Brain Research 1054 (2005) 55 – 60 www.elsevier.com/locate/brainres
Research Report
Aromatic l-amino acid decarboxylase turnover in vivo in rhesus macaque striatum: A microPET study O.T. DeJesusa,*, L.G. Floresa, D. Murali a, A.K. Converseb, R.M. Bartlett a, T.E. Barnhart a, T.R. Oakes b, R.J. Nickles a a
Medical Physics, University of Wisconsin Medical School, 1530 Medical Sciences Center, 1300 University Avenue, Madison, WI 53706, USA b Keck Imaging Laboratory, Waisman Center, University of Wisconsin, Madison, WI 53706, USA Accepted 25 June 2005 Available online 1 August 2005
Abstract The aromatic l-amino acid decarboxylase (AAAD) is involved in the de novo synthesis of dopamine, a neurotransmitter crucial in cognitive, neurobehavioral and motor functions. The goal of this study was to assess the in vivo turnover rate of AAAD enzyme protein in the rhesus macaque striatum by monitoring, using microPET imaging with the tracer [18F]fluoro-m-tyrosine (FMT), the recovery of enzyme activity after suicide inhibition. Results showed the AAAD turnover half-life to be about 86 h while total recovery was estimated to be 16 days after complete inhibition. Despite this relatively slow AAAD recovery, the animals displayed normal movement and behavior within 24 h. Based on the PET results, at 24 h, the animals have recovered about 20% of normal AAAD function. These findings show that normal movement and behavior do not depend on complete recovery of AAAD function but likely on pre-synaptic and post-synaptic compensatory mechanisms. D 2005 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Monoamine synthesis Keywords: Aromatic l-amino acid decarboxylase; Protein turnover; MicroPET imaging; Parkinson’s disease
1. Introduction The aromatic l-amino acid decarboxylase (AAAD) (EC 4.1.1.28) is the second enzyme in the biosynthetic pathway to important monoamine neurotransmitters—dopamine (DA), norepinephrine (NE) and serotonin (5-hydroxytryptamine or 5 HT). AAAD converts l-DOPA to DA which in turn is converted in NE neurons by dopamine h-hydroxylase (DhH) to NE while 5-hydroxytryptophan is converted by AAAD to 5 HT. Because the AAAD enzymic Km is higher than the normal levels of cerebral l-DOPA, conversion of l-DOPA to dopamine is rapid and has led many to believe that AAAD is not involved in dopamine
* Corresponding author. Fax: +1 608 262 2413. E-mail address:
[email protected] (O.T. DeJesus). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.06.086
regulation. However, there is now accumulating evidence that pre- and post-synaptic mechanisms alter AAAD activity via regulatory processes [6,29]. Parkinson’s disease (PD) is a movement disorder characterized by a dramatic reduction in dopamine levels in the terminals due to degeneration of nigrostriatal dopamine neurons. Since its introduction in 1967 [7], dopamine replacement using l-DOPA has remained to be the most effective therapy to ameliorate PD symptoms. Thus, AAAD is thought to play the important role as ratelimiting step in l-DOPA therapy in PD. Because changes in AAAD activity have clinical consequences [21], better understanding of AAAD function is needed. One important aspect of AAAD function which needs elucidation is its turnover rate in vivo. In this study, AAAD turnover rate in normal non-human primate striatum was assessed in vivo by monitoring recovery of AAAD function after irreversible
56
O.T. DeJesus et al. / Brain Research 1054 (2005) 55 – 60
AAAD inhibition. This study involved the administration of a single dose of an AAAD suicide inhibitor, a-monofluoromethyl-dl-3,4-dihydroxyphenylalanine (FMDOPA) [14] followed by longitudinal assessment of striatal AAAD recovery by non-invasive microPET imaging using 6[18F]fluoro-meta-tyrosine (FMT), a selective imaging agent for estimating AAAD activity [8,9].
