lesion (-84%), and another had a VOI with increased binding (⫹123%) in the vicinity. One patient with DNT had decreases in the lesion (-70%), in adjacent VOIs and remotely (-41 to -76%). One patient with PMG showed normal DPN-Vd in the lesion and adjacent VOIs but increases in two nearby regions (⫹39 and ⫹40%). One patient with TS showed a decrease of -40% in one out of three tubers; all other areas were normal. In summary, PVC showed that heterotopic GM in subcortical forms may have decreased DPN binding per unit of GM while cortex in downstream migrational paths may show increases. Findings in the cortical forms were heterogenous but compatible with known pathological features; clinical usefulness is possible in TS with single decreases despite multiple tubers. Discussion DPN-PET analysed with SPM showed abnormalities in 13/14 MCD patients. Subtypes of MCD seem to have characteristic appearances. In some forms, abnormalities are confined to the lesions seen on MRI, but particularly the subcortical forms show more widespread disturbances, in accordance with their lesser suitability for epilepsy surgery. Quantitative region-based analyses with correction for tissue type determines whether there is functional abnormality over and above structural abnormalities and gives complementary information.

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Cluster Analysis In Kinetic Modelling: Better Than Arterial Sampling? Matthew Liptrot, Karen Husted Adams, Claus Svarer, Gitte Moos Knudsen Copenhagen University Teaching Hospital, Rigshospitalet Objectives Quantification of ligand uptake and binding requires knowledge of the input function. Traditionally, this is achieved via arterial blood sampling. However, a non-invasive alternative to arterial cannulation would be preferable. Here we propose the use of a blood vessel time activity curve (TAC), extracted directly from the PET scans via cluster analysis. Methods Five healthy subjects were injected with 18F-Altanserin and subsequently scanned (3D acquisition mode, 35 slices per volume) at 39 time-points over a 2-hour period. Concurrently, blood samples were taken from the radial artery and left cubital vein. A structural MRI scan was also performed for defining regions-of-interest (ROI). A temporal cortex region TAC was extracted from the PET data set. Both one-stage and two-stage (hierarchical) K-means cluster analyses were performed on the PET time-series to extract a venous blood region. Determination of the correct number of clusters was performed by 3D visual inspection of the homogeneity of the determined clusters. For one-stage cluster analysis, 8 clusters were chosen (K⫽8), and for the two-stage analysis 4 and then 3 clusters were used (K⫽4, 3). The 4 different input curves (arterial samples, venous samples, single and hierarchical blood vessel cluster TACs) were then used to calculate the radioligand distribution volume (V D) values for the temporal cortex. Due to its deterministic, non-parametric properties, the Logan plot method was used for data analysis and extraction of V D as this reduced the amount of experimental noise compared with other kinetic modelling methods. For each subject, the ratio of the V D value obtained from each of the clusters and from the venous samples to that obtained from the arterial sample was then calculated, as shown in the table. Results The ratios show that there was a good correlation between the values of V D obtained from the arterial blood samples and those from the two K-means clustered input functions. These results are significantly different from the values obtained using the venous samples. In addition, the clustering method appears a more robust method than that using the venous samples only. Ratio of V DS, Averaged Over All Five Subjects Conclusions This work acts as a proof-of-principle that the use of cluster V (venous sample):V (arterial sample) 1.123 (⫾0.144) D D analysis on a PET dataset could obviate the requirement for V (cluster K ⫽ 8):V (arterial sample) 0.977 (⫾0.096) D D arterial cannulation when determining the input function for V (cluster K ⫽ 4,3):V (arterial sample) 1.026 (⫾0.052) D D kinetic modeling of ligand binding. It has the innate benefit of requiring no (or very little) compensation for the dispersion or delay induced by the transport through the blood vessels as both the input curves to the kinetic model are obtained from the same part of the body and have exactly the same time axis. In addition, it highlights how the use of solely the venous samples for kinetic modelling would require at least some compensation for dispersion and delay in order to obtain meaningful values. Further work needs to be done to evaluate the clustering method using other radioligands.

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Characterizing the in Vivo Binding Parameters of [F-18]Fallypride in Monkeys Using a PET Multiple-Injection Protocol Bradley Christian*†, Jogeshwar Mukherjee‡, TK Narayanan*†, Bing Shi*†, Joseph Mantil*† *Wright State University †Wallace Kettering Neuroscience Institute / Kettering Medical Center ‡University of California - Irvine Objective The goal of this work was to measure the in vivo transport and binding parameters of [ 18 F]fallypride in both the striatal (putamen, caudate, ventral striatum) and extrastriatal (thalamus, amygdala, cerebellum, temporal and frontal cortices)regions of the monkey brain. Knowledge of these parameters will aid in designing experiments for studying the D-2 dopamine receptor system and endogenous dopamine concentration for different disease processes. Methods Multiple-injection PET experimental protocols (Delforge 1995) with co-injections of radiolabeled and blocking doses of fallypride were used to measure the K 1 , k 2 , k on /V R , k off and B⬘max kinetic parameters. The experimental design was chosen using the D-optimal criterion (max{det(Hessian)}) to maximize the precision of the binding parameters. The optimization consisted of choosing the number of injections, time of injections and specific activity (SA) for each dose based on simulations using in vitro estimates of the parameters. Separate experimental protocols were designed targeting brain regions with high (striatum), medium (thalamus) and low (cortical regions) D-2 receptor density. Multiple injection (3 injections) PET studies were conducted on 2 rhesus monkeys using an ECAT EXACT HR⫹ PET scanner based on the optimal designs. Dynamic data was acquired over the 3 hour PET experiment. Arterial blood samples were drawn and assayed for parent compound, lipophilic and hydrophilic metabolites for each of the separate injections to serve as the input functions to the compartmental model. Time-activity curves were obtained by applying small circular regions of interest on the reconstructed dynamic datasets, placed over the putamen, caudate, ventral striatum, thalamus, amygdala, temporal and frontal cortices and cerebellum. The compartmental model was implemented using free (F), bound (B) and nonspecifically bound (NS) compartments for each of the 3 injections with unique specific activities. The 3 injections were linked by the saturable bound compartment which accounts for the reduction in available receptor sites (B max -B inj1 -B inj2 -B inj3 ) from the labeled and unlabeled ligand. COMKAT software (Muzik 2001) was used for the experimental design and estimation of the parameters from the experimental data. Different model configurations were tested for each brain region to examine the effects of nonspecific binding and lipophilic metabolites on the error estimates and covariance between parameters. Results Two studies have been conducted on each of the two monkeys, both optimized for parameter estimation in the high and medium D-2 receptor regions. The dose of fallypride for each of the injections is given in the table below:

Study/D-2 Region

Inj.#1 (1-2 mCi)

Inj.#2 (1-2 mCi)

Inj.#3 (0-1.5 mCi)

M1/medium M1/high M2/medium M2/high

⬍1 nmol ⬍1 nmol ⬍1 nmol ⬍1 nmol

50 nmol 102 nmol 25 nmol 159 nmol

200 nmol 300 nmol 300 nmol 300 nmol

These concentrations correspond to minimal receptor occupancy (inj #1), partial saturation (inj #2) and near saturation (inj #3), similar to that used by Delforge (1999). Various constraints were used in the estimation of the parameters for the different regions. It was found that k off was most accurately identified in the striatal regions, then fixed constant for the extrastriatal regions, with the average for both monkeys of k off ⫽ 0.038 min -1 . For all other brain regions, a total of either four (K 1 , k 2 , k on /V R , B⬘max ) or six (including k 5 ,k 6 ) parameters were allowed to float. The results for the parameter estimates averaged for the 2 monkeys are given in the table below: Parameter ⫺1

K 1 (min ) K 2 (min ⫺1 ) K ON /V R (ml/pmol min) B⬘max (pmol/ml)

Putamen

Caudate

V. striatum

Thalamus

Amygdala

0.18 0.31 0.027 35

0.16 0.37 0.025 35

0.16 0.30 0.025 20

0.14 0.35 0.11 3.2

0.11 0.20 0.081 2.0

It was found that the nonspecific binding terms (k 5 ,k 6 ) were needed only for extrastriatal regions based on the improvement of the fit. In the low D-2 receptor density regions (temporal, frontal and cerebellum) the transport parameters (K 1 ,k 2 ) could be accurately determined, however it was not possible to uniquely identify the binding parameters (k on /V R and B⬘max ) due to saturating fallypride doses from injection #2 in these regions. Further experiments are planned to measure the parameters in the low D-2 density regions. Conclusions The favorable binding characteristics of [ 18 F]fallypride make it well suited for studying the dopamine system in all regions of the brain. Knowledge of the in vivo binding characteristics are needed to understand the kinetics and guide further experimental applications such as measuring endogenous dopamine changes in extrastriatal regions.

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APPLICABILITY OF ACTIVITY RATIO METHODS TO THE ESTIMATION OF NEURORECEPTOR BINDING Rainer Hinz, Federico Tu¨ rkheimer, Vincent Cunningham Imaging Research Solutions Ltd., Hammersmith Hospital, London, UK Introduction Recently, several high affinity PET tracers for the serotonergic and dopaminergic systems have been reported which are characterised by * a very slow clearance of the parent tracer from blood plasma after bolus administration, * tissue impulse response functions that correspond with high total volumes of distribution (VD). Examples are [18F]Altanserin for the serotonin receptor (1), [11C]MDL 100,907 for the 5-HT2A receptor (2), [11C](⫹)McN5652 and [11C]DASB for the serotonin transporter (3,4) and [11C] or [76Br] FLB 457 for extrastriatal D2 receptors (5). Methods Provided that there is a reference region devoid of specific binding and that the free and non-specifically bound tracer pools in the target regions with specific binding are the same as in the reference region, the binding potential (BP) can be calculated from the ratio of the VD in a target region over the VD in the reference region minus one. However, due to the above mentioned properties, there are difficulties in obtaining unbiased local VD estimates with low variance from region of interest data of dynamic PET studies with those tracers. The compartmental analysis methods which use either a metabolite-corrected plasma input function or a reference tissue input function have to estimate the VD or the VD ratio by deconvolving the tissue response function with the input function. Because of the limited duration of a PET scan, both the measured tissue response function and the available input function are quite incomplete, resulting in a big variance of the estimated VD. Model simplifications and reduction of the number of free model parameters lead to biased parameter estimates, e.g. as reported on the graphical analysis of reversible radioligand binding after Logan where high VDs of tracers with slow kinetics are systematically underestimated (6). In this situation, radioactivity ratios can be used to estimate regional VDs and BPs, respectively. Because of the very slow clearance of those tracers from blood plasma, a transient equilibrium as discussed by Carson et al. (7) is approached in many types of tissue. Then a fairly linear relationship between the apparent VD (i.e. the ratio of tissue activity over parent tracer plasma activity) and the true equilibrium VD exists. An apparent BP can be calculated from the ratio of the tissue activity in a target region over the tissue activity in the reference region minus one which is well correlated with the true BP. Results and Conclusion Data from measurements as well as from simulations with several high VD tracers for the serotonergic and dopaminergic system have been analysed. Activities from late scan times (with respect to the half life of the radiolabel) have been used to calculate BPs. They have had a good linear correlation with the true BP throughout all considered regions. Therefore, the activity ratio BPs could been rescaled with the parameters of the regression line. Alternatively, semi-quantitave results can be obtained easily from late time PET data. This often allows the design of simplified clinical acquisition protocols. Activity ratio analysis can be employed with tracers which have a very slow plasma clearance and high VDs in tissue to obtain stable regional BP estimates with low variance. References (1) Sadzot B, Lemaire C, Maquet P, Salmon E, Plenevaux A, Degueldre C, Hermanne JP, Guillaume M, Cantineau R, Comar D, Franck G: J Cereb Blood Flow Metab 15 (1995), 787 - 797. (2) Ito H, Nyberg S, Halldin C, Lundkvist C, Farde L: J Nucl Med 39 (1998), 208 - 214. (3) Parsey RV, Kegeles LS, Hwang DR, Simpson N, Abi-Dargham A, Mawlawi O, Slifstein M, Van Heertum RL, Mann JJ, Laruelle M: J Nucl Med 41 (2000), 1465 - 1477. (4) Ginovart N, Wilson AA, Meyer JH, Hussey D, Houle S: J Cereb Blood Flow Metab 21 (2001),1342 - 1353. (5) Olsson H, Halldin C, Swahn CG, Farde L: J Cereb Blood Flow Metab 19 (1999), 1164 - 1173. (6) Slifstein M, Laruelle M: J Nucl Med 41 (2000), 2083 - 2088. (7) Carson RE, Channing MA, Blasberg RG, Dunn BB, Cohen RM, Rice KC, Herscovitch P: J Cereb Blood Flow Metab 13 (1993), 24 - 42.

