Nigrostriatal Reduction of Aromatic L-Amino Acid Decarboxylase Activity in MPTP-Treated Squirrel Monkeys: In Vivo and In Vitro Investigations

Nigrostriatal Reduction of AromaticL-Amino AcidDecarboxylase Activity in MPTP-Treated Squirrel Monkeys:

In Vivo and In Vitro Investigations

*R. E. Yee, *†S.-C. Huang, *D. B. Stout, ‡I. Irwin, †K. Shoghi-Jadid, ‡D. M. Togaski,‡L. E. DeLanney, ‡J. W. Langston, *N. Satyamurthy, §K. F. Farahani,

*M. E. Phelps, and *J. R. Barrio

Departments of*Molecular and Medical Pharmacology,†Biomathematics, and§Radiological Sciences, UCLA School ofMedicine, Los Angeles; and‡The Parkinson’s Institute, Sunnyvale, California, U.S.A.

Abstract: Aromatic L-amino acid decarboxylase (AAAD)activity was examined in vivo with positron emission to-mography (PET) using 6-[18F]fluoro-L-DOPA (FDOPA) insquirrel monkeys lesioned with graded doses of theneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP). In vitro biochemical determinations of AAAD ac-tivity in caudate, putamen, substantia nigra, and nucleusaccumbens were performed in the same animals to es-tablish a direct comparison of in vivo and in vitro mea-surements. In vivo and in vitro AAAD activities in caudate/putamen were substantially reduced in animals treatedwith the highest dose of MPTP (2.0 mg/kg). The percentchange in the striatal FDOPA uptake (Ki) and decarbox-ylation rate constant (k3) values resulting from MPTPtreatment showed highly significant correlations with invitro-determined AAAD activities. However, decarboxyl-ase rates within individual animals presented as ;10-folddifference between in vivo and in vitro values. Lower invivo k3 measurements may be attributed to several pos-sibilities, including transport restrictions limiting substrateavailability to AAAD within the neuron. In addition, reduc-tions in AAAD activity in the substantia nigra did notparallel reductions in AAAD activity within the striatum,supporting the notion of a nonlinear relationship betweennigrostriatal cell degeneration and terminal losses. Thiswork further explores the role of AAAD in Parkinson’sdisease, a more important factor than previously thought.Key Words: Aromatic L-amino acid decarboxylase—6-[18F]Fluoro-L-DOPA—Squirrel monkeys—1-Methyl-4-phe-nyl-1,2,3,6-tetrahydropyridine.J. Neurochem. 74, 1147–1157 (2000).

Akinesia and the other motor symptoms of Parkin-son’s disease and 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-induced parkinsonian syndrome areboth attributed to central dopamine deficiency (Scher-man et al., 1989; Forno et al., 1993). Two enzymes aresequentially involved in the neuronal conversion of ty-rosine to dopamine: tyrosine hydroxylase (EC 1.14.15.2)

and aromaticL-amino acid decarboxylase (AAAD; EC4.1.1.28). Because tyrosine hydroxylase is considered tobe rate-limiting (Joh et al., 1986), the focus of mostinvestigations has been on this enzyme, with relativelylittle attention being placed on AAAD. However, be-cause AAAD is the enzyme involved in the final step ofthe synthesis of dopamine in the CNS, maintenance ofdopamine levels in the brain is at least partially depen-dent on the ability of AAAD to convertL-3,4-dihydroxy-phenylalanine (L-DOPA) to dopamine.

The biochemical integrity of the dopaminergic systemusing positron emission tomography (PET) has beenassessed in vivo with 6-[18F]fluoro-L-DOPA (FDOPA) inParkinson’s disease patients and MPTP-treated monkeys(Barrio et al., 1990; Leenders et al., 1990; Gjedde et al.,1993; Pate et al., 1993; Kuwabara et al., 1995; Ishikawaet al., 1996; Cumming and Gjedde, 1998). This determi-nation is based on the AAAD-mediated conversion ofFDOPA to 6-fluorodopamine, which accumulates withinvesicles, therefore providing an assessment of AAAD-mediated decarboxylation in dopamine neurons (for re-view, see Barrio et al., 1997). No other direct measure-ments of striatal AAAD activity in Parkinson’s disease

Received August 23, 1999; revised manuscript received October 26,1999; accepted October 27, 1999.

Address correspondence and reprint requests to Dr. J. R. Barrio/Dr.S.-C. Huang at Department of Molecular and Medical Pharmacology,UCLA School of Medicine, B2-086A Center of the Health Sciences, LosAngeles, CA 90095-6948, U.S.A. E-mail: [email protected] or Dr.J. W. Langston at The Parkinson’s Institute, 1170 Morse Avenue, Sunny-vale, CA 94089-1605, U.S.A. E-mail: [email protected]

Abbreviations used:AAAD, aromaticL-amino acid decarboxylase; car-bidopa,L-a-hydrazino-a-methyl-b-(3,4-dihydroxyphenyl)propionic acid;L-DOPA, L-3,4-dihydroxyphenylalanine; FDOPA, 6-[18F]fluoro-L-3,4-di-hydroxyphenylalanine; FWHM, full width at half-maximum; LNAA,large neutral amino acid; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropy-ridine; MRI, magnetic resonance imaging; 3-OMFD, 3-O-methyl-6-[18F]fluoro-L-3,4-dihydroxyphenylalanine; PET, positron emission tomog-raphy; ROI, region of interest.

1147

Journal of NeurochemistryLippincott Williams & Wilkins, Inc., Philadelphia© 2000 International Society for Neurochemistry

or MPTP-induced parkinsonism in humans or primatesare available in the literature. This is remarkable, con-sidering that AAAD is the only enzyme responsible forthe conversion of exogenousL-DOPA in dopamine re-placement therapy and is required for alleviating thesymptoms of Parkinson’s disease (Muenter and Tyce,1971; Rossor et al., 1980). Therefore, understandingalterations in AAAD activity with Parkinson’s diseaseprogression is important therapeutically and requiresmuch further needed investigation.

The aims of this work are to address (a) the effects ofnigrostriatal lesioning on striatal AAAD activity, (b) therelationship between in vivo- and in vitro-determinedAAAD decarboxylation rates, and (c) whether the effectsof nigrostriatal lesioning as seen on AAAD activity areparalleled in both the substantia nigra and striatum. Toaccomplish these objectives, striatal AAAD activity wasdetermined in vivo with FDOPA-PET in MPTP-treatedGuyana squirrel monkeys, a model of Parkinson’s dis-ease (Langston et al., 1984), followed by subsequent invitro biochemical analysis.

MATERIALS AND METHODS

AnimalsEleven male and six female Guyana squirrel monkeys

(Saimiri sciureus), ranging in body weight from 620 to 980 g,were used in this work. All animals were purchased fromcommercial sources, quarantined, tested, and examined accord-ing to standard veterinary practices. Animals were housedindividually in standard stainless steel primate cages under a13-h light:11-h dark cycle. All animals were given free accessto water and daily diets of Teklad New World Monkey chowsupplemented with fresh fruits.