2. Materials and methods 2.1. Inhibitor synthesis and characterization FMDOPA was synthesized de novo for this study following the method of Bey et al. [3] as we previously described in the synthesis of a close analog, a-monofluoromethyl-m-hydroxyphenylalanine (FM-m-tyrosine, FMFmT) [19]. After purification, FMDOPA was authenticated by 13Cand 1H NMR spectroscopy and validated by in vitro studies using rat kidney and striatal homogenates. Consistent with the results of Bey at al. [4], our in vitro results showed that FMDOPA has IC50 value comparable to those of other AAAD inhibitors–carbidopa, benserazide and NSD 1015–in inhibiting AAAD activity in these rat tissue homogenates (unpublished results). 2.2. Radiotracer synthesis The PET tracer [18F]FMT was prepared by the method of Namavari et al. [20]. Briefly, to achieve regioselective electrophilic radiofluorination, [18F]F2, produced as previously described [24], was bubbled into a fluorodichloromethane solution of a protected stannylated m-tyrosine derivative purchased from ABX Biochemicals (Radeberg, Germany). After removal of SnF2 and other by-products using silica minicolumn, protective groups in the [18F]intermediate were removed by refluxing in 48% hydrobromic acid. The product [18F]FMT was purified using a semi-preparative HPLC (Alltech C18 column, 10 A, 250 10 mm) with a mobile phase which consisted of 0.02 M NaOAc pH 3.5 and made up with sufficient NaCl to be isotonic. The HPLC fraction containing the product was collected and sterilized by filtration through a 0.22 A filter into a vented sterile vial prior to administration into the subjects. 2.3. Animal imaging: PET and MRI studies Two male rhesus monkeys (designated as AT 97 and AU 18), both 7 years old and weighing 8 kg, were used in this study. The animals were anesthetized with ketamine (15 mg/ kg IM) to allow administration dl-FMDOPA (25 mg/kg) into the peritoneum. Both animals were scanned serially at different times ranging from 4 h up to 8 days after FMDOPA injection. The animals were fasted overnight before each PET study. On the day of the PET study,
anesthesia was initially induced with ketamine (15 mg/kg IM) to allow the transport of the animals to the Keck MicroPET Laboratory at the University of Wisconsin Waisman Center. Anesthesia during the scan was maintained with isoflurane (1% – 2% in oxygen). The protocol used in these studies was approved by the UW Animal Care and Use Committee in compliance with NIH regulations on the use of non-human primates in research. The microPET P4 scanner used in this study is a commercial dedicated small animal research scanner (22 cm bore) from Concorde Microsystems (Knoxville, TN). The calculated image resolution, using filtered back projection reconstruction, in 8 cm axial 19 cm transaxial field of view (FOV), is 2 2 2 mm3 with 2% sensitivity at center [27]. The anesthesized animal was positioned prone in the scanner bed with its head comfortably immobilized in a stereotactic holder (with ear, mouth and eye orbit bars) orienting the brain such that the PET slices paralleled the orbitomeatal (OM) line. Heart rate, SPO2, respiratory rate and temperature were monitored throughout the scan and body temperature was controlled using a loose-fitting wrap blowing warm air around the animal (Bair Hugger, Arizant Healthcare, Eden Prairie, MN). A 15-min transmission scan using a 68Ge point source was performed to verify proper positioning and for use in post-acquisition correction for photon attenuation. Data are recorded in list mode, permitting maximum post-analysis flexibility in time averaging. A 90-min dynamic 3D acquisition was begun simultaneously with the intravenous administration of 3.5–5.4 mCi [18F]FMT. Each animal had previously undergone magnetic resonance imaging (MRI) on an Advantx 1.5 T instrument (General Electric Medical Systems, Waukesha, WI) under ketamine sedation. MR imaging included a spoiled gradient-echo (SPGR) sequence, a 3D acquisition reconstructed into contiguous 1.3-mm coronal slices providing good differentiation between gray matter and white matter. 2.4. PET image and region of interest analysis After the scan, the list mode data were binned into sinograms with 20 frames (5 1 min, 5 2 min, 5 5 min and 5 10 min) and corrected for dead time and random coincidences. Images were then reconstructed with Concorde Microsystems proprietary software using OSEM, correcting for detector sensitivity and attenuation. The images were converted to Analyze format using an in-house imaging software program called SPAMALIZE [22]. Images were then smoothed with a 3 mm Gaussian filter in Spm99. The PET image was manually co-registered to each animal’s anatomical and stereotaxic MRI T1 weighted image using SPAMALIZE and a region of interest (ROI) approach was used. Striatal and cerebellar regions were identified and drawn on the MRI T1 weighed images. The ROIs were then applied to the co-registered PET images for generating time activity curves (TACs) for the subsequent Patlak analysis [23]. The Patlak Ki values obtained were
O.T. DeJesus et al. / Brain Research 1054 (2005) 55 – 60
57
Fig. 1. Time course of FMT PET images of monkey 1 (AU18) Showing recovery of AAAD Activity. Pairs of transaxial (top) and coronal (bottom) views of the striatum at different times after FMDOPA injection are shown. The head of the caudate separate from the putamen is seen at 0, 97 and 192 h images in the coronal slices. The normalized images are displayed in a hot metal scale wherein black, red, orange, yellow and white correspond to increasing levels of radioactivity.