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A Compartmental Model for the in Vivo Partitioning and Metabolism of PET Radiotracers in Blood Marie-Claude Asselin*†, Lindi M Wahl‡, Shigeko Amano*§, Claude Nahmias* *McMaster University, Hamilton, ON, Canada †Imaging Research Solutions Ltd, London, UK ‡University of Western Ontario, London, ON, Canada §Gunma University School of Medicine, Maebashi, Gunma, Japan Introduction A compartmental model combining the in vivo metabolism and partitioning of PET radiotracers in whole blood is presented. This model is used in replacement of ad hoc functions to obtain a plasma input function in terms of the time course of radioactivity concentration measured in whole blood. The applicability of the model is illustrated with blood data gathered from human PET studies with the presynaptic dopaminergic radiotracer 6-[ 18F]fluoro-L-meta-tyrosine (FmT). Theory Given a parent radiotracer and one predominant metabolite, the total radioactivity in a 1ml blood sample is distributed between four physical compartments: the parent radioactivity in plasma(C 1p) and in erythrocytes(C 1e) and similarly, the metabolite radioactivity in plasma(C 2p) and in erythrocytes(C 2e). Following a bolus injection, the parent radiotracer is exchanged between plasma and erythrocytes with rate constants k 1 and k 2, or is transformed irreversibly into its main metabolite with rate constant k 3. The metabolite is also exchanged between plasma and erythrocytes with rate constants k 1’ and k 2’. The parent radiotracer and its main metabolite leave the blood into the tissues with rate constants k 5 and k 6, respectively. This model can be simplified if either compound equilibrates rapidly between plasma and erythrocytes, or if the partition of either radiolabelled compound between plasma and erythrocytes is known independently. These models were used to fit the ratio of radioactivity in erythrocytes to that in plasma R eop⫽(C 1e⫹C 2e)/(C 1p⫹C 2p) as well as the parent contribution to the total radioactivity in plasma R fmt⫽C 1p/(C 1p⫹C 2p). The concentration of a parent radiotracer in plasma can then be inferred from knowledge of the total radioactivity concentration in whole blood and these two ratios. Data collection and analysis Blood data were collected from ten human subjects who were studied with PET following an intravenous bolus injection of 185MBq of FmT. Ten to fifteen venous blood samples were collected over two hours at increasing time intervals throughout the study. At each time point, the 5ml blood sample was taken and divided into 2ml and 3ml aliquots. The 2ml sample was used for the measurement of the haematocrit following which 1ml of blood, haemolysed by the addition of Saponin, was extracted and counted for radioactivity. The 3ml sample was centrifuged, and 1ml of plasma was pipetted and counted for radioactivity. Corrections for background radiation, detection efficiency and physical decay were applied to each measurement. The radioactivity concentration (in Bq/ml) in the erythrocytes (Ce) was calculated from that in whole blood (C w) and in plasma (C p) by using C w⫽H.C e⫹(1-H).C p, where H is the haematocrit. The concentrations C p and C e were then expressed in Bq/g of water by assuming that 1ml of plasma and 1ml of erythrocytes contained 0.84g and 0.73g of water, respectively. Throughout the study, six additional blood samples were drawn from which the plasma was extracted and analysed for metabolites of FmT. From these measurements the fraction of the parent radiotracer in plasma was determined. The measured ratios R eop and R fmt were then fitted simultaneously to those predicted by the blood model using non-linear least squares. In order to ensure that both data sets bore equal weight in the minimization procedure despite their differing size, the cost function was defined as the sum of the reduced ␹ 2 calculated separately for each ratio (both ranging from 0 to 1.5). Results In vitro studies[1] have revealed that FmT, but not its main metabolite, is equally distributed in plasma and erythrocytes, such that k 1⫽K/(1-H) and k 2⫽K/H. Furthermore, the equilibration of the main metabolite of FmT was measured in vitro to be much faster (within 5min) than that of FmT (60min)[1], thereby allowing for compartments C 2p and C 2e to be merged. The in vivo exchange of radiolabelled compounds between plasma and erythrocytes is faster than that of FmT but slower than that of its main metabolite, and has still not reached equilibrium two hours post-injection. By including the metabolism of FmT as well as the clearance of both compounds from the blood, the unified blood model provided significantly better fits (F-test, p⬍0.05) to the blood partition data than the in vitro models[1]. Comments The unified compartmental model for blood data reduces the influence of outliers on the fitted curves by forcing the two data sets to be physiologically consistent. The reduced variability in the extrapolated values of the blood partition ratio and the parent fraction allows for population-based curves for these quantities to be derived. Coupled with image-derived blood time-activity-curves, plasma input functions can be obtained without the need for invasive blood sampling and elaborate blood analyses. Reference [1] Nahmias C, Wahl LM, Amano S, Asselin M-C and Chirakal R (2000) J.Nucl.Med. 41: 1636-41.

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[11C]Flumazenil kinetic analysis using image-derived input function S Sanabria*, A Maes†, P Dupont†, G Bormans†, T De Groot†, A Coimbra*, W Eng*, T Laethem‡, I De Lepeleire‡, J Gambale*, JM Vega*, HD Burns* *Merck Research Laboratories, West Point, USA. †University Hospital of Leuven PET Center, Leuven, Belgium. ‡Merck Research Laboratories, Brussels, Belgium. Introduction The kinetic analysis of [11C]flumazenil (FMZ) data for benzodiazepine receptor (BZR) quantification relies on the estimation of the [11C]FMZ plasma concentration from arterial blood sampling during data acquisition. In this work, the shape of the arterial plasma curve is extracted from the time activity curve (TAC) measured in cerebral arteries. Validation was assessed by comparing the volume of distribution (DV) obtained using the image-derived and the experimental plasma curve. Method Kinetic data were obtained using a ⬍ 60-sec bolus [11C]FMZ injection in 6 normal subjects. Studies were performed using the PET scanner ECAT EXACT HR⫹. A sequence of 20 frames was obtained after injection (4⫻15 s, 4⫻60 s, 2x150 s and 10⫻300 s), and 63 contiguous transaxial slices were reconstructed with a voxel size of 2-mm in the tomographic direction and 2.4-mm in the axial direction. Arterial blood sampling was performed during the experiments to measure total 11C activity in plasma. The relative FMZ fraction in plasma (fFMZ) was estimated using 6 blood samples (2, 5, 10, 20, 40 & 60 min). PET frames acquired during the first minute after injection were summed and regions of interest (ROI) drawn on the arteries. Arterial ROIs were drawn on the carotid arteries in the lower planes of the images to minimize the spillover effect from the surrounding tissue (mainly the cerebral trunk and the lower temporal lobe). An arterial volume of interest (VOI) was obtained by assembling 2D ROIs drawn on consecutive planes that were automatically projected onto all frames to obtain the corresponding arterial TAC. The shape of the plasma [11C]FMZ concentration curve was obtained from the product of the arterial TAC and fFMZ. The resulting curve was “calibrated” using 3 late [11C]FMZ plasma samples (20, 40 and 60 min). This procedure assumes that the ratio between the activity concentrations in whole blood and plasma and the spillover contribution from the surrounding tissue are constant. When comparing the image-derived curve to the experimental plasma curve from peripheral blood sampling, the initial peak appears underestimated (figure 1) due to partial volume effects (PVE) associated to the small size of the arterial VOI and to the low-sampling frequency of the scanner. Assuming that all brain regions share the same input curve, simultaneously fitting of several tissue TACs to a 2-compartment model were applied. The missing fast frequency component and its amplitude of the image-derived plasma were used as coupling parameters. Results A good agreement was found between the image-derived and the experimental plasma curves for all subjects (figure 1). DV estimates using both curves were highly correlated (r 2⬃1.00 in all cases, figure 2). The slope and intercept values of the linear regression were in the interval [0.9, 1.0] and [– 0.12, 0.06], respectively. Conclusion The proposed method is suitable for BZR quantification. This approach requires only a few blood samples to measure fFMZ and to calibrate the image-derived curve. The method can be simplified further by substituting arterialized venous blood for arterial blood.

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Effect of Scatter Correction on Kinetic Analysis in I-123 IBF SPECT Study Kyeong Min Kim*, Hiroshi Watabe*, Yoshihiro Onishi†, Yoshiharu Yonekura‡, Hidehiro Iida* *Department of Investigative Radiology, National Cardiovascular Center - Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka, 565-8565, Japan †Nihon Medi-Physics, Hyogo, Japan ‡Fukui Medical School, Fukui, Japan Introduction Scatter correction (SC) is essential for the accurate quantitation of brain perfusion by SPECT using I-123 and Tc-99m labeled compounds with SPECT [1,2]. Dopamine studies using kinetic analysis and dynamic images have been performed, which showed a tracer distribution of having a high activity accumulation in a small region with a low activity in the background of the brain. However, the contribution of SC remains to be examined yet. We investigated the impact of SC on the kinetic parameters derived from SPECT using a D2 receptor ligand and various kinetic models. Methods Dynamic SPECT acquisitions (41 frames for 2 hours) were performed on healthy subjects (n ⫽ 8), with serial arterial blood sampling, following the intravenous injection of I-123 iodobezofuran (IBF). All subjects also underwent MRI scans to obtain uniform attenuation maps. The transmission dependent convolution subtraction (TDCS) technique [1] was employed for SC. Images were reconstructed by OSEM, which included the attenuation correction (AC), with (ACSC) and without SC (NOSC(I)). FBP with Butterworth filter and post AC without SC (NOSC(II)) was also performed for image reconstruction. Time-activity curves (TACs) of the striatum (STR) and the occipital lobe (OCC) were generated for the three types of reconstructed dynamic images, ACSC, NOSC(I), and NOSC(II). The quantitative values of binding potential (BP) were estimated for four kinetic models, including the 3-compartment model (3COM) [3], the linear graphic plot (GRP) [4], the reference tissue method (RTM) [5], and the multiregression method (MLR) [6]. The values of kinetic parameters from 3COM and BP from four models were compared among three reconstruction methods. Results TACs of both STR and OCC were varied with the three reconstructions, which caused significant changes in K 1 and k 2 in 3COM (p ⬍ 0.05). SC increased BP by 33.3 ⫾ 2.1% and 13.3 ⫾ 3.1%, compared to NOSC(I) and NOSC(II), in four kinetic models, respectively. The sensitivity to SC was great in the 3COM. Conclusion SC induced a significant change in the shape of TACs in both STR and OCC, which resulted in the increase of BP with all four kinetic methods. The contribution of SC depended on the kinetic method employed. Therefore, similar to the brain perfusion study, SC should be required for the quantitative SPECT study with D2 receptor ligands. References (1) Iida H et al., J Nucl Med 1998:39:181-189. (2) Shidahara M et al, IEEE Trans Nucl Sci 2001 (in press). (3) Laruelle et al, J Nucl Med 1994:35:743-754. (4) Logan et al, J Cereb Blood Flow Metab 1990:10:740747. (5) Lammertsma et al, J Cereb Blood Flow Metab 1996: 16:42-52. (6) Ichise et al, J Nucl Med 1997:38:31-37.

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Estimation of arterial input function for [ 123I]CNS 1261 from venous blood samples Kjell Erlandsson*, Rachel S. Mulligan*, Rodrigo A. Bressan†, Ian D. Cullum*, Lyn S. Pilowsky† *Institute of Nuclear Medicine, University College London, Middlesex Hospital, Mortimer Street, London W1T 3AA, UK †Institute of Psychiatry, KCL, London, UK Introduction We have previously characterised the NMDA receptor tracer [ 123I]CNS 1261 in healthy volunteers, using kinetic modelling and full arterial sampling [1]. Since NMDA receptors are ubiquitously distributed in the brain it is not possible to use a reference tissue model for this tracer, so the arterial input function is needed for quantification of bolus studies. Sometimes it is not practicable to take arterial samples, whereas it might be possible to obtain venous samples. We have developed a method to derive an approximate arterial input function from venous samples, which could be used in such cases. Methods Dynamic SPET scans were performed on 6 healthy volunteers in a Prism 3000XP scanner after i.v. injection of ⬃185 MBq of [ 123I]CNS 1261, with both arterial and venous blood sampling. The initial peak in the arterial time-activity curve, C a(t), was absent from the venous curve, C v(t), but the two curves were in good agreement from 1-2 h p.i. There was a high degree of variation between different subjects in the time course of the fraction of unchanged parent compound, but it was very similar in arterial and venous plasma. In order to find a way of generating C a(t) from C v(t) we investigated the integrals of the two curves and found a direct linear relationship: I a ⫽ a⫻I v ⫹ b, where I x is the integral of C x(t) (left graph below). An approximate arterial input function could then be generated as follows: C a’(t) ⫽ C v(t) ⫹ ␰⫻C p(t), where ␰ ⫽ (a-1)I v⫹b and C p(t) is a standard peak function of unity integral. To test the method we analysed time-activity curves from different brain regions using the 1-tissue compartment model and calculated the total volume of distribution (V T⫽K 1/k 2). This was done using both the true, measured arterial input function and the approximate one, generated from venous samples. Results There was a good correlation between the V T values obtained using the true and the approximate input function (slope⫽1.01, R 2⫽0.95, right graph below). Discussion Our new method obviates the need for arterial sampling. An alternative way is to use a bolus/infusion protocol, but with the present method it is not necessary to wait for equilibrium conditions, which is advantageous in cases where the subject becomes uncooperative after the start of the study. Conclusions We have developed a method for estimating an “arterial” input function in [ 123I]CNS 1261 studies from venous blood samples. Inter-individual variations in plasma clearance can be taken into account, since this can be measured in venous plasma. Reference [1] Erlandsson K, et al., (Abstract), Shizophrenia Research, 49:193, 2001.