Study designAnimals were randomly placed into one of four treatment

groups. The groups were injected as follows: group I (n5 4;1.0 mg/kg MPTP), group II (n5 5; 1.5 mg/kg MPTP), groupIII (n 5 5; 2.0 mg/kg MPTP); and group IV (n5 3; saline).FDOPA PET and magnetic resonance imaging (MRI) scanningwere performed on groups I–III before MPTP treatment. Onemonth after scanning, animals were given a single subcutane-ous injection of 1.0, 1.5, or 2.0 mg/kg MPTP as previouslydescribed (Irwin et al., 1990). Three months after MPTP treat-ment, animals from groups I–III were rescanned using PETwith FDOPA. After the end of the fourth month, all animals(groups I–IV) were killed, and the brains were processed forbiochemical analyses. Animals and tissue samples were codedso that all investigators and analysts were blinded to the treat-ment conditions until all data were acquired. All procedureswere approved by the Parkinson’s Institute and the Universityof California at Los Angeles Animal Research Committees.

Quantitative in vivo determination with PETEmission tomography.Animals were scanned using a

FDOPA PET protocol as described in detail elsewhere (Stout,1999). Peripheral decarboxylation of FDOPA was minimizedby pretreating animals with 5–15 mg/kgL-a-hydrazino-a-methyl-b-(3,4-dihydroxyphenyl)propionic acid (carbidopa);30min before tracer injection (Hoffman et al., 1992; Chan et al.,1995). Animals were maintained under anesthesia with eitherpentobarbital or ketamine and midazolam as previously de-

scribed (Barrio et al., 1990; Stout, 1999). Scanning was per-formed on a Siemens ECAT 713 scanner with a 5-cm axial fieldof view for a 120-min session with interleaved bed positionsafter the first 30 min. After intravenous injection of 2.0 mCi/kgFDOPA (specific activity, 2–5 Ci/mmol), arterial blood sam-ples were drawn throughout the scan, so that the total bloodvolume withdrawn from each animal did not exceed 5 ml.Plasma time–activity curves of FDOPA and 3-O-methyl-FDOPA (3-OMFD) were constructed based on the total radio-activity determined in plasma from all blood samples taken(Huang et al., 1991b). The percentages of FDOPA and metab-olites were also determined from blood samples drawn andused to adjust plasma time–activity curves (Huang et al.,1991b) using previously reported methods (Melega et al.,1991a,b). In addition, plasma large neutral amino acid (LNAA)concentrations were determined from blood samples taken 7min after tracer injection as described in detail elsewhere (Keenet al., 1989).

Each animal was also scanned by MRI. Scanning was per-formed on a 1.5-T Vision Siemens Medical System. Protondensity andT2-weighted turbo spin echo (TSE) images wereobtained. The images were acquired with a 150-3 200-mmfield of view and a 2563 256 acquisition matrix, as previouslydescribed (Stout, 1999). The sequence resulted in 16 planes, 3mm thick. Animals were imaged in a plane parallel to theAC–PC line using scout midsagittal images as a reference.

Image processing.All PET images were reconstructed usingfiltered backprojection and measured attenuation correction.Dynamic images were reconstructed using a Hann filter (0.3Nyquist cutoff) with a resulted in-plane resolution of 5.5 mmfull width at half-maximum (FWHM). Summed images (30–120 min) were reconstructed using a Shepp 0.5 filter (4.7 mmFWHM).

The contours of the basal ganglia were more evident inimages obtained from the summation of the dynamic emissiontomography sequence. Therefore, striatal regions of interest(ROIs) were defined on lower noise summed images and thenoverlaid on dynamic images to compute average striatal ROIvalues on pre-MPTP animals. Owing to nigrostriatal damage,striatal ROI values on post-MPTP animals were computed byoverlaying pre-MPTP ROIs onto resliced post-MPTP dynamicimages after coregistration (Lin et al., 1996) of post-MPTPimages to pre-MPTP images. Because of the small size of thecaudate and putamen in the squirrel monkey, PET determina-tions are based on measurements for the entire striatum (Stout,1999). However, owing to the larger size of the putamenrelative to the caudate in tomographic cross-sections, the puta-men has, overall, a greater contribution to the PET slices.

Cerebellum was used as a reference region because of thenear absence of AAAD activity (Mackay et al., 1978), whichmeans that the presence of a specific binding compartment canbe neglected. Owing to poor anatomical definition of cerebel-lum regions in functional PET images, cerebellum ROIs weredetermined by locating the sagittal and coronal coordinates ofthe center of the cerebellum as defined by MRI and thenconstructing a circular ROI around the cerebellum with a 3-mmradius. The defined ROI was overlaid onto resliced PET dy-namic image to compute the average cerebellum ROI value.Dynamic images were resliced based on registration parametersderived from the coregistration of summed PET images tomagnetic resonance images (Lin et al., 1994). Pre- and post-MPTP data were resliced to MRI coordinates for the definitionof cerebellar ROIs but not for the determinations of striatalROIs.

J. Neurochem., Vol. 74, No. 3, 2000

1148 R. E. YEE ET AL.

Determination of in vivo kinetic rate constants.Parameterestimates were obtained by fitting the striatal tissue time–activity curves with a five-compartment model using plasmaFDOPA and 3-OMFD time–activity curves as input functions.Kinetic analysis procedures and model constraints were previ-ously described (Huang et al., 1991a,b). In this model, theforward and reverse transport rate constants of FDOPA acrossthe blood–brain barrier are represented byK1 andk2, respec-tively. Subsequent decarboxylation of FDOPA by striatalAAAD is described by the rate constantk3, whereas clearanceof decarboxylated metabolites is denoted byk4. Blood–brainbarrier transport of 3-OMFD, the major peripheral metaboliteof FDOPA formed in the presence of carbidopa (Hoffmanet al., 1992), is described by the rate constantsK5 andk6. Thedistribution volume of 3-OMFD in the striatum (K5/k6) wasconstrained to the cerebellum-to-plasma ratio (Cb/Cp) (Huanget al., 1998; Stout et al., 1998),k4 was set to a minimal valueof 0.000001, and a fixed blood volume of 5% was subtractedfrom striatal time–activity curves to account for intravascularradioactivity.