taken from a linear least squares fit of the time frames 15 – 20 corresponding to 41 –90 min of the scan. Patlak Ki values are the model outcome measures related to AAAD activity [9].
3. Results Both animals were scanned twice before FMDOPA treatment. Baseline Ki values of 0.01025 min 1 and 0.01030 min 1 for one animal (AU97) and 0.01112 min 1 and 0.00959 min 1 for the other (AU18) were obtained and the mean test – retest reproducibility is about 5.4%. Four hours after dl-FMDOPA treatment, microPET results
Fig. 2. Patlak plots calculated from PET data showing AAAD recovery for monkey 1 (AU18). The slope, Ki, is the striatal uptake constant (using cerebellum as reference tissue) for the irreversible tracer [18F]FMT and is a model outcome measure related to AAAD activity.
showed complete inhibition of [18F]FMT uptake in subject AU18. The PET images for subject AU18 (Fig. 1) show the time course of [18F]FMT uptake in the caudate-putamen regions in two views, transaxial (parallel to the OM line) and coronal. Patlak plots based on these images for monkey AU18 are shown in Fig. 2. The fractional recovery of AAAD activity in both animals is plotted in Fig. 3, where recovery in each animal is expressed as fraction of the measured Ki relative to the mean baseline Ki for that subject. Fitting the data in Fig. 3 to a simple exponential equation showed good correlation (r = 0.94) and the halflife of AAAD recovery is estimated to be 86 h after treatment. Further, in Fig. 3, about 80% recovery was observed within 192 h while complete AAAD recovery was estimated to take about 16 days after FMDOPA treatment. After recovering from anesthesia after dl-FMDOPA treatment, both animals were bradykinetic and assumed
Fig. 3. Fractional AAAD recovery, calculated as ratios of Ki(t) at time = t to baseline Ki(t = 0) for each animal, as a function of time after FMDOPA administration for the 2 animals (circles for AU-18 and squares for AT-97). Fitting the data to a simple exponential show a half-life of recovery of 86.4 h (r = 0.94).
58
O.T. DeJesus et al. / Brain Research 1054 (2005) 55 – 60
crouched postures but were able to feed. However, both animals appeared normal in their behavior and movement within 24 h.