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Precision of Coregistration Techniques for F18-Altanserin PET Images Peter Willendrup, Claus Svarer, Steen G. Hasselbalch, Gitte M. Knudsen Copenhagen University Hospital, Neurobiology Research Unit Objectives Determination of the precision of four coregistration methods. The methods Air 3.0 [1], SPM [2] and two methods developed at Neurobiology Research Unit (NRU) were used for coregistration of F18-Altanserin (5HT-2A receptor) PET images and T1 weighted MR images (MPRAGE). Methods Four methods (Air 3.0 [1], SPM [2], Coreg (NRU), Registrate (NRU)) were used to coregister F18-Altanserin PET images and T1 MR images. The first NRU method is inspired by Pfluger et al., [3]. Initially the centre of mass of the two images are registered, after which the user interactively rotates/translates three transparent slices and contours from the PET image displayed on top of the MR. The second NRU method is based on manual definition of landmarks in both images, point sets x and x’. From the point sets, a 6 parameter linear transformation A is found by minimising E(r,␪)ⴝS(A(r,␪ )xj-x’j) 2, with respect to translational parameters r and angular parameters ␪. Once A is estimated, the user is presented with a performance-measure for each of the defined coordinates e jⴝ|Ax j-x’ j|, which can be used to decide for redefinition of the points. The non-NRU methods [1]-[2], are carried out with deshelled MR images using standard settings. The deshelling procedure used is ‘BET’, [4]. Results The pseudo precision of the parameter set from one method F w. resp. to the parameter set G from another method is defined as e Fⴝ|x-x’|ⴝ|x-G -1Fx|, where x is the coordinates of a brain voxel. e F is evaluated for all brain voxels, and means calculated. Figure 1 shows results for a single scan set and a single user. Left: Mean e F, with F and G set to method all four methods. (Air ⬃ 1, Coreg ⬃ 2, Registrate ⬃ 3, SPM ⬃ 4). The maximum e F found is approximately 4.58 mm. Right: e F with F set to method 1-4, and G the average of all F (as in [3]). Conclusion The data indicate that all four methods give reasonable results [3],[5] with F18-Altanserin scans, even though cerebellum is almost invisible in the PET scans. Air (Woods) is closest to the average transformation, indicating that if deshelling of the MR data can be done accurately, and no problems occur due to limited binding, this method should be preferred with F18-Altanserin. This might not be the case with more localized images, suggesting that the NRU methods (2,3 in the figure) could be useful in these cases. The reproducibility of the two manual methods (2,3) will be investigated. References [1] Woods RP, Mazziotta JC, Cherry SR. MRI-PET registration with automated algorithm. J Comput Assist Tomogr 1993; 17(4):536-46. [2] Ashburner J, Friston K. Multimodal image coregistration and partitioning—a unified framework. Neuroimage 1997; 6(3):20917. [3] Pfluger T, Vollmar C, Wismuller A et al. Quantitative comparison of automatic and interactive methods for MRI- SPECT image registration of the brain based on 3-dimensional calculation of error. J Nucl Med 2000; 41(11):1823-9. [4] Smith, S. Robust automated brain extraction. In Sixth Int. Conf. on Functional Mapping of the Human Brain, page 625. [5] West J, Fitzpatrick JM, Wang MY et al. Comparison and evaluation of retrospective intermodality image registration techniques. J Comput Assist Tomogr 1997; 21(4):554-66.

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Three-dimensional probabilistic atlas of the human brain Alexander Hammers*†, Richard Allom*, Samantha L. Free†, Ralph Myers‡, Louis Lemieux†, Tejal N. Mitchell†, David J. Brooks*, Matthias J. Koepp*†, John S. Duncan† *MRC Clinical Sciences Centre and Division of Neuroscience, Faculty of Medicine, Imperial College, Hammersmith Hospital, DuCane Road, London †Department of Clinical and Experimental Epilepsy, Institute of Neurology, UCL, Queen Square, London, and National Society for Epilepsy MRI Unit, Chalfont St Peter ‡Imaging Research Solutions Ltd., MRC Cyclotron Building, Hammersmith Hospital, London Probabilistic or frequency-based, label-based atlases of neuroanatomy are more representative of population anatomy than single brain atlases. They allow the statistical assessment of normal ranges for structure volumes and extents within the group used for their creation and, ultimately, the comparison of patient groups against this standard. No such manually constructed atlas is currently available for the frequently studied group of young adults. We investigated 20 normal subjects (10 male, 10 female, median age 31 years) with high resolution MRI scanning. Images were nonuniformity corrected and reorientated along both the anterior-posterior commissure (ACPC) line horizontally and the midsagittal plane sagittally. Building on our previous work, we have expanded and refined existing algorithms for the subdivision of MRI datasets into anatomical structures. 49 structures were interactively defined as three-dimensional volumes-of-interest (VOIs). The resulting 20 individual atlases were spatially transformed (normalized) into standard stereotactic space, using standard software (SPM99) and a widely used template (MNI/ICBM 152). We evaluated volume data for all structures both in native space and after spatial normalization. We then used the normalized superimposed atlases to create a maximum probability map in stereotactic space which retains quantitative information regarding intersubject variability. Its many potential applications range from the automatic labeling of new scans to the detection of anatomical abnormalities in patients. Further data can be extracted from the atlas for the detailed analysis of individual structures.

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Region of Interest Analysis: One for All Claus Svarer, Peter Willendrup, Karen Husted Adams, Gitte Moos Knudsen Neurobiology Research Unit, Rigshospitalet, Copenhagen, Denmark Introduction The aim of this study is to create a method for automatic application of sets Regions of Interests (ROIs) to functional brain images on the basis of structural Magnetic Resonance (MR) scans. The development of such a method would enable a less time consuming and more user independent approach in the analysis of new data. Methods High resolution (ⱁ1.2 mm) T1 weighted MR scans and corresponding 18F-altanserin PET scans were acquired for 13 subjects and the images were carefully aligned using a manual landmark based method. Manual delineation of 24 anatomically relevant ROIs were done on each MR scan by one evaluator. All the other MR scans were then co-registered to each subject’s MR scan using first the AIR 12 parameter homogenous transformation (Woods, R. P. et al.1993) and then a non-linear warping method (Kjems, U. et al.1999). For each subject, the transformation parameters identified by the transformation procedures were then applied to the ROIs so they became co-registered to the PET scan. Hence, each subject had 13 sets of ROIs, one defined directly on the subject’s own MR scan, the other 12 sets transferred from the other subjects. Results The average intensity of the manually defined PET-voxels were calculated for each set of 24 ROIs and this average value was then compared to the median of the averages for the ROIs from the 12 other subjects. The mean of this difference is 2.75⫾0.90%. Calculating the variation directly the mean of the normalized standard deviation for all 24 regions and for all 13 subjects is 5.29⫾4.85%. An alternative evaluation approach would be to generate a set of new ROIs for each subject based alone on voxels that were present in more than half of the 13 ROI sets. This is done for all 13 subjects and these new ROI sets (meat-ROIs) which are not as dependent of the delineations done on each subject are then transformed to each of the subjects. Using this approach the mean normalized standard deviation is 1.60⫾2.13%. Conclusion The proposed method enables future automatic generation of functional imaging ROI sets in subjects on the basis of their high resolution MR scans. It builds on manually delineation of regions on previously aquired MR-data sets from 13 subjects. Comparing the results from the two methods based on individual delineations of ROIs or based on the meta-ROIs and then transformation of these to each subject shows that the methods based on only one delineation show significant more variation in the calculated mean values. That is, for this method to be applicable it is essential that the manual delineation of the ROIs has been carefully conducted. Eventually they can be based on the proposed meta-ROI method. To evalutate whether the proposed method yields better and more reproducible results in terms of the functional images, a reproducibility study will need to be conducted where the same evaluator repeatedly defines ROIs in different subjects and where different evaluators define ROIs in the same subjects. References 1. Kjems, U., Strother, S. C., Anderson, J., Law, I., and Hansen, L. K. Enhancing the multivariate signal of [15O] water PET studies with a new nonlinear neuroanatomical registration algorithm. IEEE Trans Med Imaging 18(4), 306-19. 99. 2. Woods, R. P., Mazziotta, J. C., and Cherry, S. R. MRI-PET registration with automated algorithm. J Comput Assist Tomogr 17(4), 536-46. 93.

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“NO FREE LUNCH” THEOREM FOR NEUROIMAGING F. E. Turkheimer*, J. A. D. Aston†, V. J. Cunningham* *IRSL, Cyclotron Building, Hammersmith Hospital, London, UK †MRC-CSC, Hammersmith Hospital, London, UK

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Voxel Based and ROI Based Statistical Analysis of Benzodiazepine Receptor with PET as a Guide for Surgical Treatment of Intractable Mesial Temporal Lobe Epilepsy Yoshihisa Ohta*, Tadashi Nariai*, Kinji Ishii†, Kiichi Ishiwata†, Masahira Mishina†, Kikuo Ohno* *Neurosurgery, Department of Brain Medical Science, Tokyo Medical and Dental University, Tokyo, Japan †Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan Background and Purpose Analysis of central benzodiazepine receptors (cBDZ-R) with positron emission tomography (PET) is a useful tool to locate the epileptic focus of patients with mesial temporal lobe epilepsy (MTLE). However, the objective method to determine the area with abnormally decreased binding of cBDZ-R in each patient is need to be established with validation by the clinical outcome. In this study, we investigated patients with MTLE using PET measurement of cBZD-R. We compared the result obtained by voxel based analysis using the statistical parametric mapping (SPM) and that obtained by placing regions of interest (ROIs) on PET image in order to examine if these can offer the predictive information to foresee the surgical outcome of each patient. The same analysis was applied to cerebral blood flow (CBF) and glucose metabolism (CMRGlc) image to clarify the significance of cBDZ-R analysis Methods Eleven patients with intractable MTLE underwent PET measurement of cBDZ-R using C-11 flumazenil. CBF and CMRGlc was simultaneously measured using [O-15]H2O-15 and [F-18]FDG respectively. All the PET images were co-registered to patients’ own MRI. All the patients were treated with anterior temporal lobectomy and their outcome were classified according to Engel’s classification (Class 1 (seizure free), 8; Class 3 (90 % reduction of seizure), 3) after following them up more than one year. In voxel based analysis, PET data of each patient were analyzed using SPM99, by statistically comparing the voxel value of PET parameters between single patient and twelve normal volunteers. Voxels having significantly decreased value than normal control were mapped on standard brain atlas. P ⬍ 0.05 was considred as significant. In ROI based analysis, ROIs were placed on the mesial, basal and lateral temporal cortex over MRI which is co-registered to PET. In order to depict the ROIs with abnormally decreased value than contralateral side, asymmetry index (AI ⫽ (lt - rt) x 2 / (lt ⫹ rt)) was calculated and AI outside the mean ⫹/- 2SD range of normal control was considered as abnormal. Results 1) In both SPM and ROI analysis, CBF did not have predictive value in determining the side of epilepsic focus. 2) In ten out of eleven patients, the resected temporal lobe bore more voxels or ROIs with abnormally decreased CMRGlc than the contralateral side. However, in only three out of eleven patients, the resected temporal lobes bore more voxels or ROIs with abnormally decreased cBDZ-R than contralateral side. 3) In all patients, the voxels or ROIs with abnormally decreased CMRGlc extended beyond the resected area even in the Class 1 patients. The voxels or ROIs with abnormally decreased cBDZ-R (Fig. 1) represented the resected area more accurately than CMRGlc study (Fig. 2). 4) The voxel based analysis with SPM was more specific than ROI analysis to delineate the area with decreased cBDZ-R in epileptic focus. Conclusions In either ROI or voxel based statistical analysis, the side of epileptic focus was more sensitively detected by CMRGlc analysis than by cBDZ-R analysis. However, cBDZ-R analized by SPM was the most specific method to delineate the accurate epileptic focus to the others in the present study.