Owing to the competitive effects of LNAAs on blood–brainbarrier transport of FDOPA,K1 values were examined in rela-tion to plasma LNAA levels (Stout et al., 1998). A correlationof K1 5 0.073 2 7.46 3 1025 [LNAA] with R2 5 0.42resulted. Individually measuredK1 values were adjusted basedon the above relationship and an average plasma LNAA con-centration of 376.3mM using the methods previously reported(Stout et al., 1998), whereK1,adjusted 5 7.46 3 1025

(LNAA measured2 376.3)1 K1,measured.The parameterKi is defined as the overall FDOPA uptake

constant and represents the overall uptake and decarboxylationof FDOPA in the brain relative to plasma FDOPA levels.Ki

was calculated based on the following relationship ofKi

5 K1,adjustedp k3/(k2 1 k3) (Huang et al., 1991a).

Quantitative in vitro decarboxylase determinationDissection technique.Four months after MPTP administra-

tion, 1 month after PET scans, animals were killed by intrave-nous injection with a lethal dose of pentobarbital (100 mg/kg).This was in accordance with recommendations of the AmericanVeterinary Association. After each animal was killed, the brainwas rapidly removed and transferred to a glass plate on ice. Thebrain was then dissected into two hemispheres. In one hemi-sphere, the striatum was dissected as previously described(Irwin et al., 1990). In this approach, 2-mm-thick coronal slicesof the hemisphere were cut beginning at the anterior border ofthe head of the caudate. Six slices were obtained, with theanterior commissure appearing in the third and fourth slice. Thecaudate and putamen were dissected from each slice. Thenucleus accumbens was dissected from the slice immediatelyanterior to that containing the commissure. The midbrain wasisolated, and the substantia nigra was dissected. When dissec-tion was completed, tissue samples were immediately wrappedin foil, frozen on dry ice, and stored at280°C until analyzed.Biochemical analyses were performed on striatal tissue, fromthe slice containing the anterior commissure and the sliceimmediately anterior, from the nucleus accumbens and thesubstantia nigra.

AAAD assay.The dissected tissue samples were homoge-nized by sonication in 100 volumes of 50 mM sodium phos-phate (pH 7.4). Tissue homogenate (100ml) was preincubatedwith a 200-ml mixture of 50 mM sodium phosphate (pH 7.4),0.04 mM pyridoxyl phosphate, and 0.2 mM pargyline for 5 minat 37°C. The reaction was initiated by addition ofL-DOPA toa final concentration of 1 mM. Samples were further incubated

for 20 min at 37°C. The reaction was terminated by addition of20ml of concentrated perchloric acid. Samples were transferredto an ice bath for 5 min and then centrifuged. Supernatant fromeach sample was assayed by reversed-phase HPLC for dopa-mine using a modification (Irwin et al., 1990) of the methodreported by Kilpatrick et al. (1986). Protein concentration wasdetermined from the remaining pellet using a BCA ProteinReagent Assay Kit (Pierce, Rockford, IL, U.S.A.). AAADactivity was calculated as picomoles of dopamine formed perminute per milligram of protein.

Unit conversion of in vitro decarboxylation rates.The invitro-measured decarboxylations rates were converted to equiv-alent units used to measure in vivo decarboxylation rates toallow for more direct comparisons between the two. The invitro enzyme activity per gram of protein was multiplied by theprotein fraction of wet tissue, to calculate the activity per gramof tissue. The amount of dopamine produced per minute pergram of wet tissue was then divided by the concentration ofL-DOPA in the reaction to give the fraction ofL-DOPA usedper minute. Therefore, the in vitro decarboxylation rate, mea-sured in picomoles per minute per milligram of protein, wasconverted to the equivalent units of milliliters per minute pergram of tissue or per minute, based on the approximation of a1-ml volume per gram of tissue.

Amine determination.Dopamine concentrations were deter-mined by homogenization of caudate, putamen, and substantianigra tissue samples in 0.4M perchloric acid by sonication,followed by centrifugation. Supernatants were collected andassayed for dopamine concentrations by reversed-phase HPLCas described above. Protein concentration from the remainingpellet was determined as mentioned above.

Statistics.Data are reported for each individual animal orgrouped according to MPTP dose administered and representedas average6 SEM values. Animals served as their own controlfor PET-derived kinetic data by comparing kinetic parameterestimates before and after MPTP lesioning. A separate group ofsaline-injected animals served as controls for biochemical anal-ysis. To identify significant differences between treatmentgroups, data were subjected to a one-way ANOVA at a signif-icance level of 0.05. Linear regression analysis was performedto detect relationships between changes in kinetic parameterestimates to in vitro-determined decarboxylase activity. Thepattern of percent loss of parameter magnitudes after MPTPbetween brain regions was examined by testing whether theintercept of the regression line is significantly different from 0.This was performed by using anF test that compares theresidue sum of squares of the data around the regression line tothe residue sum of squares around a fitted line that goes throughthe origin (Keeping, 1995). A significant difference from 0 forthe intercept indicates a nonparallel relationship between thechanges of the two variables due to MPTP treatment.

RESULTS

The estimated striatal kinetic rate constants after ad-justments for the competitive effects of plasma LNAAsfor each animal before MPTP treatment and the corre-sponding kinetic values for each animal after MPTPlesioning are reported in Table 1. BBB transfer ofFDOPA, as described by the rate constantK1, was un-affected by MPTP lesioning at all doses administered.The in vivo decarboxylation rate (k3) and the overallFDOPA uptake constant (Ki) were both reduced by;70% after treatment with 2.0 mg/kg MPTP. This re-


1149STRIATAL REDUCTIONS OF AAAD ACTIVITY

duction was highly significant when compared with the1.0 mg/kg dose group fork3 andKi ( p , 0.0004 andp, 0.005, respectively) and was even significant whencompared with the 1.5 mg/kg dose group (p , 0.001 andp , 0.01, respectively). MPTP lesioning had little effecton k3 and Ki for animals in the low-dose (1.0 mg/kg)group compared with their baseline and had some sig-nificant effects in animals receiving 1.5 mg/kg MPTPcompared with the 1.0 mg/kg dose group (p , 0.03 andp , 0.04, respectively).

In vitro biochemical determinations indicate a sub-stantial reduction (65%) of AAAD activity in the caudateand putamen also after treatment with 2.0 mg/kg MPTP(Table 2) compared with controls (p , 0.003 andp, 0.002, respectively) and with the 1.0 mg/kg dosegroup (p , 0.0004 andp , 0.001, respectively). Thisreduction in the 2.0 mg/kg dose group was also signifi-cant when compared with the 1.5 mg/kg dose group forboth the caudate and putamen nucleus (p , 0.04 andp, 0.003, respectively). When compared with controls,striatal AAAD activity remained relatively unchanged atlow doses of MPTP (1.0 and 1.5 mg/kg dose groups)with a slight reduction (20%;p , 0.06) occurring only inthe putamen at 1.5 mg/kg doses of MPTP. The averageAAAD activity in the substantia nigra was gradually

reduced with increasing doses of MPTP, with a signifi-cant reduction of 40% (p , 0.05) after 2.0 mg/kg MPTPcompared with controls (Table 2). Selective lesioning ofthe nigrostriatal dopamine system was demonstrated be-cause AAAD activity found in the nucleus accumbenswas unchanged, even after 2.0 mg/kg MPTP (Table 2).Therefore, mesolimbic dopaminergic neurons of the ven-tral tegmentum were less susceptible to MPTP toxicitythan those of the nigrostriatal pathway, in agreementwith previously reported data (Burns et al., 1983;Elsworth et al., 1987; German et al., 1988).