4. Discussion The use of PET imaging to assess enzyme turnover in vivo in monkeys was first reported by Arnett et al. [2] in the study of MAO B turnover by irreversible inhibition with ldeprenyl, a selective MAO B suicide inhibitor. This method was later extended to assessing MAO A and MAO B turnover in humans [11]. In the present study, a similar approach was used to assess the turnover rate of striatal AAAD enzyme in vivo in the primate brain after suicide inhibition. While the study of Arnett et al. [2] used the same compound, [11C]-l-deprenyl, both as PET imaging agent and as suicide inhibitor, in the present study, an AAAD substrate, [18F]FMT, was used as imaging agent but a different compound, FMDOPA, was used as suicide inhibitor. A subtle difference between these two approaches arises from the tracer kinetic properties of the imaging agents utilized, with each assessing a different aspect of enzyme function. The tracer kinetics measured with covalently bound [11C]-l-deprenyl reflects enzyme protein levels while the tracer kinetics of a substrate such as [18F]FMT, wherein the [18F]-labeled product formed is essentially trapped, measures enzyme activity. The mechanism for the suicide inhibition of AAAD by FMDOPA was proposed by Maycock et al. [18] to involve adduct-formation between the FMDOPA-derived inhibitor and the AAAD enzyme. Recent crystal structural analysis of AAAD by Burkhard et al. has provided additional insights into the mechanism of FMDOPA suicide inhibition [5]. AAAD is a tightly associated a2-dimer belonging to the afamily of pyridoxal 5V-phosphate (PLP)-dependent enzymes [1]. In the first step in enzyme action, the co-factor PLP is anchored in the active site of AAAD by an extended hydrogen bond network through its phosphate group while the protonated pyridine nitrogen stabilizes the carbanionic Schiff base intermediate formed with the substrate [5]. FMDOPA, like l-DOPA, is thought to form this Schiff base intermediate with the enzyme-bound PLP co-factor, with the carboxylate group forming hydrogen bonds with His 192 in the enzyme active site [5]. Elimination of carbon dioxide yields a decarboxylated FMDOPA quinoid intermediate which, instead of being released as FMdopamine analogous to dopamine release after l-DOPA decarboxylation, undergoes fluoride elimination leaving a reactive alkylating electrophile which then forms a covalent bond at the enzyme’s active site. This alkylation reaction essentially blockades the AAAD active site thereby incapacitating the enzyme. In order to recover decarboxylation function, synthesis of new AAAD protein in the dopamine cell bodies becomes necessary. New protein synthesis requires gene transcription of new AAAD mRNA followed by
translation into new AAAD enzyme proteins as demonstrated by Li et al. in vitro in PC12 cells using the dopamine inhibitor NSD 1015 [17]. New AAAD enzyme protein synthesized in the nigral cell bodies is then delivered by axoplasmic transport to the striatal terminals [12]. Monitoring recovery of striatal enzyme activity using a substrate as tracer allows the estimation of the combined rates of AAAD protein synthesis and axonal transport. The inhibitor dose used in this study was selected based on the report by Jung et al. evaluating the effects of dlFMDOPA in mice [14]. In mice, 4 h after the administration of 25 mg/kg FMDOPA, AAAD activity in the kidney and heart was almost completely inhibited while whole brain AAAD was inhibited about 28% of control [14]. In contrast, after administration of 25 mg/kg FMDOPA, both macaques in this microPET study showed complete striatal AAAD inhibition at 4 and 26 h, respectively. Although peripheral AAAD activity was not assessed in this study, evidence for significant peripheral AAAD inhibition, which would slow [18F]FMT plasma clearance and allow more unmetabolized [18F]FMT to enter the brain, can be inferred from the normalized PET images shown in Fig. 1 wherein images obtained at 4 h and 49 h after FMDOPA treatment showed higher background radioactivity compared to the images at the three other time points. Consistent with the notion that AAAD protein synthesis occurs in the nigral dopamine cell bodies, Gardner and Richards found that AAAD recovery after FMDOPA inhibition in synaptosomal fractions (P2 fraction) in the mouse brain lagged the recovery in sub-cellular fractions rich with cell bodies (S2 fraction) by about 2 days [12]. Based on axonal length consistent with the mouse brain, axonal transport rate of 1– 6 mm/day was estimated [12]. Although, we can identify the substantia nigra in the PET images