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The presence of 3-O-methyl-[18F]fluoro-DOPA (3OMFD) influences the evaluation of the 18F-fluorodopa tissue input uptake rate constant in a disease dependent way: a study in Parkinson’s Disease Vesna Sossi*, James E. Holden†, Raul de la Fuente-Fernandez‡, Thomas J. Ruth*, A. Jonathan Stoessl‡ *University of British Columbia/TRIUMF, Vancouver, B.C. Canada †University of Wisconsin, Madison, Wisconsin, USA ‡University of British Columbia, Vancouver, B.C. Canada Introduction Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons, thus decreasing the system ability to produce and store dopamine (DA). Such ability is often investigated using 18F-fluorodopa (FD) Positron Emission Tomography (PET) and quantified with a variety of different models. A commonly used model is the modified Patlak graphical approach (1,2,3). This approach allows for a plasma and a tissue input function yielding the respective uptake rate constants Ki and Kocc. This method requires the presence of an irreversible compartment and the absence of any non-trapped tracer metabolite. This last assumption is violated by the presence of the FD metabolite 3OMFD. For the plasma input function this violation can be overcome (3). The tissue input function however includes an indistinguishable component due to the 3OMFD thus making the evaluation of Kocc susceptible to a downward bias. Nevertheless, Kocc has been used extensively to quantify DA synthesis and storage. The disease discriminating and quantifying ability of Ki and Kocc have been already widely investigated with discrepant results (4,5,6). In this study we show that contrary to the value of Ki, the value of Kocc depends on the time interval used for its evaluation and that the magnitude of the 3OMFD derived bias is disease dependent. Methods Six normal subjects (N) and fifteen mild PD patients (motor component of the Modified Columbia Scale score 14.3 ⫾ 5.7) underwent a four hour FD scan (9x 10 min, 30 min interval, 12x 10 min). 23 blood samples were taken and selected ones analyzed for metabolites. The first 90 min of data were considered for this study. Ki and Kocc were evaluated from data points acquired between 20 and 70 min (Ki70, Kocc70 ) and 20 and 90 min (Ki90 and Kocc90) for the caudate and putamen separately. The ratio Ki/Kocc was calculated in each case. The comparisons between Ki70 and Ki90 and between Kocc70 and Kocc90 were used to estimate the bias as a function of the time interval. The comparison of Ki/Kocc between the N and the PD group was used to estimate the bias disease dependence. The ratio Ki/Kocc is mathematically predicted to be equal to K1/k2 (the clearance rates from plasma into tissue and from tissue into plasma). Since K1/k2 would not be expected to vary appreciably between normals and PD patients (6), any difference in the Ki/Kocc ratio between the N and the PD group can be attributed to the bias introduced by the presence of the 3OMFD in the tissue input function. Results Results are presented in table 1 (* values expressed in 10-2).

Table 1 Caudate

Putamen

Caudate

Putamen

KI90* KI70* Normal 1.89 ⫾ 0.26 1.81 ⫾ 0.19 1.89 ⫾ 0.24 1.81 ⫾ 0.17 1. The value of Ki does not change as a funcPD 1.44 ⫾ 0.33 1.00 ⫾ 0.36 1.41 ⫾ 0.36 1.03 ⫾ 0.37 tion of time interval for either group. It KOCC90* KOCC70* exhibits the typical rostrocaudal gradient Normal 1.19 ⫾ 0.11 1.07 ⫾ 0.09 1.38 ⫾ 0.11 1.22 ⫾ 0.09 for the PD group. The value for Kocc70 is PD 0.91 ⫾ 0.12 0.51 ⫾ 0.18 1.02 ⫾ 0.16 0.59 ⫾ 0.20 larger than Kocc90 for both groups. This is KI90/KOCC90 KI70/KOCC70 indicative of the downward bias introduced Normal 1.58 ⫾ 0.17 1.70 ⫾ 0.19 1.37 ⫾ 0.14 1.41 ⫾ 0.16 by 3OMFD. The bias is present even for the PD 1.59 ⫾ 0.33 2.05 ⫾ 0.59 1.38 ⫾ 0.26 1.83 ⫾ 0.52 normal group even for evaluation times ⬍ 90 min. 2. The Ki/Kocc values are appreciably higher compared to K1/k2 (6), which is indicative of the bias due to 3OMFD. 3. Ki/Kocc changes with time interval for both groups as expected from the Kocc results, but while it is the same between the two groups for the caudate (the less affected part of the striatum), it increases significantly in the PD group compared to the N group (p ⬍ 0.05) for the putamen, thus demonstrating a progressive underestimate of Kocc for increased disease severity.

Observations

Conclusion These results show that the presence of 3OMFD in the tissue input function causes a downward bias in the Kocc estimate and the magnitude of the bias is disease dependent. Kocc may therefore be a good disease discriminator, but may not an accurately quantify changes in DA synthesis and storage capability. References 1. 2. 3. 4. 5. 6.

Patlak CS, Blasberg RG, Fenstermacher JD (1983). J Cereb Blood Flow Metab 3: 1-7 Patlak CS, Blasberg RG (1985) J Cereb Blood Flow Metab 5: 584-590 Martin WRW et al.,(1989) Ann Neurol 26: 535-542 Hoshi H et al., (1993) J Cereb Blood Flow Metab 13:57-69 Takikawa S et al., (1994) J Nucl Med 35: 955-963 Dhawan et al., (1996) Quantification of brain function, Academic Press 219-223

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Binding Potential Image Based Statistical Mapping for Detection of Dopamine Release by [11C]raclopride Dynamic PET Yun Zhou*, James Brasic*, Christopher J. Endres*, Hiroto Kuwabara‡, Alane Kimes†, Carlo Contoreggi†, Atul Maini*, Monique Ernst†, Dean F. Wong* *Department of Radiology, Johns Hopkins University School of Medicine, Baltimore, MD, 21287 †NIDA-Intramural Program, Baltimore, MD ‡University of Pittsburgh, PA Changes in binding potential (BP) are widely used to estimate the changes in dopamine release with[ 11C]raclopride ([ 11C]RAC) dynamic PET. In cue-elicited craving studies, based on conventional regional of interest (ROI) kinetic analysis, we have found that the BP differences between the cocaine-specific stimulus and neutral stimulus are small for both cocaine abusers and control subjects. The purpose of this study is to develop a BP image based statistical mapping method for improving the sensitivity in detection of dopamine release. PET scans on 10 healthy controls and 10 cocaine dependent subjects were performed on a GE Advance scanner in 3D mode after the intravenous injection of 22 ⫾ 3.78 mCi (mean ⫾ SD) of high specific activity (⬎1.5 Ci/␮mol)[ 11C]RAC. In each subject 2 PET studies were performed on the same day using bolus injection, and on a separate day 2 PET studies were performed using bolus plus infusion (B/I). For each pair of PET studies performed on a single day, a neutral video (arts and crafts) and a video of cocaine usage and paraphernalia for cue-induced craving, were viewed during the first and second PET study, respectively. Each dynamic image set consisting of 30 frames over 90 min and 35 planes (matrix size 128x128, pixel size 2mmx2mm, plane separation 4.25 mm) were reconstructed using filtered back projection with ramp-0.5 filter. Metabolite-corrected arterial plasma time radioactivity was used as input function. A two-compartment three-parameter (K 1, k 2, V p) model was used to describe pixel tracer kinetics. The operational equations: were used to estimate K 1, and distribution volume (DV) (⫽K 1/k 2) directly without division. Our linear parametric imaging with spatial constraint algorithm was applied to generate images of K 1 and DV. Since PET-to-PET intrasubject image registration is more stable and reliable than intersubject PET-to-PET image registration, PET-to-PET parametric image registration within subject was first performed before spatial normalization. Because more structural information is contained in the images of K 1 than DV, both PET-to-PET image registration and spatial normalization using PET template were based on K 1 images by using statistical parametric mapping software (SPM99). The ROIs of the cerebellum were defined on the standard stereotactic (Talairach) space and applied to all spatial normalized DV images for estimating DV of cerebellum (DV(cerebellum)) in each dynamic PET study. The BP image was calculated as DV(pixel)/ DV(cerebellum)-1. Based on the BP images, paired (baseline v.s. stimulation) T statistics images were generated for both control and cocaine user group. Eleven ROIs across the whole brain were applied to images of T statistics. Results showed that the parametric images of K 1, DV and BP are of good image quality. The procedure of PET-to-PET intrsubject registration and spatial normalization with SPM99 used in this study was stable. In the control group, the mean of BP (caudate, putamen) for baseline and stimulation studies are (2.419, 2.987) and (2.396, 2.945), respectively. In the cocaine dependent group, the mean of BP (caudate, putamen) for baseline and stimulation group are (2.361, 2.813) and (2.328, 2.696), respectively. The ROI values calculated on T images are illustrated in Table 1. Statistical testing shows there is no significant difference in BP between baseline and stimulation study for control group. For the cocaine dependent group, BP was significantly decreased due to cue-induced dopamine release in the putamen, and no significant differences in BP between the two study conditions occurred in the caudate and other brain tissues. Due to partial volume effects and accuracy in PET registration and spatial normalization, any inferences related to BP are more robust in putamen compared to smaller structures like the caudate. In conclusion, the difference of BP between baseline and stimulation studies was small for both health control and cocaine dependent subjects. The quantification method developed in this study is suitable for detecting the dopamine release during cue-elicited craving with [ 11C]RAC dynamic PET, and is applicable to other stimuli evoking DA release/inhibition.

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EVALUATION OF AMISULPRIDE D 2/D 3 RECEPTORS LIMBIC SELECTIVITY—A REFERENCE TISSUE MODELLING STUDY OF [ 123I]-EPIDEPRIDE Rodrigo A. Bressan*, Kjell Erlandsson†, Hugh M. Jones*, Peter J. Ell†, Lyn S. Pilowsky*† *Institute of Psychiatry,Kings College London, De Crespigny Park, Denmark Hill, London, SE5 8AF, UK †Institute of Nuclear Medicine, UCL Medical School, London, UK Background Limbic selective action at temporal cortical over striatal D 2/D 3 receptors by atypical antipsychotics is a putative mechanism for their therapeutic efficacy without extrapyramidal side effects 1. We report SPET scans of schizophrenic patients (n ⫽ 7) using the high affinity radioligand [ 123I]-epidepride to evaluate the action of amisulpride at striatal and limbic D 2/D 3 receptors in vivo. Methods Seven schizophrenic patients were prospectively evaluated at baseline, prior to amisulpride treatment, and on the day of the scan (at least 4 weeks of amisulpride treatment). Dynamic SPET sequences were obtained for 5 hours postinjection of the ligand (151 MBq), using a high-resolution triple-headed brain scanner (Prism 3000XP Philips/Marconi). Full and simplified reference tissue modelling (with cerebellum as a reference region) was performed to obtain the binding potential in striatum, thalamus and temporal cortex. D 2/D 3 receptor occupancy was estimated relative to a normal volunteer control group, scanned with the same protocol. Results The amisulpride dose range was 1 patient on 150 mg/day; 4 on 400 mg/day; 2 on 600 mg/day. Binding potential (BP) values obtained with the two reference tissue models were highly correlated (slope ⫽ 0.98, R 2 ⫽ 0.99). The mean D 2/D 3 receptor occupancy (SD) was 81.7% (15.3) in temporal cortex, 76.2% (13.0) in thalamus, and 58.9% (18.1) in striatum (Figure). There was a significant higher occupancy values in limbic regions (temporal cortex and thalamus) when compared to striatum (p ⬍ 0.01). No significant difference was seen between temporal cortex and thalamus (p ⫽ 0.08). Discussion These data substantiate Xiberas et al 2 findings suggesting limbic selective occupancy of D 2/D 3 receptors in low to moderate doses of amisulpride. Limbic selectivity was observed during treatment with various atypical antipsychotics, such us clozapine 1, olanzapine 3, quetiapine 4 and risperidone 5, but not during treatment with typical antipsychotics 6. Preferential binding at D 2/D 3 limbic regions is an explanatory hypothesis for antipsychotic efficacy without side effects, though action at 5HT 2a receptors has also been proposed as critical for an atypical clinical profile. Amisulpride has negligible affinity for 5HT 2a receptors, and a highly selective action at dopamine D 2-like receptors. These data suggest limbic selective action at dopamine D 2/D 3 receptors in and of itself can account for atypical antipsychotic drug action. References 1. 2. 3. 4. 5. 6.

Pilowsky LS et al., Lancet, 1997; 350:490-491. Xiberas X et al., J Clin Psychopharmacol 2001;21(2):207-14. Bigliani V et al. Psychopharmacol 2000;150(2):132-40. Stephenson CME et al. Br J Psychiatry. 2000; 177:408-15. Bressan RA et al. Eur J Nucl Med. 2001; 28(8) 1034. Bigliani V et al. Br J Psychiatry 1999;175:231-8.