Changes in the FDOPA uptake constant (Ki) due toMPTP lesioning were significantly correlated with invitro-determined caudate (R 5 0.55, p , 0.04) andputamen (R 5 0.68,p , 0.007) AAAD activities (Fig.1A and B). A relatively stronger relationship was foundwhen comparing changes in the in vivo decarboxylationrate (k3) with in vitro caudate (R 5 0.65,p , 0.01) andputamen (R 5 0.93,p , 0.0001) AAAD activities (Fig.1C and D). This is consistent with previous findings thatthe composite nature ofKi predicts that its magnitudeshould not decline as a simple linear function ofk3(Cumming and Gjedde, 1998). Although a highly signif-icant relationship exists between in vivo- and in vitro-determined striatal decarboxylation rates, quantitative

TABLE 1. Corrected striatal kinetic rate constants for FDOPA after LNAA concentration adjustments in Guyana squirrelmonkeys before and after MPTP treatment

Dose(mg/kg)

Animalno.

K1 (ml/min/g) k2 (/min) k3 (/min) Ki (ml/min/g)

Baseline MPTP % Baseline Baseline MPTP % Baseline Baseline MPTP % Baseline Baseline MPTP % Baseline

1.0 219 0.0526 0.0560 106 0.0255 0.0482 189 0.0118 0.0128 108 0.0167 0.0117 70316 0.0181 0.0365 202 0.0279 0.0385 138 0.0147 0.0127 86 0.0062 0.0091 147427 0.0398 0.0291 73 0.0348 0.0345 99 0.0131 0.0140 107 0.0109 0.0084 77429 0.0448 0.0372 83 0.0326 0.0222 68 0.0105 0.0116 110 0.0109 0.0128 117

Average 0.0388 0.0397 116 0.0302 0.0359 124 0.0125 0.0128 103 0.0112 0.0105 103SEM 0.0064 0.0050 25 0.0018 0.0047 23 0.0008 0.0004 5 0.0019 0.0009 16

1.5 182 0.0595 0.0411 69 0.0209 0.0299 143 0.0133 0.0107 80 0.0232 0.0108 47359 0.0457 0.0355 78 0.0269 0.0324 120 0.0143 0.0144 101 0.0159 0.0109 69314 0.0394 0.0355 90 0.0327 0.0331 101 0.0102 0.0075 74 0.0094 0.0065 69361 0.0625 0.0653 104 0.0330 0.0682 207 0.0169 0.0143 85 0.0212 0.0113 53360 0.0555 0.0497 90 0.0224 0.0279 125 0.0164 0.0124 76 0.0234 0.0153 65

Average 0.0525 0.0454 86 0.0272 0.0383 139 0.0142 0.0119 83a 0.0186 0.0110 61a

SEM 0.0039 0.0050 5 0.0023 0.0067 16 0.0011 0.0011 4 0.0024 0.0012 4

2.0 225 0.0515 0.0450 87 0.0469 0.0798 170 0.0290 0.0035 12 0.0197 0.0019 10233 0.0569 0.0345 61 0.0568 0.0641 113 0.0268 0.0046 17 0.0182 0.0023 13428 0.0469 0.0484 103 0.0625 0.0504 81 0.0232 0.0065 28 0.0127 0.0055 43366 0.0586 0.0404 69 0.0236 0.0305 129 0.0115 0.0066 57 0.0191 0.0072 38424 0.0412 0.0348 84 0.0390 0.0400 103 0.0164 0.0078 48 0.0122 0.0057 47

Average 0.0510 0.0406 81 0.0458 0.0530 119 0.0214 0.0058 32b 0.0164 0.0045 30b

SEM 0.0029 0.0025 7 0.0061 0.0078 13 0.0029 0.0007 8 0.0015 0.0009 7

Individual Guyana squirrel monkeys (n5 14) were scanned using FDOPA with PET. Afterward, animals were given a single subcutaneousinjection of 1.0 (n5 4), 1.5 (n5 5), or 2.0 (n5 5) mg/kg MPTP and then rescanned 3 months after MPTP treatment. Kinetic rate constants wereestimated by iterative fitting to a five-compartment model for the striatum with data acquired during scanning. LNAA concentrations were determinedfor each animal, and the kinetic rate constantsK1 andKi were adjusted to account for competitive effects of LNAAs on FDOPA transport across theblood–brain barrier. The percentage of baseline was determined by comparing striatal kinetic values before and after MPTP treatment. Data are alsorepresented as average6 SEM values. No change was apparent inK1 after MPTP treatment for all dose groups.

a After treatment with 1.5 mg/kg MPTP,k3 andKi were significantly reduced by one-way ANOVA compared with animals given 1.0 mg/kg (p, 0.03 andp , 0.04, respectively).

b One-way ANOVA revealed a significant reduction ink3 andKi after 2.0 mg/kg MPTP compared with animals given 1.0 mg/kg MPTP (p , 0.0004andp , 0.005, respectively), and compared with animals given 1.5 mg/kg MPTP (p , 0.001 andp , 0.01, respectively).



comparison of individual in vivo rates with their corre-sponding in vitro rates revealed an;10-fold discrepancyin their calculated values (in vitro. in vivo; Tables 1and 2).

F test analysis of the percent loss in in vitro-measuredAAAD activity in the substantia nigra compared withthat of the caudate and putamen [F(12,1) # 9.33, p5 0.01] revealed a nonparallel relationship betweenchanges of the two variables due to MPTP treatment(Fig. 2A and B). This was also apparent when comparingsubstantia nigra AAAD loss with the reduction in PETstriatal kinetic values ofk3 [F(12,1) # 9.33,p 5 0.01]andKi [F(12,1)# 4.75,p 5 0.05] byF test analysis (Fig.2C and D).

The percent change in in vitro dopamine concentra-tions with MPTP dose determined in the caudate andputamen nuclei was significantly correlated with the per-cent change in striatal AAAD activities determined invitro (R 5 0.86,p , 0.0001 andR 5 0.85,p , 0.0001;Fig. 3).

DISCUSSION

Large reductions (70 –90%) in striatal dopaminelevels in symptomatic Parkinson’s disease patientshave been well documented in postmortem determina-tions (Hornykiewicz, 1966). Similar observations weremade in nonhuman primates after selective lesioningwith relatively high doses of the neurotoxin MPTP(Elsworth et al., 1987; Pifl et al., 1991). In this work wehave demonstrated that these reductions in striatal dopa-mine levels are linked to striatal AAAD activities. In-deed, equivalent reductions (;60–70%) in total striatalAAAD content present were apparent after administra-tion of a high dose (2.0 mg/kg) of MPTP (Table 2). Thisresulted in a strong linear correlation (R 5 0.85, p, 0.0001) between striatal dopamine and in vitro-deter-mined AAAD activities in the same animals for bothcaudate and putamen nuclei (Fig. 3).

Reductions in in vitro-measured AAAD activitywithin the caudate and putamen were matched with in

TABLE 2. In vitro striatal AAAD activities in Guyana squirrel monkeys treated with MPTP

MPTP (mg/kg) Animal no.