and calculate nigral Ki values, the sample of two animals did not provide sufficient power to obtain statistically significant measure of AAAD recovery which would have been useful in estimating axoplasmic transport rate under these conditions. Although complete AAAD recovery was not observed until an estimated 16 days after the total enzyme blockade by suicide inhibition (Fig. 3), both animals displayed normal movement and behavior within a day. This is not unexpected because of known plasticity of the dopamine system and suggests that compensatory mechanisms are involved [30]. In mice, 6 h after the highest single FMDOPA dose used (500 mg/kg) was given wherein complete AAAD inhibition is assumed, dopamine levels on a per gram whole brain basis dropped to less than 40% of control. Because of higher dopamine concentration in the striatal terminals, the drop in synaptic dopamine levels in the striatum is likely larger. In this study, based on the PETestimated half-life of AAAD recovery of 86 h, 24 h after treatment, one can estimate the recovered AAAD activity to be about 20% of control. The observed behavioral and motor recovery at this level of AAAD activity suggests that
O.T. DeJesus et al. / Brain Research 1054 (2005) 55 – 60
the dopamine levels under this condition are sufficient to allow normal function. It is likely that compensatory mechanisms similar to those reported in dopamine deficient mice wherein normal locomotor activity can be restored with striatal dopamine levels amounting to 9% of normal values [26] are involved. Reduced dopamine levels can trigger upregulation of the rate-limiting conversion of ptyrosine to l-DOPA by tyrosine hydroxylase (TH) by posttranslational changes, e.g., phosphorylation [10]. However, because of much reduced AAAD activity after FMDOPA inhibition, the rate-limiting step may have shifted from the TH step to the AAAD step. Another pre-synaptic compensatory response may be the downregulation of metabolism involving re-uptake by the dopamine transporter (DAT) similar to that seen in pre-clinical PD [16]. On the postsynaptic side, dopamine receptors may become supersensitive because of reduced dopamine levels as first reported in 6-OHDA-lesioned animals [28] and recently found in the TH-deficient dopamine depleted mice [15]. Such supersensitivity is likely related to increased levels of the high affinity states of D2 receptors [25]. It is interesting to note that the symptomatic threshold for PD is associated with declines in striatal dopamine levels in the order of about 20– 40% of normal levels [13]. A recent PET study with [18F]FDOPA estimated the PD symptomatic threshold to be at 47– 62% of normal putaminal uptake values [16]. The recovery of both animals within 24 h after complete AAAD inhibition may therefore be similar to the pre-clinical stage of PD but since normal brains are involved in this study the observed faster motor and behavioral recovery is a reasonable outcome. A third animal given the same dose of FMDOPA in the afternoon and whose PET scan was planned for the following day was found to be surprisingly alert and displayed normal movement and behavior the next morning. The seeming lack of FMDOPA effect on this animal prompted the misguided decision to forego its PET study. In summary, the in vivo turnover rate of AAAD enzyme protein in the rhesus macaque striatum was assessed by monitoring recovery of enzyme activity after suicide inhibition by microPET imaging using the tracer [18F]FMT. Results showed that the AAAD turnover half-life is about 86 h and total recovery is estimated to be within 16 days after complete inhibition. However, normal movement and behavior were observed within 24 h at which time AAAD function was shown by the PET data to be about 20% of normal levels. This suggests that pre- and post-synaptic compensatory mechanisms allow recovery of normal movement and behavior earlier than recovery of AAAD function. FMDOPA inhibition and microPET imaging, using other pre- and postsynaptic dopamine imaging agents, in non-human primates may be useful in elucidating these compensatory mechanisms. Furthermore, a possible use of AAAD suicide inhibition is in generating a reversible non-human primate model of pre-symptomatic Parkinson’s disease wherein the consequences of dopamine depletion on dopamine receptors
59
and transporters and other dopamine-interacting neurotransmitter systems such as serotonin and GABA, may be investigated in vivo longitudinally using the appropriate PET imaging agents.
Acknowledgment The support of NIH Grant NS 26621 is gratefully acknowledged.