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Dopamine transporters in early onset Parkinson’s disease: a PET study with [11C]FE-CIT Clara Gobbo*, Angelo Antonini†, Rosa Maria Moresco‡, Giovanni Lucignani§ ¶, Roberto De Notaris†, Andrea Panzacchi*, Assunta Carpinelli㥋, Antonio Pezzoli†, Ferruccio Fazio*‡㥋 *Scientific Institute H San Raffaele †ICP Milan ‡University of Milan-Bicocca §University of Milan ¶

Ospedale L. Sacco Milan 㛳INB-CNR

Introduction Parkinson’s disease (PD) is characterised by a progressive loss of dopaminergic neurones in the substantia nigra and of dopamine nerve terminals in the striatum. A reduction of dopamine transporter (DAT) has been reported in the caudate and putamen of parkinsonian patients and DAT tracers are currently used to differentiate PD from PSP or essential tremor. However only few studies focused on the in vivo assessment of DAT in parkinsonian patients carrying a parkin gene mutation have been reported (Hilker et al. 2001, Broussolle et al 2000). Aim of this study was to evaluate striatal DAT binding in early onset PD patients with or without parkin gene mutation, using PET and [11C]FE-CIT (Laihinen et al. 2000, Ginovart et al. 2000). Patients and methods: 12 patients with early onset Parkinson’s disease (6 women,mean age:39⫾5 years;UPDRS:15⫾8, duration:3.2⫾3 years) were enrolled. Three of these patients carried a parkin gene mutation. Eleven healthy volunteers (6 men, mean age: 47⫾18 years) were studied as control. Each subjects underwent 3D PET scan (GE Advance) after the injection of 3.7⫾1.9 MBq/Kg of [11C]FE-CIT. PET images acquired between 60 to 90 min. were normalised in the Talairach and Tournoux space using a template of [11C]FE-CIT distribution built from the brain of 11 normal subjects. Normalised images were divided pixel by pixel to cerebellar mean activity concentration, to transform radioactivity images in binding images, and then smoothed with a Gaussian kernel (8 mm FWHM). [11C]FE-CIT binding images were analysed using both ROIs and SPM (SPM96). Results ROIs analysis showed a severe reduction of tracer binding in the basal ganglia of early PD patients relatively to normal controls. In particular a reduction of dopamine transporter was observed in the caudate and putamen of PD patients that did not carried a parkin mutation (18.24%,p⫽0.002 and 51.94%, p⬍0.0001 respectively). Interestingly in the 3 patients carrying the parkin mutation we observed, both in the caudate and putamen, the lowest mean specific uptake of [11C]FE-CIT (47.34%,p⬎0.0001 and 70%,p⬍0.0001 respectively). These data were confirmed by the SPM analysis (fig. 1), which showed a reduction of [11C]FE-CIT binding both in the caudate and in the putamen of PD patients, more evident in patients with parkin gene mutation (P threshold ⫽0.05). Conclusion The results of this study suggest a different degree of striatal degeneration in patients with early onset PD carrying or not carrying a parkin mutation. The rate of neuronal degeneration is under investigation in longitudinal studies.

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Alcoholics exhibit spatially distinct changes in D2-receptor-binding dependent on drinking habits and craving: a PET correlation analysis Thomas Siessmeier*, Andreas Heinz†, Jana Wrase†, Derik Hermann†, Hans-Georg Buchholz*, Frank Roesch‡, Mathias Schreckenberger*, Gerd Gruender§, Karl Mann†, Peter Bartenstein* *Department of Nuclear Medicine, University of Mainz †Zentralinstitut fu¨ r seelische Gesundheit, Mannheim ‡Department of Nuclear Chemistry University of Mainz §Department of Psychiatry University of Mainz Aims Dopamine release and turnover probably plays a key role in development and maintenance of alcoholism. Aim of this study was to correlate dopamine D2 receptor binding with a D2 receptor ligand with affinity similar to dopamine and psychological parameters reflecting drug induced craving (ACQ) and amount of alcohol intake (LDH) in abstinent alcoholics. Methods 9 male (age 35-57) alcoholics fulfilling DSM-IV and ICD-10 criteria who were abstinent for 2 to 4 weeks were examined with PET and evaluated extensively with neuropsychological questionnaires. Special emphasis was put on craving scales (ACQ) and on alcohol intake during the drinking period (LDH). Their data were compared with 8 age matched volunteers. PET data were acquired dynamically with an ECAT EXACT PET scanner after administration of 194 ⫹/-27 MBq 18F-Desmethoxyfallypride to image dopamine-D2-receptor binding. Following extensive model evaluation the pixelwise simplified reference tissue model using the cerebellum as D2 receptor free region (Gunn et al. 1997) was found to be suitable to quantify D2-receptor binding with this radiopharmaceutical. This model was applied to the dynamic PET-data yielding parametric images of the binding potential (BP). After stereotactic normalisation to the Talairach space a categorical comparison between the two groups and statistic parametric correlations were performed using SPM99. Results Categorical comparisons did not yield statistically significant differences in D2-receptor binding between the patients and volunteers. The severity of alcohol craving was significantly associated with reductions in D2 receptor availability exclusively in the bilateral ventral striatum and the caudate nucleus. The strongest correlation was observed in the right nucleus accumbens (xyz 6/14/-6; p⬍0.005, z-score 3.42) and the ventral left caudate nucleus (xyz -14/16/-2; p⬍0.005, z-score 4.5). An exclusive positive correlation between D2-receptor binding of the amygdalo-hippocampal complex and alcohol intake was observed as well (xyz – 6/22/14; p⬍0.005, z-sore 3.12). Conclusion The data suggest that the dopaminergic system is involved in development and maintenance of alcoholism. The association between decreased D2-receptor availability in nucleus accumbens /ventral striatum (Fig. 1) and alcohol craving in abstinent alcoholics supports the hypothesis that stimulus induced dopamine release may trigger the attention towards alcohol associated stimuli and may be associated with compensatory changes in postsynaptic D2 receptors. These findings support the hypothesis that dopaminergic transmission in nucleus accumbens and ventral caudate plays a key role in drug craving. Gunn RN, Lammertsma AA, Hume SP, Cunningham VJ. Parametric imaging of ligand-receptor binding in PET using a simplified reference region model. Neuroimage 1997 Nov;6(4):279-87

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DOES OPIOID INDUCED DOPAMINE RELEASE IN HUMANS REQUIRE A SUBJECTIVE “HIGH”? A PET STUDY WITH 11C-RACLOPRIDE Mark Daglish*†, Anne Lingford-Hughes*†, Tim Williams*†‡, Sue Wilson*, David Brooks†, Judith Myles‡, Paul Grasby†, David Nutt* *Psychopharmacology Unit, University of Bristol, UK †MRC Clinical Sciences Centre, Hammersmith Hopsital, London, UK ‡Bristol Specialist Drug Service, Blackberry Hill Hospital, Bristol, UK Introduction Release of dopamine in the meso-cortico-limbic pathway of the brain is thought to be critical in mediating pleasurable effects of drugs of abuse and to drive further use. Opioids have been shown to release dopamine in this pathway in animal models. Positron emission tomography (PET) and 11C-Raclopride have successfully been used to demonstrate changes in extra-cellular dopamine levels in humans. This study aimed to test the hypothesis that dopamine release can be measured in response to opioids in dependent individuals and that this release would be associated with subjective ‘pleasure’. Subjects: 8 male methadone maintained opiate dependent subjects (age 25-35 years) were recruited. Subjects had no history of dependent use of other drugs (except nicotine) or neurological disease, and no other concurrent major medical or psychiatric illness. Methods All subjects underwent two 11C-Raclopride PET scans. The first 2 subjects received 50% of their usual methadone dose on the morning of the scan. The remaining 6 subjects received no methadone until after the scan. In a double-blind randomised placebo-controlled design subjects received either 10mg hydromorphone SC, (equivalent to 35mg diamorphine) or saline 15 minutes prior to each scan. The subjects were informed that they may receive the active drug on 0, 1 or 2 occasions. The PET images were analysed to produce Binding Potential (BP) images using the RPM software package developed in-house. Mean BP values for each scan were calculated for 3 bilateral regions – caudate, putamen & ventral striatum. These regions were compared to look for changes in brain dopamine levels in response to hydromorphone. During the scans subjects completed visual analogue scales (VAS) to measure subjective drug effects and had saccadic eye movements (SEM) recorded to assess objective effects. Results No changes in 11C-Raclopride BP were observed in any of the 3 bilateral regions: Caudate (drug 2.8 ⫹/- 0.2 vs. placebo 2.9 ⫹/0.4) Putamen (drug 2.8 ⫹/- 0.3 vs. 2.7 ⫹/- 0.4) or ventral striatum (drug 2.6 ⫹ 0.2 vs placebo 2.5 ⫹/- 0.3). The VAS showed small non-significant increases in “rush”, “high”, “gouched” & “sleepy” as well as decreases in “crave” and “urge” for heroin. The SEM showed a small reduction in peak velocity following hydromorphone. Conclusions No changes of dopamine levels could be inferred in response to this substantial opioid dose. Interestingly, although this dose of hydromorphone has produced pronounced effects in an identical population in an out-patient setting, the expected subjective and objective effects were less in this scanning study. We are thus unable to conclude whether dopamine release is key to mediating pleasurable effects of opioids in dependent users. In order to further explore this possibility, we are currently challenging subjects with diamorphine, which more reliably induces subjective effects in the scanning session. This work was funded by an MRC Programme grant.

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D 2-like dopamine receptor occupancy in amisulpride-treated schizophrenic patients in relationship to plasma-levels and side-effects: A PET-study using 18F-Desmethoxyfallypride IB Vernaleken, T Siessmeier, HG Buchholz, Y Zhou, S Haertter, C Hiemke, DF Wong, F Roesch, P Bartenstein, G Gruender Introduction The selective D 2-like dopamine receptor antagonist amisulpride (ASP) is an atypical antipsychotic agent frequently used in European countries. Receptor occupancy studies using 76Br-FLB 457 and a simplified binding index method published recently found preferential antagonism in extrastriatal regions, while comparably low receptor occupancies in striatal areas were observed. Aim of this investigation was to determine receptor occupancies in amisulpride-treated schizophrenic patients in relationship to plasma levels and side-effects by positron emission tomography with several established analytical methods. We used the substituted benzamide 18F-Desmethoxyfallypride (DMFP)as the radioligand, which has been proven to be a suitable agent for D 2-like dopamine receptor mapping, although its’ moderate affinity does not reliably allow quantification of extrastriatal dopamine receptors. Methods Nine patients suffering from DSM-IV diagnosis of schizophrenia or schizoaffective disorder were included. All patients received a long-term amisulpride-treatment with stable oral daily doses (1 x 200 mg/d; 3 x 400 mg/d; 1 x 600mg/d; 2 x 800 mg/d; 1000 mg/d). Benzodiazepines in low doses were allowed as concomitant medication. Eleven healthy and drug-free volunteers served as control group. PET-Scans were started approximately four hours after the last drug-ingestion on a Siemens ECAT EXAT whole-body scanner (FWHM: 5.4 mm; 47 slices). 165-308 MBq [18F]DMFP was injected intravenously as a bolus, followed by a scanning-period of 124 min (30 time-frames). Most patients underwent arterial plasma sampling for determination of plasma input function and labeled metabolites. After movement correction, coregistration with a T1-weighted MRI and subsequent normalization, polygonal volumes of interest were defined according to the anatomical structures. Time activity curves were plotted for caudate nucleus, putamen, medial thalamus und cerebellum. Data analysis was carried out using pseudoequilibrium method, graphical methods, simplified reference tissue model and compartmental models as well as analysis via parametric spectral analysis. Plasma levels of amisulpride were determined closely before the last ingestion of amisulpride and during the scan-period in 30 minute-intervals. Additionally, side-effect ratings, psychopathological ratings and neuropsychological tests were conducted. Results Control subjects show binding potentials (BP) in putamen between 2.19 ⫾ 0.32 (pseudoequilibrium method) and 2.44 ⫾ 0.40 (Logan-plot), depending on the analytical method. Caudate nucleus yielded lower BPs between 1.61 ⫾ 0.34 (Logan plot with reference region) and 1.80 ⫾ 0.41 (2-compartment model). All methods were significantly correlated with each other. Preliminary data analysis (pseudoequilibrium method) in amisulpride-treated patients revealed BPs between 0.36 and 2.18 for the putamen and between 0.12 and 1.27 for caudate nucleus, respectively. From the comparison with BPs of the control group occupancy-rates between 13% and 83% were calculated (putamen), which were significantly correlated with extrapyramidal side effect rating scores (Simpson-Angus-Scale). Morning plasma levels of ASP ranged from 67 ng/ml to 426 ng/ml, mean intra-scan-levels ranged from 177 ng/ml to 608 ng/ml. No correlation could be found between plasma levels and receptor occupancy. During the 124 min. lasting scanning procedure a considerable decline of plasma levels could be observed in most cases (up to 41%). Conclusions The D 2-receptor selective DMFP is a suitable tracer for D 2-like receptor mapping in striatal regions. The highly significant correlations between various analytical methods show that the applied methods reveal valid and reliable binding potentials. In contrast to previous investigations we found much higher D 2 receptor occupancies in putamen (average occupancy: 49 ⫾ 23% under mean dose of 622 ⫾ 291 mg/d), with blockade even exceeding 80% in one subject. This is in accordance with the significant correlation with extrapyramidal side effect-scores. These differences to previously reported data may be due to a more accurate methodological approach in comparison to static calculation of a binding index, which is being used by others. Although DMFP does not provide estimates of extrastriatal D 2 receptor densities, our results question the hypothesis of the preferential extrastriatal action of amisulpride. Furthermore, the marked decline of plasma levels during the scan is noteworthy. Thus, when reporting relationships between plasma levels and receptor occupancies, the time of blood sampling for determination of plasma levels as well as the time of last drug-intake in relation to time of PET scanning are of critical importance. Finally, the absence of significant correlations in our sample between occupancy and morning plasma level or daily dose, respectively, may be critically considered when providing dichotomic dosing-recommendations.