Activity (ml/min/g)

Caudate Putamen Substantia nigra Nucleus accumbens

0.0 315 0.2339 0.2577 0.2709 0.1455325 0.3044 0.2820 0.3809 0.0952347 0.3313 0.2940 0.5001 0.1772

Average 0.2899 0.2779 0.3840 0.1393SEM 0.0290 0.0107 0.0662 0.0239

1.0 219 0.2953 0.2796 0.2742 0.1229316 0.3209 0.2216 0.4098 0.0949427 0.3115 0.3204 0.2714 0.1644429 0.2530 0.2774 0.3186 0.1811

Average 0.2952 0.2748 0.3185 0.1408SEM 0.0150 0.0203 0.0323 0.0196

1.5 182 0.1768 0.2082 0.2939 0.1344359 0.1718 0.2146 0.3202 0.1888314 0.1950 0.1738 0.3186 0.1290361 0.3594 0.2671 0.2867 0.2179360 0.5298 0.2559 0.2930 0.1747

Average 0.2866 0.2239a 0.3025 0.1690SEM 0.0700 0.0169 0.0070 0.0168

2.0 225 0.0432 0.0379 0.1939 0.1651233 0.0441 0.0432 0.1603 0.0786428 0.1190 0.1321 0.1896 0.1481366 0.1639 0.1370 0.3415 0.1552424 0.1311 0.1533 0.2592 0.1274

Average 0.1003b 0.1007b 0.2289b 0.1349SEM 0.0243 0.0248 0.0325 0.0154

In vitro AAAD activity was determined in Guyana squirrel monkeys (n5 17) 4 months after MPTP treatment. Homogenates from caudate,putamen, substantia nigra, and nucleus accumbens were incubated with 1 mM L-DOPA for 20 min. Supernatants were assayed for dopamine formationby reversed-phase HPLC. In vitro AAAD activity units were converted from pmol/min/mg of protein to equivalent units of ml/min/g of tissue or /minbased on the approximation of 1 ml/g of tissue. Data are also represented as average6 SEM values. No change was apparent in the nucleus accumbensAAAD activity for all dose groups (p , 0.9).

a Putamen AAAD activity was reduced by 20% in animals receiving 1.5 mg/kg MPTP compared with animals not treated with MPTP (p , 0.06).b One-way ANOVA revealed a significant reduction in caudate and putamen AAAD activities in animals given 2.0 mg/kg MPTP compared with

animals not treated with MPTP (p , 0.003 andp , 0.002, respectively); the 1.0 mg/kg dose group (p , 0.0004 andp , 0.001, respectively); andthe 1.5 mg/kg dose group (p , 0.04 andp , 0.003, respectively). Substantia nigra AAAD activity was significantly reduced after 2.0 mg/kg MPTPcompared with controls (p , 0.05).



vivo determinations performed in the same animals withPET (Tables 1 and 2). In vivo decarboxylation rateconstant (k3) values and AAAD activities were signifi-cantly correlated (Fig. 1C and D). As expected, changesin k3 more closely corresponded with in vitro AAAD

activity in the putamen than in the caudate nucleus.Because putamen appears more sensitive than the cau-date nucleus to the effects of MPTP (Nyberg et al., 1983;Kish et al., 1988; see also Table 2) and because of therelatively higher contributions of the putamen in the

FIG. 1. Correlation analysis of FDOPA PET striatalkinetic rate constants with in vitro striatal AAADactivities in MPTP-treated Guyana squirrel mon-keys. Linear regression analysis revealed a signif-icant relationship between the percentage of con-trol for in vitro striatal AAAD activity and Ki (A andB) and k3 (C and D). FDOPA PET correlations withputamen AAAD activities (B and D) were moresignificant than with caudate AAAD activities (Aand C) for both Ki and k3. Best-fit (black lines) andthe 95% confidence limits (gray lines) are shown.

FIG. 2. Correlation between reductions in sub-stantia nigra AAAD activities and reductions instriatal AAAD activities and FDOPA PET striatalkinetic rate constants in MPTP-treated Guyanasquirrel monkeys. The relationship between thepercent loss in substantia nigra AAAD activityand (A) caudate and (B) putamen in vitro-mea-sured AAAD activities was examined in animalsafter treatment with 1.0 (n 5 4), 1.5 (n 5 5), and2.0 (n 5 5) mg/kg MPTP. The relationship of thepercent loss in substantia nigra AAAD withFDOPA PET striatal kinetic parameters (C) Ki and(D) k3 was also examined. A comparison ofslopes by F test analysis showed that changes inAAAD activity in the substantia nigra comparedwith those in the caudate or putamen, and withPET striatal kinetic values, did not parallel oneanother. Solid lines represent the best-fit lines,and dotted lines represent the best-fit linesthrough the zero. Data are from individual ani-mals treated with 1.0 (■), 1.5 (F), or 2.0 mg/kgMPTP (�).



tomographic sections of the brain (Stout, 1999), putamenin vitro AAAD activity follows more closelyk3 measure-ments obtained in vivo with PET (Fig. 1D). Even thoughthe correlations established above are strong, aquanti-

tative comparisonof in vivo (k3) with in vitro ratesclearly demonstrates that the latter was at least 10-foldlarger than the in vivo decarboxylation rates determinedin the same squirrel monkey (Tables 1 and 2) and aspreviously observed in ex vivo experiments in rats(Cumming et al., 1994). Literature precedence reveals asimilar apparent discrepancy between in vivo and in vitrodeterminations of AAAD activities in control animals(Tables 3 and 4), but in vitro information is lacking as tothe effects of MPTP. Most previously determined in vivoAAAD activities have been assayed in humans and mon-keys using FDOPA and PET, whereas most in vitroAAAD activity determinations were performed in ro-dents. Cross-species determinations of AAAD activitiesare frequently equivocal because comparable in vitrodeterminations obtained across mammalian species, suchas rats (Broch and Fonnum, 1972; Rahman et al., 1981),rabbits (McCaman et al., 1965), cats (Kuntzman et al.,1961), monkeys (Goldstein et al., 1969), and humans(Lloyd and Hornykiewicz, 1972; Mackay et al., 1978),are substantially different. Therefore, this work offers adirect quantitative comparison of in vivo decarboxyl-ation rate (k3) values with in vitro-determined AAADactivities in nonhuman primates and in the same set ofanimals. It also investigates the effects of the neurotoxinMPTP on these parameters.

Several possible explanations may exist for the dis-crepancy between in vivo- and in vitro-determined de-carboxylation rates. They may be summarized as fol-lows:

(a) Because of the small size (23–29 g) of the squirrelmonkey brain and the limited resolution of the PETscanner, partial volume effects would impact estimatedk3 values. Corrections in spillover may increasek3 bytwo- to threefold (Cumming and Gjedde, 1998). Theeffects of partial volume in squirrel monkeys have beendiscussed in detail elsewhere (Stout, 1999), but it cannotaccount for.50% in the underestimation ofk3 deter-

TABLE 3. Summary of reported in vivo striatal AAAD rates

Subject Assay descriptionAAAD activity

[k3 (/min)] Reference

Human FDOPA PET 0.024 Gjedde et al. (1991)Human FDOPA PET 0.041 Huang et al. (1991a)Human FDOPA PET 0.083 Hoshi et al. (1993)Human FDOPA PET 0.021 Ishikawa et al. (1996)Human FDOPA PET 0.080 Kuwabara et al. (1995)Human FDOPA PET 0.012 Nahmias et al. (1996)Human 6-FMT PETa 0.011 Nahmias and Wahl (1995)Monkey (Macaca nemestrina) FDOPA PET 0.021 Barrio et al. (1990)Monkey (vervet) FDOPA PET 0.015 Barrio et al. (1996)Monkey (rhesus) L-[b-11C]DOPA PET 0.012 Hartvig et al. (1993)Monkey (rhesus) L-[b-11C]DOPA PET 0.011 Tsukada et al. (1996)Monkey (vervet) 6-FMT PET 0.033 Barrio et al. (1996)Rat (Sprague–Dawley) FDOPA 0.010 Reith et al. (1990)b

Rat (Wistar) [3H]DOPA 0.260 Cumming et al. (1995)b

Rat (Long–Evans) FDOPA 0.170 Cumming et al. (1994)b

a 6-FMT, 6-[18F]fluoro-L-m-tyrosine.b Ex vivo experiments.