References [1] F.W. Alexander, E. Sandmeier, P.K. Mehta, P. Christen, Evolutionary relationships among pyridoxal-5V-phosphate-dependent enzymes. Regio-specific alpha, beta and gamma families, Eur. J. Biochem. 219 (1994) 953 – 960. [2] C.D. Arnett, J.S. Fowler, R.R. MacGregor, D.J. Schlyer, A.P. Wolf, B. Langstrom, C. Halldin, Turnover of brain monoamine oxidase measured in vivo by positron emission tomography using l[11C]deprenyl, J. Neurochem. 49 (1987) 522 – 527. [3] P. Bey, J.P. Vevert, V. Van Dorsselaer, M. Kolb, Direct synthesis of ahalogenomethyl-a-amino acids from the parent a-amino acids, J. Org. Chem. 44 (1979) 2732 – 2742. [4] P. Bey, M.J. Jung, J. Koch-Weser, M.G. Palfreyman, A. Sjoerdsma, J. Wagner, M. Zraika, Further studies on the inhibition of monoamine synthesis by a-monofluoromethyldopa, Br. J. Pharmacol. 70 (1980) 571 – 576. [5] P. Burkhard, P. Dominici, C. Borri-Voltattorni, J.N. Jansonius, V.N. Malashkevich, Structural insight into Parkinson’s disease treatment from drug-inhibited DOPA decarboxylase, Nat. Struct. Biol. 8 (2001) 963 – 967. [6] S. Cho, N.H. Neff, M. Hadjiconstantinou, Regulation of tyrosine hydroxylase and aromatic l-amino acid decarboxylase by dopaminergic drugs, Eur. J. Pharmacol. 323 (1997) 149 – 157. [7] G.C. Cotzias, M.H. Van Woert, L.M. Schiffer, Aromatic amino acids and modification of parkinsonism, N. Engl. J. Med. 276 (1967) 374 – 379. [8] O.T. DeJesus, C.J. Endres, S.E. Shelton, R.J. Nickles, J.E. Holden, Evaluation of fluorinated m-tyrosine analogs as PET imaging agents of dopamine nerve terminals: comparison with 6-fluoroDOPA, J. Nucl. Med. 38 (1997) 630 – 636. [9] O.T. DeJesus, C.J. Endres, S.E. Shelton, R.J. Nickles, J.E. Holden, Noninvasive assessment of aromatic l-amino acid decarboxylase activity in aging rhesus monkey brain in vivo, Synapse 39 (2001) 58 – 63. [10] P.R. Dunkley, L. Bobrovskaya, M.E. Graham, E.I. von NagyFelsobuki, P.W. Dickson, Tyrosine hydroxylase phosphorylation: regulation and consequences, J. Neurochem. 91 (2004) 1025 – 1043. [11] J.S. Fowler, R.R. MacGregor, A.P. Wolf, C.D. Arnett, S.L. Dewey, D. Schlyer, D. Christman, J. Logan, M. Smith, H. Sachs, et al., Mapping human brain monoamine oxidase A and B with 11C-labeled suicide inactivators and PET, Science 235 (1987) 481 – 485. [12] C.R. Gardner CR, M.H. Richards, Use of d,l-alpha-monofluoromethyldopa to distinguish subcellular pools of aromatic amino acid decarboxylase in mouse brain, Brain Res. 216 (1981) 291 – 298. [13] O. Hornykiewicz, Biochemical aspects of Parkinson’s disease, Neurology 51 (1998) S2 – S9. [14] M.J. Jung, M.G. Palfreyman, J. Wagner, P. Bey, G. Ribereau-Gayon, M. Zraika, J. Koch-Weser, Inhibition of monoamine synthesis by irreversible blockade of aromatic amino acid decarboxylase with alpha-monofluoromethyldopa, Life Sci. 24 (1979) 1037 – 1042. [15] D.S. Kim, M.S. Szczypka, R.D. Palmiter, Dopamine-deficient mice
60
[16]
[17]
[18]
[19]
[20]
[21]
[22] [23]