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PET imaging of mesolimbic D2 receptors and the subjective effects of cocaine Diana Martinez*†, Allegra Broft*†, Dah-Ren Hwang*†, Mark Slifstein*†, Osama Mawlawi*†, Yiyun Huang*†, Audrey Perez*†, Ingrid Stokes*†, Ronald Van Heertum*, Richard Foltin*†, Marian Fischman*†, Marc Laruelle*† *Columbia University †New York State Psychiatric Institute Objective Previous PET studies have demonstrated that cocaine dependence is associated with a reduction in striatal dopamine D2 receptors (Volkow et al, Am. J. Psych 1993 and Nature 1997). A recent study in non-human primates suggests that this reduction in D2 receptors is a risk factor for cocaine self administration, rather than a sequelae of chronic cocaine exposure (Nader et al, NIDA Res Mono 2001). Furthermore, a study in healthy controls has shown that subjects with a low D2 receptor availability are more likely to report positive subjective effects in response to a psychostimulant compared to subjects with a higher D2 receptor availability. These findings have led to the hypothesis that low D2 receptor availability may correlate with increased psychostimulant-induced euphoria and predispose certain individuals toward cocaine abuse. Therefore, the goal of this study was twofold: 1) to measure D2 receptor availability in chronic cocaine abusers compared to healthy controls, and 2) to investigate an association between D2 receptor binding and the reinforcing effects of cocaine in the group of cocaine abusers. Methods Nine healthy control subjects and 13 chronic cocaine abusers (CCA) were scanned using [C-11]raclopride and the ECAT EXACT HR⫹ PET camera. Exclusion criteria for both groups included the presence of serious medical or neurologic conditions and any Axis I psychiatric disorder other than cocaine dependence in the cocaine group. Studies were approved by the institutional review boards and informed consent obtained. All subjects were scanned with a bolus plus constant infusion of [C-11]raclopride and emission data was collected at equilibrium over 40 to 90 minutes. The regions of interest analyzed included the ventral striatum (VS), dorsal caudate (DC), and dorsal putamen (DP), each defined on the individual subjects’ MRI. The CCA were admitted for monitored abstinence 14 days prior to scanning and participated in cocaine self administration sessions following the PET scans. In the self administration sessions, CCA were initially given a low dose of smoked cocaine (0,6,or 12 mg) and asked about the positive subjective effects of each dose. Following this, subjects were then given a priming dose of cocaine (0,6, or 12 mg) followed by 5 choices. The choices were between the same dose of cocaine or a $5 gift certificate. All CCA underwent lab sessions with each dose of cocaine (0,6,12 mg) in random order. Results The CCA had a significantly lower D2 receptor availability in each of the striatal subregions as follows: 2.27 ⫹/- 0.50 (controls) vs. 1.77 ⫹/- 0.25 (CCA) for the VS (p ⫽ 0.02), 3.20 ⫹/- 0.30 (controls) vs. 2.71 ⫹/- 0.21 (CCA) for the DP (p ⫽ 0.004), and 2.5 ⫹/- 0.24 vs. 2.50 ⫹/- 0.24 vs. 2.25 ⫹/- 0.21 (CCA) for the DC (p ⫽ 0.01). In the CCA self administration sessions, there was no significant difference in the ratings of positive subjective effects between the 0 and 6 mg dose of cocaine, while the positive effects of the 12 mg dose were significantly greater (p ⫽ 0.02). However, there was a significant increase in the choice to take the 6 mg dose vs. $5 compared to the 0 mg dose. Thus, we investigated the subjective effects of the 12 mg dose in association with D2 receptor availability and found no significant association (r2 ⫽ 0.01, p ⫽ 0.70). We also investigated the choice for the 6 mg dose vs $5 and D2 receptor binding and found no association (r2 ⫽ 0.01, p ⫽ 0.67). Conclusion We found a significant decrease in D2 receptor availability in the subregions of the striatum of 22% in the VS, 15% in the DP, and 10% in the DC. However, we found no association between the decrease in [C-11]raclopride binding and the positive subjective effects of cocaine reported by chronic cocaine abusers. Furthermore, we found that D2 receptor availability did not correlate with the choice for cocaine vs a monetary reward.

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Dose-Occupancy Relationship of Five Selective Serotonin Reuptake Inhibitors Using [11C] DASB PET Jeffrey Meyer, Alan Wilson, Nathalie Ginovart, Sandra Sagrati, Anna Carella, Sylvain Houle PET Centre, Centre for Addiction and Mental Health, Department of Psychiatry, University of Toronto [11C] DASB PET may be applied to determine the serotonin transporter (5-HTT) occupancy of antidepressants during treatment in humans. We now extend our previous work to investigate the relationship between dose and occupancy for five different selective serotonin reuptake inhibitors (SSRI). Thirty drug free depressed subjects and 32 healthy subjects were recruited. To obtain test-retest data, the striatal 5-HTT binding potential (BP) was found twice with a four week interval using [11C] DASB PET. In the remaining subjects, the striatal 5-HTT BP was found before and after four weeks of medication administration. Subjects received fluoxetine, sertraline, venlafaxine XR, citalopram, or paroxetine. Healthy subjects received subtherapeutic doses and depressed subjects received therapeutic doses. Percent occupancy for each medication and for each dose was calculated. Occupancy⫽(5-HTT BP scan1 minus 5-HTT BP scan2)/5-HTT BP scan1. As compared to test-retest data, each drug at minimum therapeutic dose (fluoxetine - 20mg, sertraline - 50mg, venlafaxine XR - 75mg, citalopram - 20mg, paroxetine - 20mg) had a highly significant effect upon striatal 5-HTT BP. The mean occupancy at minimum therapeutic dose ranged from 75-83 per cent across the different SSRIs. For each drug, as the dose (or serum level) increased, occupancy increased in a non-linear relationship such that some degree of plateau occurred for higher doses (hyperbolic curve of form f(x)⫽a*x/(b⫹x) significantly fit the data for each drug). The minimum therapeutic doses are known to distinguish SSRI treatment of depression from placebo. It is interesting that all of these doses were associated with a 5-HTT occupancy of approximately 80 per cent. Measurement of 5-HTT occupancy should play an important role in future antidepressant development. References 1. Meyer JH, Wilson AA, Ginovart N, Goulding V, Hussey D, Hood K, Houle S. Occupancy of Serotonin Transporters by Paroxetine and Citalopram During Treatment of Depression: A [11C] DASB PET Imaging Study. Am J Psychiatry 2001; 158: 1843-1849. 2. Wilson AA, Ginovart N, Schmidt M, Meyer JH, Threlkeld PG, Houle S. Novel Radiotracers for Imaging the Serotonin Transporter by Positron Emission Tomography: Synthesis, Radiosynthesis, and in Vitro and ex Vivo Evaluation of 11C-Labelled 2-(Phenylthio)araalkylamines. J Med Chem 2000; 43(16): 3103-3110. 3. Houle S, Ginovart N, Hussey D, Meyer JH, Wilson AA. Imaging the Serotonin Transporter with Positron Emission Tomography: Initial Human Studies with [11C]DAPP and [11C]DASB. Eur J Neuroimaging 2000; 27: 1719-1722. 4. Ginovart N, Wilson AA, Meyer JH, Hussey D, Houle S. Positron Emission Tomography Quantification of [11C] DASB Binding to the Human Serotonin Transporter: Modelling Strategies. JCBF 2001; 21: 1342-1353.

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Quantification of [ 11C]Ro15-4513 Binding; Possible Subtype of GABA A/Benzodiazepine Receptor Complex Yoshiyuki Asai*†‡, Yoko Ikoma§ ¶, Tetsuya Suhara*‡, Akihiro Takano*†‡, Jun Maeda*‡, Hinako Toyama ¶, Hiroshi Ito㥋, Fumihiko Yasuno*‡, Tetsuya Ichimiya*‡ *Brain Imaging Project, National Institute of Radiological Sciences, Chiba, Japan †Department of Psychiatry, Division of Neurological Science, Hokkaido University Graduate School of Medicine, Sapporo, JAPAN ‡CREST, Japan Science and Technology Corporation (JST), Saitama, JAPAN §Department of Science and Engineering, Waseda University, Tokyo, JAPAN ¶

Medical Information Processing Office, Reseach Center of Charged Particle Therapy, National Institute of Radiological Sciences, Chiba, JAPAN 㛳Department of Radiology and Nuclear Medicine, Akita Research Institute of Brain and Blood Vessels, Akita, JAPAN

The central benzodiazepine (BZ) binding sites are located in the GABA A/BZ receptor chloride channel complex and play important roles in several disorders such as epilepsy, anxiety and insomnia. Several ligands have been developed to visualize BZ receptors in vivo, with [ 11C]flumazenil and [ 123I]iomazenil being commonly used at various centers. The distributions of [ 11C]flumazenil and [ 123I]iomazenil are reported to be similar. However, a different distribution pattern has been reported for the binding of [ 11C]Ro15-4513 compared to that of [ 11C]flumazenil. The accumulation of [ 11C]flumazenil is widespread in the cerebral cortex but in the occipital cortex it is relatively higher than in other cortical regions. On the other hand, [ 11C]Ro15-4513 accumulates at relatively higher levels in the hippocampus, anterior cingulate gyrus and insular cortex compared to other cortical regions. The mechanisms for these different distribution patterns have not been fully examined, but the role of a subtype of BZ receptor has been suggested. The aim of this PET study was perform a quantitative analysis of [ 11C]Ro15-4513 in the living human brain. Nine healthy male volunteers (aged 21 to 31 years) participated after giving their informed consent. [ 11C]Ro15-4513 (196.47415.51 MBq) was injected intravenously and radioactivity was measured using Siemens ECAT Exact HR⫹ in 3D mode for 60 min. Specific radioactivity was 13.87-174.96 GBq/␮mol at the time of injection. Arterial blood samples were taken manually to obtain arterial input function and determine the metabolites in plasma. Regions of interest (ROIs) were drawn on the anatomical images of MRI and transferred to reconstructed PET images. ROIs were delineated for the pons, cerebellar cortex, hippocampus, amygdala, anterior cingulate gyrus, insular cortex, lateral temporal cortex and occipital cortex. Kinetic data analysis was performed with the 2- and 3-compartment models. The Akaike information criterion (AIC) and Schwarz criterion (SC) were used to evaluate the fitting. [ 11C]Ro15-4513 binding was also analyzed by graphical analysis without blood sampling (Logan et al. 1996) and simplified reference tissue model analysis (Lammertsma et al. 1996). The pons was used as reference region because it is almost devoid of GABA A/BZ receptor complex. The effects of noise on the estimation reliability of these two methods were simulated. The accumulations of [ 11C] Ro15-4513 in the hippocampus, amygdala, anterior cingulate gyrus and insular cortex were relatively higher than in other cortical regions and the cerebellum. AIC and SC of the 3-compartment model were smaller than those of the 2-compartment model in cortical regions. The 3-compartment model was statistically better than the 2-compartment model to describe [ 11C]Ro15-4513 binding in cortical regions. On the other hand, AIC and SC of these two models were similar in the pons, and specific binding of the pons appeared to be negligible. The binding potentials were obtained by graphical analysis without blood sampling and by simplified reference tissue model analysis, and the values correlated well with those from the 3-compartment model. In the simulation study, the estimated parameters by graphical analysis without blood sampling were more reliable than those by simplified reference tissue model analysis at a high noise level.