FIG. 3. Relationship between in vitro-determined striatal AAADactivity and striatal dopamine (DA) concentrations in MPTP-treatedGuyana squirrel monkeys. A: Linear regression analysis reveals asignificant relationship between the percent of control of caudatedopamine concentrations and caudate AAAD activity. B: A signifi-cant linear relationship between putamen dopamine concentra-tions and AAAD activity was also apparent in animals treated withMPTP. Black lines represent the best-fit lines; 95% confidencelimits (gray lines) are also shown.



mined in vivo with PET scanned in the Siemens ECAT713.

(b) The stability of MPTP lesioning over time, as wellas dose and route of MPTP administration, may contrib-ute to the variations seen between individual animals.The stability of MPTP lesioning may also contribute tothe differences observed between in vivo and in vitroresults because in vitro analysis could not be performedimmediately after PET scanning. However, the selectionof squirrel monkeys was based on minimizing this con-sideration because MPTP-treated squirrel monkeys ex-hibited permanent selective damage to the pars compactaof the substantia nigra (Langston et al., 1984).

(c) An overestimation of in vitro AAAD activity mea-sured in striatal tissue may result from the release ofAAAD from nondopaminergic neurons (Arai et al.,1994; Mura et al., 1995); however, only 15–20% ofAAAD in the striatum has been estimated to originate innondopaminergic sources, such as serotonergic neurons,interneurons, and efferent neurons (Melamed et al.,1981). This overestimation may be a more importantfactor in the lesioned striatum because these sources maybe less susceptible to MPTP toxicity. In addition, thisresidual AAAD activity is particularly evident whenmeasured in vitro because the accumulation of18F inPET recordings requires both the decarboxylation ofFDOPA and its vesicular storage (Gjedde et al., 1991;Cumming et al., 1994).

(d) Differences inKm andVmaxbetween FDOPA (usedin vivo) and L-DOPA (used in vitro) as substrate forAAAD could be considered a contributing factor. How-ever, this is unlikely becauseKm andVmaxdetermined forFDOPA against AAAD (101mM and 150 nmol/min/g,respectively) are well within the range determined forL-DOPA [40–200mM and 33–150 nmol/min/g, respec-tively (Awapara and Saine, 1975; Hefti et al., 1980;

Reith et al., 1990)]. Moreover, in vivo PET determina-tions of k3 with L-[b-11C]DOPA (Hartvig et al., 1993;Tsukada et al., 1996) are also within the range ofk3values obtained with FDOPA (Table 3), and investiga-tions involving the direct comparison of the kinetics ofFDOPA andL-DOPA decarboxylation showed no differ-ence in either substrate affinity orVmax (Cumming et al.,1988).

(e) The catalytic role of pyridoxal phosphate in thedecarboxylation of aromatic amino acids has been estab-lished (Bowsher and Henry, 1986). It has been demon-strated that AAAD contains one molecule of pyridoxalphosphate for every molecule of protein (Christensonet al., 1970; Borri Voltattorni et al., 1971). Therefore, notall AAAD may be active in vivo owing to limitations onthe availability of this essential enzyme cofactor or thepresence of other inactivating mechanisms for the en-zyme. For example, the degradation of ornithine decar-boxylase by 26S proteasome in the presence of antizyme(Hayashi et al., 1996; Murakami et al., 1996) demon-strates a mechanism by which enzyme activity can beinhibited in vivo.

(f) As discussed previously (Kish et al., 1995), enzymeregulation may possibly play a role in the apparentdiscrepancy between PET results and in vitro findings.The rate of DOPA decarboxylation can be rapidly mod-ulated by pharmacological stimulus (Cumming et al.,1995, 1997; Cho et al., 1999). Activation or blocking ofdopamine D1 and D2 receptors can diminish or enhanceAAAD activity, in which several biochemical mecha-nisms, such as second messengers, phosphorylation, orprotein synthesis, seem responsible for its modulation(Neff and Hadjiconstantinou, 1995).

(g) An intriguing possibility to account for restrictionsin FDOPA in vivo decarboxylation is that the substrate(FDOPA) may have limited accessibility to AAAD be-cause of multiple compartmentation of dopamine (andAAAD) within the neuron (Doteuchi et al., 1974; Grop-petti et al., 1977), a subject of controversy in the litera-ture. Two main hypotheses are proposed for the state oftransmitter stores in the neuron: (a) catecholamines arestored in a single open compartment, where they arereleased; or (b) catecholamines are stored in more thanone compartment, consisting of a functional pool, wherethe transmitter is quickly synthesized and released, and astorage pool. There is considerable evidence for oragainst each one of these hypotheses (for review, seeGlowinski, 1973).

(h) Although the possibility that not all AAAD isactive in vivo or that the enzyme is in multiple neuronalpools cannot be entirely dismissed, an alternative expla-nation as to why in vitro AAAD measurements do notmimic in vivo AAAD determinations is the limitation ofsubstrate access, e.g., FDOPA, across the neuronal mem-brane to intraneuronal AAAD in in vivo determinations(Barrio et al., 1997). Restrictions due to membrane trans-port into the neuron are not present in in vitro assayprocedures that involve mechanical dissection and ho-mogenization of tissue samples taken from the striatum.

TABLE 4. Summary of reported in vitro AAAD

SubjectBrainregion

AAADactivity

(ml/min/g) Reference

Cat Caudate 0.215a Kuntzman et al. (1961)Human

(postmortem) Caudate 0.057 Mackay et al. (1978)Monkey (green) Putamen —b Goldstein et al. (1969)Rat (Donryu) Caudate 0.107 Rahman et al. (1981)

Rat (Wistar) Striatum 0.447Broch and Fonnum(1972)

Rat (Sprague–Dawley) Striatum 0.075

Awapara and Saine(1975)

Rat (Sprague–Dawley) Striatum 0.090 Hefti et al. (1980)

Rat (hooded) Striatum 1.800 Cumming et al. (1994)Rabbit Caudate 0.155 McCaman et al. (1965)

a Unit conversion was carried out by dividing AAAD activity deter-mined by the averageKm for AAAD determined from pig kidneyenzyme. The averageKm used was 165mM as determined previously(Reith et al., 1990).

b In vitro AAAD activity was determined to be 753 cpm/h/mg ofprotein.