O.T. DeJesus et al. / Brain Research 1054 (2005) 55 – 60 are hypersensitive to dopamine receptor agonists, J. Neurosci. 20 (2000) 4013 – 4405. C.S. Lee, A. Samii, V. Sossi, T.J. Ruth, M. Schulzer, J.E. Holden, J. Wudel, P.K. Pal, R. de la Fuente-Fernandez, D.B. Calne, A.J. Stoessl, In vivo positron emission tomographic evidence for compensatory changes in presynaptic dopaminergic nerve terminals in Parkinson’s disease, Ann. Neurol. 47 (2000) 493 – 503. X.M. Li, A.V. Juorio, A.A. Boulton, NSD-1015 alters the gene expression of aromatic l-amino acid decarboxylase in rat PC12 pheochromocytoma cells, Neurochem. Res. 18 (1993) 915 – 919. A.L. Maycock, S.D. Aster, A.A. Patchett, Inactivation of 3-(3,4 dihydroxyphenyl) alanine decarboxylase by 2-(fluoromethyl)-3-(3,4dihydroxyphenyl)alanine, Biochemistry 19 (1980) 709 – 718. D. Murali, L.G. Flores, A.D. Roberts, R.J. Nickles, O.T. DeJesus, Aromatic l-amino acid decarboxylase (AAAD) inhibitors as carcinoid tumor-imaging agents: synthesis of 18F-labeled alphafluoromethyl-6-fluoro-m-tyrosine (FM-6-FmT), Appl. Radiat. Isot. 59 (2003) 237 – 243. M. Namavari, N. Satyamurthy, M.E. Phelps, J.R. Barrio, Synthesis of 6-[18F] and 4-[18F]fluoro-l-m-tyrosines via regioselective radiofluorodestannylation, Appl. Radiat. Isot. 44 (1993) 527 – 536. N.H. Neff, M. Hadjiconstantinou, Aromatic l-amino acid decarboxylase modulation and Parkinson’s disease, Prog. Brain. Res. 106 (1995) 91 – 97. T.R. Oakes, (http://www.keck.waisman.wisc.edu/oakes/spam/spam_frames. htm). C.S. Patlak, R.G. Blasberg, Graphical evaluation of blood-to-brain
[24] [25]
[26]
[27]
[28]
[29]
[30]
transfer constants from multiple-time uptake data. Generalizations, J. Cereb. Blood Flow Metab. 5 (1985) 584 – 590. A.D. Roberts, T.R. Oakes, R.J. Nickles, Development of an improved target for [18F]F2 production, Appl. Radiat. Isot. 46 (1995) 87 – 91. P. Seeman, D. Weinshenker, R. Quirion, L.K. Srivastava, S.K. Bhardwaj, D.K. Grandy, R.T. Premont, T.D. Sotnikova, P. Boksa, M. El-Ghundi, B.F. O’Dowd, S.R. George, M.L. Perreault, P.T. Mannisto, S. Robinson, R.D. Palmiter, T. Tallerico, Dopamine supersensitivity correlates with D2High states, implying many paths to psychosis, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 3513 – 3518. M.S. Szczypka, M.A. Rainey, D.S. Kim, W.A. Alaynick, B.T. Marck, A.M. Matsumoto, R.D. Palmiter, Feeding behavior in dopaminedeficient mice, Proc. Natl. Acad. Sci. U. S. A. (1999) 12138 – 12143. C. Tai, A. Chatziioannou, S. Siegel, J. Young, D. Newport, R.N. Goble, R.E. Nutt, S.R. Cherry, Performance evaluation of the microPET P4: a PET system dedicated to animal imaging, Phys. Med. Biol. 46 (2001) 1845 – 1862. U. Ungerstedt, Postsynaptic supersensitivity after 6-hydroxy-dopamine induced degeneration of the nigro-striatal dopamine system, Acta Physiol. Scand., Suppl. 367 (1971) 69 – 93. M.Y. Zhu, A.V. Juorio, I.A. Paterson, A.A. Boulton, Regulation of striatal aromatic l-amino acid decarboxylase: effects of blockade or activation of dopamine receptors, Eur. J. Pharmacol. 238 (1993) 157 – 164. M.J. Zigmond, Do compensatory processes underlie the preclinical phase of neurodegenerative disease? Insights from an animal model of parkinsonism, Neurobiol. Dis. 4 (1997) 247 – 253.