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First Results of a Novel Adenosine A1 Receptor PET Ligand Andreas Bauer*, Marcus Holschbach†, Christian Boy*, Heinz Coenen†, Karl Zilles*‡ *Institute of Medicine, Research Centre Juelich, Germany †Institute of Nuclear Chemistry, Research Centre Juelich, Germany ‡C & O Vogt Brain Research Institute, Duesseldorf, Germany Adenosine plays an important role in neurotransmission. It exerts a potent feedback inhibitory effect on excitatory, esp. glutamatergic neurotransmission and is thus regarded as a “natural” neuroprotective compound. Most of its effects are mediated by the activation of A1 receptors. The occupancy of these receptors is, therefore, regarded as a potential indicator of neuronal stress. In vivo imaging of A1 receptors has now become feasible with a novel PET ligand for A1 receptors ([18F]CPFPX), which has been developed at the Research Center Juelich. Quantitative dynamic receptor PET was performed in healthy subjects and patients with diverse neurological disorders. In vivo distribution of [18F]CPFPX is in accordance with in vitro studies on the regional pattern of A1 receptors of human post mortem brains. Highest binding potentials were found in striatum and thalamus, followed by cortical regions with an accentuation of frontal and parietal lobes with exception of the central region, which is characterized by a relatively lower binding potential. Medium receptor densities are observed in the midbrain and brainstem. The cerebellum shows a very low binding capacity. As a preliminary result [18F]CPFPX binding potential is found to be reduced in the dorsal striatum of patients suffering from Parkinsons disease. In contrast, cases of multiple system atrophy show broader reductions of binding also within neocortical areas. In temporal lobe epilepsy there is a loss of binding potential in the epileptic focus. In single cases A1 receptors are upregulated in the vicinity of the epileptic focus. The novel adenosinergic PET ligand [18F]CPFPX has great potentials as an indicator and progression marker of neurological disorders. Especially in degenerative and epileptic disorders the usefulness of in vivo imaging with [18F]CPFPX has been substantiated.

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Effect of Fluvoxamine on cortical 5HT2A receptors assessed by PET and [11C]MDL100907 in drug naive obsessive compulsive patients Rosa Maria Moresco*, Marco Locatelli†, Daniela Perani‡§, Clara Gobbo†, Andrea Panzacchi†, Giovanna Rizzo‡, Marta Henin†, Mario Matarrese‡, Lorena Bonaldi‡, Laura Bellodi†, Ferruccio Fazio*†‡ *University of Milan-Bicocca †Scientific Institute H San Raffaele ‡INB-CNR §University Vita e Salute Changes in 5HT2 receptor binding during antidepressants in patients with major depressive disorder have been reported using PET and 5HT2 ligands. Aim of this study was to evaluate whether a similar modifications in 5HT2A receptors occur in patients affected by Obsessive Compulsive Disorder (OCD). For this purpose we measured the in vivo binding of the selective 5HT2A receptors antagonist [11C]MDL100907 in the brain of a group of OCD patients before and after the repeated administration of the serotonin reuptake sites inhibitor (SSRI) fluvoxamine. To avoid confounding effect of previous treatment, the study was performed in a group of drug naı¨ve patients. Eight OCD patients completed the study undergoing the second PET scan (5 males, mean age 31⫹7 yrs., range: 20 to 45 yrs.). Each patient underwent a 90 min. PET scan (GE Advance), before and after 8 to 10 weeks of treatment with fluvoxamine. In order to minimized possible biases in patients selection, a semi-quantitative protocol, that avoid arterial blood sampling, was applied. Radioactivity concentration images were transformed in binding potential (BP) images using the simplified reference tissue model developed by R Gunn et al., (1998) and the cerebellum as a reference tissue. Parametric PET images were normalized in the Talairach and Tournoux space using a template of [11C]MDL100907 BP images, and then smoothed (8 mm FWHM). The effect of fluvoxamine on BP images was evaluated using SPM96. The presence of modifications in tracer uptake were also evaluated using ROIS on radioactivity concentration images divided for the injected dose. SPM analysis revealed that fluvoxamine treatment significantly increased [11C]MDL100907 BP in the cortex of OCD patients (p ⫽ 0.01), increase that was particularly evident in the middle temporal gyrus (p ⫽0.001; Figure 1). Similar modifications were also observed in radioactivity concentration images. Fluvoxamine administration increased tracer uptake in different cortical regions, particularly in frontal and temporal aspects (Orbitofrontal Cortex: mean relative change: 21⫹24%; range –5%⫹61%; p⫽ 0.04) but not in the basal ganglia, thalamus or cerebellum. In this last region a mean 9%⫹11% (range -30% - ⫹5%) reduction in radioactivity concentration was observed. In conclusion chronic treatment with fluvoxamine significantly increases [11C]MDL100907 uptake and BP in the cortex of previously drug naı¨ve OCD patients. The reduction in cerebellar radioactivity concentration did not correlate with the increase in BP measured in parametric images, suggesting that the increased in tracer BP induced by fluvoxamine is not related to modifications in reference region. The increase of the in vivo binding of [11C]MDL100907 observed may reflect a modification in 5HT2A binding capacity secondary to changes in cortical serotonin activity. Whether these modifications are related to patients responsiveness need be further investigated.

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Gabaergic system in Prader Willi Syndrome: a PET-[11C]flumazenil study Giovanni Lucignani*, Andrea Panzacchi†, Rosa Maria Moresco‡, Laura Bosio†, Laura Ravasi†, Ferruccio Fazio†‡§, G. Chiumello* *University of Milan †Scientific Institute H San Raffaele ‡University of Milan-Bicocca §INB-CNR Introduction Prader Willi Syndrome (PWS) is a multi-system defect characterized by delayed mental and physical development, hyperphagia and gross obesity, bizarre antisocial and maladaptive behaviors, self injury and reduced sensibility to pain. Neuropathologic abnormalities include cortical nerve loss, poor myelination, cerebellar heterotopia, and calcifications. PWS patients have a typical deletion of chromosome 15 (15q11-13) or maternal disomy, entailing a deletion of the gene encoding the GABAA receptor beta3 subunit. Mean plasma GABA levels in PWS patients are 2-3 folds higher than in non retarded moderately obese control subjects. Aim of this study was to evaluate the expression of central benzodiazepine (BDZ) receptors with PET and [11C]flumazenil (FMZ), in a group of patients affected by PWS. Method 6 subjects with genetic diagnosis of PWS (mean age 22.8, range 18.8-28.8 yrs) and 9 age matched normal controls were recruited for the study. All [11C]FMZ PET studies were performed with a eighteen-ring whole body PET scanner (GE Advance: General Electric Medical System, Milwaukee, WI USA). Each subjects underwent a 60 min. 3D dynamic scan after the injection of approximately 1.8-3.6 MBq/kg of [11C]FMZ. Parametric images were built starting from radioactivity distribution images using the simplified reference region model (Gunn et al., 1997) and the pons as a reference region. RI and BP images were normalized within a stereotactic space using SPM96. Differences between PWS and normal controls were evaluated using SPM96. Results No differences in whole brain BP or RI values were found between PWS and normal controls. However, using whole brain BP values as a covariate, a decrese of [11C]FMZ BP in different cortical region of PWS patients (superior frontal, pre-central gyrus, cyngulus, insula and hippocampus) was observed. Conclusion An altered [11C]FMZ BP suggest the presence of modFig. 1. ANCOVA SPM ANALYSIS: BP [11C]FMZ BP valification in GABAA receptor density and/or composi- ues PWS ⬍ C tion due to the deletion of the beta3 subunit. PWS patients displayed regional GABAergic impairment in cerebral areas involved in the processing of emotional content. These alterations may be related to the abnormality in self perception, eating, social behaviors or pain response observed in these patients.

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Nicotinic acethylcholine receptors in Alzheimer’s disease: 5IA-SPECT study Hidefumi Yoshida*‡, Manabu Inoue*‡, Chisako Oyanagi*‡, Yukinori Katsumi*‡, Takahiro Mukai†‡, Koichi Ishizu†‡, Kazuo Hashikawa*‡, Hidenao Fukuyama*‡ *Human Brain Research Center †Nuclear Medicine and Diagnostic Imaging ‡Kyoto University Graduates School of Medicine Objective Nicotinic acethylcholine receptors (nAChRs) have been implicated to play an important role in neuropsychological functions as learning and memory. In Alzheimer’s disease, which has cognitive impairment as one of the main symptoms, the pathological process might involve the functions of nAChRs. This study aims to investigate the density of nAChRs in the patients with Alzheimer’s disease by using a newly developed radioligand for nAChRs and to compare the imaging of nAChRs with other functional imaging modalities, with regard to pathological and clinical significance. Methods The study involved five patients, clinically diagnosed as Alzheimer’s disease by the NINDS criteria (1 male and 4 females, from 67 to 77 years old). Six normal volunteers were also examined similar to the patients. Approximately 150 MBq of 123I(S)-5-iodo-3-(2-azetidinylmethoxy) pyridine, 123I 5-iodo-A-85380 (5IA), a specific radioligand for the ␣4␤2 subtype of the nAChRs was injected intravenously at a constant rate with an infusion pump for 1 min. Data acquisition was performed in 60 min (2min/frame) with a triple head rotating gamma camera system (Prism 3000, Picker USA) equipped with low-energy, high resolution, fan beam collimators resulting 30 images with 64 x 64 matrices. Radioactivities of 5IA in the arterial plasma were simultaneously obtained with metabolite correction for each subject. The late images (50 to 60 min) were added and compared by visual inspection with the images of [I-123]-N-isopropyl-piodoamphetamine (IMP) or those of [F-18]-2-deoxy-2-fluoro-D-glucose (FDG) taken by PET. Regions of interest were determined on 6 different brain regions in each hemisphere of transaxial SPECT images: the frontal, temporal, parietal and occipital lobes, the thalamus and the cerebellum and a time activity curve was generated for each ROI. The distribution volume (DV) values of 5IA were calculated by ROI based kinetic analysis of non-linear curve fitting method on two-compartment model and compared with those of the control group. Results By visual inspection, there was revealed a same pattern in the cortex among the late added images of 5IA (shown in the figure), and the images of IMP and FDG; they all showed decreased uptake in the cerebral cortices, particularly in the temporo-parietal areas. The DV values of the patients with Alzheimer’s disease were significantly reduced in the cerebral cortices, compared with those of the control group. The DV values in the thalamus and the cerebellum were also decreased in the patient group. The DV of those structures has relatively large values over those of the cerebral cortices; the tendency was observed not only in the normal controls, but in the patients group. Conclusion These results indicate that 5IA-SPECT has a possibility to evaluate the pathogenesis of cognitive impairment in Alzheimer’s disease and that it may add clinical usefulness of SPECT in the diagnosis of Alzheimer’s disease.

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Quantitative analysis of Benzodiazepine receptor by I-123 iomazenil SPECT in Alzheimer’s disease Kazuo Hashikawa*, Yujiro Seike†, Naohiko Oku†, Hiroshi Moriwaki†, Kazuki Fukuchi‡, Hidenao Fukuyama*, Tsunehiko Nishimura§ *Human Brain Research Center, Kyoto University Graduate School of Medicine †First Department of Internal Medicine, Osaka University Graduate School of Medicine ‡Division of Tracer Kinetics, Osaka University Graduate School of Medicine §Department of Radiology, Kyoto Prefectural University Objective The central type of benzodiazepine receptor (BZR) was coupled with GABA receptors in cortices and their density might be proportional to neuronal density. The purpose of this study was to measure binding potential (BP) of BZR quantitatively by using I-123 iomazenil SPECT and to evaluate its significance in Alzheimer’s disease. Subjects and Methods Eight patients with probable Alzheimer’s disease, who were diagnosed by NINCDS-ADRDA criteria, were studied. The average age was 57.5⫾2.3(mean⫾sd) years old. The mean score of MMSE(/30) was 20.4⫾1.7 and that of RCPM(/36) was 19.9⫾6.1. For control, five male CVD patients (56.4⫾3.3 years old) were studied, who had silent lacunars in MRI, but had no abnormal findings in neurological, cognitive and CBF studies. Dynamic SPECT acquisition was performed after an injection of 165 MBq of I-123 iomazenil (Nihon Medi-physics). At same time, the arterial samplings were performed. The values of K1 and BP (binding potential) were calculated on 3-compartment model by using chloroform fraction of arterial plasma as an input function. Moreover, we evaluated the late images of I-123 iomazenil, those were acquired at 3 hrs after an injection, in comparison with Tc-99m HMPAO SPECT images visually. Results K1 and BP of Alzheimer’s patients (K1 0.105⫾0.005 ml/min/ml, BP 33.2⫾2.0 ml/ml) were lower significantly than those of control (K1 0.123⫾0.013, p⬍0.01; BP 46.6⫾2.4, p⬍0.0001), respectively in the parietal lobe. Compared with Tc-99m HMPAO images, late images of I-123 iomazenil showed more severe and extensive involved areas. Conclusion I-123 iomazenil SPECT might provide more sensitive information than perfusion SPECT in Alzheimer’s disease.