Indeed, we have shown using isolated nerve terminals(synaptosomes) prepared from rat striatum that uptake ofL-DOPA into synaptosomes could be decreased by com-peting LNAAs for substrate transport (Yee et al., 1998).This is in agreement with earlier observations by Katz(1980), who found that AAAD activities measured indisrupted synaptosomes were substantially higher (atleast fivefold) than decarboxylation rates observed inmembrane intact synaptosomes. In addition, the func-tional expression of a neuronal transporter protein withspecificity for L-DOPA (Yee et al., 1998) provides mo-lecular evidence and a possible mechanism as to howsubstrate access may be restricted.

Another important observation of this work is thatAAAD activities measured in the substantia nigra did notparallel reductions in striatal AAAD activities as deter-mined in vitro (Fig. 2A and B) or in vivo with FDOPA-PET (Fig. 2C and D). The differential sensitivity of thenigrostriatal pathway to MPTP neurotoxic effects isclearly visible with various dopaminergic indices. Spe-cifically, AAAD reductions in the nigra with increasingMPTP doses are also paralleled by reductions in nigraldopamine cell number and substantia nigra dopamineand dopamine metabolite concentrations measured in thesame animals (authors’ unpublished data). Therefore,reductions in substantia nigra indicesseem toprecedereductions in the striatumin animals treated with gradeddoses of MPTP at 1.0 and 1.5 mg/kg (Table 2 andauthors’ unpublished data). These early changes in thesubstantia nigra may have relevance on the therapeuticactions ofL-DOPA. Evidence indicates that dopaminerelease in the substantia nigra can influence basal ganglianeurotransmission by activating dopamine D1 receptorson striatonigral nerve terminals and D2 receptors ondopamine cell bodies and dendrites (Robertson et al.,1991). Activation of nigral D2 receptors may serve anautoinhibitory role to decrease striatal release of dopa-mine (Cheramy et al., 1981), whereas activation of D1receptors has been shown to increase the release ofGABA (Reubi et al., 1977; Kozlowski et al., 1980), amajor inhibitory neurotransmitter in the brain. Therefore,in the case of Parkinson’s disease, the activation ofreceptors to decrease neuronal firing would not be desir-able. Indeed, the substantia nigra dopamine levels inthese animals were substantially reduced (data notshown).

Previous work with symptomatic MPTP-treated mon-keys (Burns et al., 1983; German et al., 1988) and withdopamine neurons grown in culture (Mytilineou et al.,1985) indicates that terminal damage precedes cell bodydeath following MPTP insults. In agreement with thisobservation, animals treated with 2.0 mg/kg MPTP(present study) exhibit reductions in caudate/putamenAAAD activity (65%) that are greater than the reductionobserved in the substantia nigra (40%) (Table 2). How-ever, at low MPTP doses (1.0 and 1.5 mg/kg), as previ-ously observed in squirrel monkeys (Irwin et al., 1990),the results appear to contradict the hypothesis that ter-minal loss precedes cell body degeneration as a result of

MPTP toxicity. This discrepancy may be only apparent,however, because the initial site of MPTP action mayvery well be the terminal end. The appearance of com-pensatory mechanisms, like axonal sprouting, enzymeup-regulation, or modulation of autoinhibitory responses,as a result of MPTP’searly insult, e.g., with 1.0 mg/kg,may explain this apparent discrepancy. Thus, in the earlystages of the neuronal insult, functional activity is main-tained even though cell body indicators in the substantianigra are reduced. However, when substantial damage isinflicted to the dopaminergic pathway, e.g., MPTP dosesof 2.0 mg/kg, neuronal terminal compensatory mecha-nisms cannot be maintained, neurochemical indicatorsfall precipitously, and symptoms become apparent.FDOPA central kinetics and striatal dopaminergic indi-ces are thus sensitive to this process in neuronal termi-nals.

Three major conclusions can be drawn from this work:(a) Intraneuronal AAAD activities, and not just dopa-mine levels, are severely reduced in the MPTP monkeymodel of Parkinson’s disease. (b)k3 accurately indicatesstriatal AAAD activity in vivo with FDOPA PET butmay be severely restricted by factors absent in vitro, suchas neuronal transport. The presence of substrate transportrestrictions across the neuron could alter interpretation ofin vivo FDOPA kinetic determinations. Limitations onAAAD accessibility because of neuronal transport andthe regulation of AAAD activity in vivo also raise ques-tions on their effects ofL-DOPA therapy for Parkinson’sdisease patients. (c) The nigrostriatal pathway presentsdifferential sensitivity to neurotoxic MPTP insults. Ap-parent compensatory mechanisms in dopaminergic ter-minals mask early declines in neurochemical indexes inthe substantia nigra, which may occur in Parkinson’sdisease. The understanding of this differential sensitivitymay permit the development of the necessary tools todetect early signs of dopamine cell degeneration and alsopreclinical detection of Parkinson’s disease.

Acknowledgment: The authors give thanks to the cyclotronstaff and R. Sumida, B. Morelos, J. Edwards, W. Ladno, and P.Chan for their excellent technical support. This work was madepossible by financial support from the National Institutes ofHealth (grant RO1 NS 33356), the Department of Energy (grantDE FC0387-ER60615), and the United States PharmacopeialConvention by providing a fellowship to R.E.Y.

REFERENCES

Arai R., Karasawa N., Geffard M., Nagatsu T., and Nagatsu I. (1994)Immunohistochemical evidence that central serotonin neuronsproduce dopamine from exogenousL-DOPA in the rat, withreference to the involvement of aromaticL-amino acid decarbox-ylase.Brain Res.667,295–299.

Awapara J. and Saine S. (1975) Fluctuations in DOPA decarboxylaseactivity with age.J. Neurochem.24, 817–818.

Barrio J. R., Huang S.-C., Melega W. P., Yu D.-C., Hoffman J. M.,Schneider J. S., Satyamurthy N., Mazziotta J. C., and Phelps M. E.(1990) 6-[18F]Fluoro-L-DOPA probes dopamine turnover rates incentral dopaminergic structures.J. Neurosci. Res.27, 487–493.

Barrio J. R., Huang S.-C., Yu D.-C., Melega W. P., Quintana J., CherryS. R., Jacobson A., Namavari M., Satyamurthy N., and Phelps



M. E. (1996) RadiofluorinatedL-m-tyrosines: new in-vivo probesfor central dopamine biochemistry.J. Cereb. Blood Flow Metab.16, 667–678.

Barrio J. R., Huang S.-C., and Phelps M. E. (1997) Biological imagingand the molecular basis of dopaminergic disease.Biochem. Phar-macol.54, 341–348.

Borri Voltattorni C., Minelli A., and Turano C. (1971) Spectral prop-erties of the coenzyme bound to dopa decarboxylase from pigkidney.FEBS Lett.17, 231–235.