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

POSTERS - SESSION II

Altered 5HT-1A Binding In Major Depression: An [11C]WAY100635 PET Study J. John Mann*†‡, Maria A. Oquendo*†, Norman Simpson*‡, Yiyuan Huang‡, Ronald Van Heertum*‡, Victoria Arango*, Ramin V. Parsey*† *Department of Neuroscience, New York State Psychiatric Institute †Department of Psychiatry, Columbia University ‡Department of Analytical Psychopharmacology, New York State Psychiatric Institute Introduction Two studies (Drevets et. al 1999, Sargent et al 2000) have demonstrated lower 5-HT1A binding in depressed subjects using [11C]WAY100635 PET. Such an abnormality suggests a post-synaptic receptor deficiency model of serotonergic hypofunction in major depression. Quantification of binding in these studies was based on the specific to nonspecific equilibrium partition coefficient (V3? ⫽ k3/k4) derived using the simplified reference tissue model (SRTM). This method uses the cerebellum as the input function and is more susceptible to confounding effects when compared with full quantification using a three compartment kinetic model. We have found 3 compartment modeling to be the method of choice for this ligand in humans (Parsey et al. 2000). We therefore studied depressed patients with [11C]WAY100635 PET and an arterial input function. Methods 25 healthy volunteers and 24 subjects who met DSM-IV criteria for a major depressive episode were scanned in an ECAT EXACT HR⫹ camera for 110 minutes. All subjects were currently depressed and had been off or all medications for at least 2 weeks prior to the scan. All subjects had metabolite corrected arterial input functions. Regions of interest (ROIs) were drawn on individual MRIs except for the raphe ROI which was a fixed volume placed on each individual?s mean PET scan. Time activity curves were generated from MRI coregistered PET images. A three compartment kinetic model was used with the K1/k2 ratio in the region of interest constrained to the total distribution volume of the cerebellum. The primary outcome measure was binding potential calculated directly as K1*k2/k3*k4. Results No significant differences were detected between the healthy volunteers and patients in age (40.3 ⫾ 15.3 vs. 40.2 ⫾ 11.4), % female (48% vs. 58%), injected mass (8.2 ⫾ 5.1 nmole vs. 7.1 ⫾ 3.6 nmole), or clearance of the compound (141 ⫾ 38 L/hr vs. 155 ⫾ 29 L/hr). Subjects had significantly lower free plasma (f1) tracer compared with controls (6.8 ⫾ 3.0% v. 9.4 ⫾ 2.3%, p ⫽ 0.001). Therefore in all subsequent analyses we covaried for inverse f1. Elsewhere we have reported a significantly higher BP in females compared with males and no age related effect in any brain region. We found significantly lower 5-HT1A binding potential in the depressed group compared to the control group in all brain regions when covarying for inverse f1 and sex. This was demonstrated by principal component analysis. 76% of the cumulative variance in the ROI binding potential is explained by the first principal component, 89% by the first and second, and 96% by the first three components combined. MANOVA on the first three principal components with sex and the inverse of the free fraction as covariates, showed a significant diagnosis effect (p⫽0.0485). Post-hoc analysis confirmed significantly lower binding potential in the anterior cingulate cortex, cingulate cortex, and medial prefrontal cortex. Conclusions We have demonstrated lower 5-HT1A binding potential in depressed subjects using [11C]WAY100635 when accounting for sex and free fraction of the tracer. Our method rules out potential artifacts of a reference tissue method as an explanation of the results. Future studies need to determine the reason for lower 5-HT1A binding and the effect of antidepressant treatment and clinical recovery on binding. PHS grant MH40965, MH62185, and NARSAD.

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

POSTERS - SESSION II

Effects of Sex, Age, and Aggressive Traits in Man on Brain Serotonin 5-HT1A Receptor Binding Potential using [11C]WAY-100635 Ramin V. Parsey*†, Maria A. Oquendo*†, Norman Simpson*‡, Ronald Van Heertum*‡, Victoria Arango*†§, J. John Mann*†‡ *Department of Neuroscience, New York State Psychiatric Institute †Department of Psychiatry, Columbia University ‡Department of Radiology, Columbia University §Department of Anatomy and Cell Biology, Columbia University Introduction Serotonin (5-HT) 1A receptors have been implicated in a variety of conditions including, depression, suicidal behavior, and aggression. Postmortem brain studies and in vivo imaging studies report a variety of age and sex effects on brain 5-HT1A binding. Behavioral data from 5-HT1A specific pharmacological challenges suggest a role for the 5-HT1A receptor in aggression. The goal of the present study was to determine age, sex, and aggression effects on 5-HT1A binding potential (BP) in vivo using positron emission tomography (PET) and the high affinity 5-HT1A antagonist [11C]WAY-100635. Methods We studied twelve healthy females (age 41.0 ⫾ 15.7) and 13 healthy males (age 39.6 ⫾ 15.5). Axis I psychopathology was ruled out using a SCID-NP structured clinical interview. Measures of lifetime aggression were obtained using the Brown Goodwin Aggression History Scale. We computed the correlation of binding with severity of life-time aggression. Subjects were scanned for 110 minutes after bolus injections of [C-11]WAY100,635 in 3D mode on an ECAT EXACT HR⫹ camera. A metabolite corrected plasma input function was obtained on each subject to permit full kinetic modeling. Binding potential corrected for the free fraction was determined directly from a three compartment kinetic model whose K1/k2 ratio was constrained on the total volume of distribution of the cerebellum. Regions of interest including the anterior cingulate cortex, cingulate cortex, hippocampus, amygdala, medial prefrontal cortex (PFC), and orbital PFC, were drawn on MRIs and transferred to coregistered PET data, with the exception of the dorsal/median raphe nuclei which was drawn directly on the PET scans. Results No significant correlation between age and BP was detected in any brain region. The free fraction was not significantly different between males and females. MANOVA of the first three principle components demonstrates a significantly higher BP in females compared to males (p⫽0.0127). Post-hoc tests demonstrate significant sex differences (p⬍0.05) in the following regions: dorsal raphe, amygdala, anterior cingulate, cingulate body, medial PFC, and orbital PFC (Figure). The cerebellar volume of distribution was also significantly higher in women. Because of this difference in cerebellar binding, if we use V3? (k3/k4) as the outcome measure the differences in regional binding are masked. There is a significant negative correlation between binding in several regions and aggression (range: orbital PFC R ⫽ -0.551, p ⫽ 0.005, hippocampus R ⫽ -0.328, p ⫽ 0.011). Males and females were not statistically significantly different in their aggression scores (13.6 ⫾ 14.2 females, 14.9 ⫾ 13.4 males, p ⫽ 0.41) Conclusions We have replicated our postmortem findings of higher 5-HT1A binding in healthy females compared to males. We did not detect an age dependent decrease in binding in males or females. BP and not V3? should be the outcome measure because of differences in binding in the cerebellum, the reference region. Since lower serotonin is associated with more aggression, perhaps the negative correlation of BP with aggression suggests an upregulation of 5HT1A receptors in response to lower levels of synaptic serotonin. These results suggest that careful attention to sex and the method used to quantify binding in clinical studies are warranted. NIMH grants MH40695 and MH62185, The Georgia & Glenn H. Greenberg Foundation, and NARSAD

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

POSTERS - SESSION II

Imaging the 5-HT 1A receptor in patients with schizophrenia and healthy controls using [ 11C]WAY-100635 Ilise Lombardo*, Mark Slifstein*, Catherine Fullerton*, Roberto Gil*, Dah-Ren Hwang*, John Martin*, William Frankle*, Christine Fairbank*, Ronald Van Heertum†, Jack Gorman*, Marc Laruelle*, Anissa Abi-Dargham* *Columbia University/NYSPI †Columbia University Objective Abnormalities in the density of the 5-HT 1A receptor for various brain regions have been reported in postmortem studies of schizophrenia. These include increases in cortical receptor density, and abnormal persistence of receptors in the cerebellar vermis. The goal of the present study was to examine the density of the 5-HT 1A receptor using the PET radiotracer [ 11C]WAY-100635 in patients with schizophrenia and healthy controls. Methods The sample consisted of 12 patients with schizophrenia, mean age 34 ⫹/- 7 y, and 12 matched healthy controls, mean age 35 ⫹/11 y. At the time of the study, 7 of the patients had been drug free for more than 3 weeks and 5 were drug naı¨ve. Subjects underwent a 90-minute PET study using the ECAT EXACT HR⫹ after [ 11C]WAY-100635 injection. Regions of interest (ROIs) were drawn on the co-registered MRI. ROIs included dorsolateral prefrontal cortex, medial cortex, orbital cortex, anterior cingulate, temporal cortex, insula, parietal cortex, occipital cortex, medial temporal cortex, amygdala, hippocampus, striatum, thalamus, cerebellar vermis and dorsal raphe. Regional distribution volumes (V T) were derived by kinetic analysis (2 tissue compartment model) using the arterial input function. Binding potential (BP) was calculated as the difference between V T in the ROI and V T in the cerebellar lobes. V⬙3 was calculated as BP/V 2. Results A significant decrease in [ 11C]WAY-100635 BP and V⬙3 was observed in the anterior cingulate in patients with schizophrenia compared to controls. No between-group differences were found in other regions examined, including the cerebellar vermis. There was no correlation between either BP or V⬙3 and severity of clinical symptoms. Conclusion This study suggests that schizophrenia might be associated with a significant decrease in 5-HT 1A receptors in anterior cingulate. This finding if replicated, suggests a potential abnormality that might be alleviated by the 5-HT 1A receptor agonism property of clozapine. On the other hand, this study failed to confirm alterations in receptor density of the cortex and cerebellum previously reported in postmortem samples.

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NeuroImage 16, Number 3, July 2002, Part 2 of 2 Parts

POSTERS - SESSION II

Patients with Obsessive-Compulsive Disorder Have Increased 5-HT 2A Receptor Binding in the Right Caudate Nucleus Karen Husted Adams*, Elsebeth Steno Hansen†, Lars Hagemann Pinborg*, Steen G. Hasselbalch*, Claus Svarer*, Søren Holm‡, Tom G. Bolwig†, Gitte Moos Knudsen* *Neurobiology Research Unit, University Hospital of Copenhagen, Denmark †The Dept. of Psychiatry, University Hospital of Copenhagen, Denmark ‡PET and Cyclotron Init, University Hospital of Copenhagen, Denmark Introduction Obsessive-Compulsive Disorder (OCD) is a psychiatric anxiety disorder of unknown cause. However, the pharmacological effect of the Selective Serotonin Re-uptake Inhibitors in the treatment of OCD suggests that it is caused by a dysfunction in the serotonin system. Furthermore, studies of OCD patients showing abnormal cerebral blood flow and metabolism along specific frontosubcortical brain circuits suggest that these regions are involved. This study aims to determine whether the 5-HT 2A receptor binding is affected in OCD patients as compared to healthy volunteers. Subjects and Method Thirteen OCD patients and 13 age and gender matched volunteers were recruited. Subjects underwent a Magnetic Resonance Imaging (MRI) scan and a Positron Emission Tomography (PET) scan. For PET, [ 18F]-altanserin (3.7 MBq/kg) was administered as a bolus injection followed by continuous infusion (B/I 1.75 hrs). Dynamic PET imaging was initiated 120 mins after administration of [ 18F]-altanserin. Venous blood samples were drawn and plasma samples were counted in a well counter and analysed for radioactive metabolites. PET and MRI images were coregisterred, and Regions of Interest (ROI) defined on MRI images were transferred to PET images. Time-activity curves were generated for the PET images and Volume of Distribution (DV’3 ⫽ (C ROI - C reference )/C plasma) were calculated for all ROI’s using cerebellum as a reference. Results DV’3 was calculated for: orbito-frontal cortex, ventral lateral frontal cortex, hippocampus, cingulate, insula, caudate nucleus, lentiformis nucleus, thalamus, temporal cortex, parietal cortex, and dorsal lateral prefrontal cortex. The DV’3 was on average 13% higher for all ROI’s in OCD patients. Furthermore, when comparing specific frontosubcortical brain regions, DV’3 is significantly higher for the right caudate nucleus in OCD patients (DV’3: 0.28 ⫾ 0.16) when compared to healthy volunteers (DV’3: 0.18 ⫾ 0.12) (paired t-test, P⬍0.05). Conclusion In conclusion we find that OCD patients show an increased 5-HT 2A receptor binding in the right caudate nucleus. This is in accordance with other studies indicating this region as being specifically affected in OCD.

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