Bowsher R. R. and Henry D. P. (1986) Aromatic-L-amino acid decar-boxylase: biochemistry and functional significance, inNeurometh-ods, Series 1: Neurochemistry, Neurotransmitter Enzymes(Boul-ton A. A., Baker G. B., and Yu P. H., eds), pp. 33–77. HumanaPress, Clifton, New Jersey.

Broch O. J. Jr. and Fonnum F. (1972) The regional and subcellulardistribution of catechol-O-methyltransferase in the rat brain.J. Neurochem.19, 2049–2055.

Burns R. S., Chiueh C. C., Markey S. P., Ebert M. H., JacobowitzD. M., and Kopin I. J. (1983) A primate model of parkinsonism:selective destruction of dopaminergic neurons in the pars com-pacta of the substantia nigra byN-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine.Proc. Natl. Acad. Sci. USA80, 4546–4550.

Chan G. L.-Y., Doudet D. J., Dobko T., Hewitt K. A., Schofield P., PateB. D., and Ruth T. J. (1995) Routes of administration and effect ofcarbidopa pretreatment on 6-[18F]fluoro-L-DOPA/PET scans innon-human primates.Life Sci.21, 1759–1766.

Cheramy A., Nieoullon A., and Glowinski J. (1978) In vivo changes indopamine release in cat caudate nucleus and substantia nigrainduced by nigral application of various drugs including GABAer-gic agonists and antagonists, inInteractions Between PutativeNeurotransmitters in the Brain(Garattini S., Pujol J. F., andSaminin R., eds), pp. 175–190. Raven Press, New York.

Cho S., Duchemin A. M., Neff N. H., and Hadjiconstantinou M. (1999)Tyrosine hydroxylase, aromaticL-amino acid decarboxylase anddopamine metabolism after chronic treatment with dopaminergicdrugs.Brain Res.830,237–245.

Christenson J. G., Dairman W., and Udenfriend S. (1970) Preparationand properties of a homogeneous aromaticL-amino acid decar-boxylase from hog kidney.Arch. Biochem. Biophys.141, 356–367.

Cumming P. and Gjedde A. (1998) Compartmental analysis of DOPAdecarboxylation in living brain from dynamic positron emissiontomograms.Synapse29, 37–61.

Cumming P, Hau¨sser M., Wayne Martin W. R., Grierson J., AdamM. J., Ruth T. J., and McGeer E. G. (1988) Kinetics ofin vitrodecarboxylation and thein vivo metabolism of 2-18F- and 6-18F-fluorodopa in the hooded rat.Biochem. Pharmacol.2, 247–250.

Cumming P., Kuwabara H., and Gjedde A. (1994) A kinetic analysis of6-[18F]fluoro-L-dihydroxyphenylalanine metabolism in the rat.J. Neurochem.63, 1675–1682.

Cumming P., Kuwabara H., Ase A., and Gjedde A. (1995) Regulationof DOPA decarboxylase activity in brain of living rat.J. Neuro-chem.65, 1381–1390.

Cumming P., Ase A., Laliberte´ C., Kuwabara H., and Gjedde A. (1997)In vivo regulation of dopa decarboxylase by dopamine receptorsin rat brain.J. Cereb. Blood Flow Metab.17, 1254–1260.

Doteuchi M., Wang C., and Costa E. (1974) Compartmentation ofdopamine in rat striatum.Mol. Pharmacol.10, 225–234.

Elsworth J. D., Deutch A. Y., Redmond D. E., Sladek J. R., and RothR. H. (1987) Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropy-ridine (MPTP) on catecholamines and metabolites in primate brainand CSF.Brain Res.415,293–299.

Forno L. S., DeLanney L. E., Irwin I., and Langston J. W. (1993)Similarities and differences between MPTP-induced parkinsonismand Parkinson’s disease. Neuropathologic considerations.Adv.Neurol.60, 600–608.

German D. C., Dubach M., Askari S., Speciale S. G., and BowdenD. M. (1988) 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-in-duced parkinsonian syndrome inMacaca fascicularis:which mid-brain dopaminergic neurons are lost?Neuroscience24, 161–174.

Gjedde A., Reith J., Dyne S., Le´ger G., Guttman M., Diksic M., EvansA., and Kuwabara H. (1991) Dopa decarboxylase of the humanbrain.Proc. Natl. Acad. Sci. USA88, 2721–2725.

Gjedde A., Leger G. C., Cumming P., Yasuhara Y., Evans A. C.,Guttman M., and Kuwabara H. (1993) StriatalL-DOPA decarbox-ylase activity in Parkinson’s disease in vivo: implications for theregulation of dopamine synthesis.J. Neurochem.61, 1538–1541.

Glowinski J. (1973)Brain, Nerves and Synapses, Vol. 4: Pharmacol-ogy and the Future of Man,pp. 204–214. Karger, Basel.

Goldstein M., Anagnoste B., Battista A. F., Owen W. S., and NakataniS. (1969) Studies of amines in the striatum in monkeys with nigrallesions. The disposition, biosynthesis, and metabolites of [3H]do-pamine and [14C]serotonin in the striatum.J. Neurochem.16,645–653.

Groppetti A., Algeri S., Cattabeni F., Di Giulio A. M., Galli C. L.,Ponzio F., and Spano P. F. (1977) Changes in specific activity ofdopamine metabolites as evidence of multiple compartmentationof dopamine in striatal neurons.J. Neurochem.28, 193–197.

Hartvig P., Tedroff J., Lindner K. J., Burling P., Chang C.-W., TsukadaH., Watanabe Y., and Långstro¨m B. (1993) Positron emissiontomographic studies on aromaticL-amino acid decarboxylase ac-tivity in vivo for L-DOPA and 5-hydroxy-L-tryptophan in themonkey brain.J. Neural Transm.94, 127–135.

Hayashi S., Murakami Y., and Matsufuji S. (1996) Ornithine decar-boxylase antizyme: a novel type of regulatory protein.TrendsBiochem. Sci.21, 27–30.

Hefti F., Melamed E., and Wurtman R. J. (1980) Partial lesions of thedopaminergic nigrostriatal system in rat brain: biochemical char-acterization.Brain Res.195,123–137.

Hoffman J. M., Melega W. P., Hawk T. C., Grafton S. C., Luxen A.,Mahoney D. K., Barrio J. R., Huang S.-C., Mazziotta J. C., andPhelps M. E. (1992) The effects of carbidopa administration on6-[F-18]fluoro-L-dopa kinetics in PET studies.J. Nucl. Med.33,1472–1477.

Hornykiewicz O. (1966) Dopamine (3-hydroxytyramine) and brainfunction.Pharmacol. Rev.18, 925–964.

Hoshi H., Kuwabara H., Le´ger G., Cumming P., Guttman M., andGjedde A. (1993) 6-[18F]Fluoro-L-DOPA metabolism in livinghuman brain: a comparison of six analytical methods.J. Cereb.Blood Flow Metab.13, 57–69.

Huang S.-C., Yu D.-C., Barrio J. R., Grafton S., Melega W. P.,Hoffman J. M., Satyamurthy N., Mazziotta J. C., and Phelps M. E.(1991a) Kinetics and modeling ofL-6-[18F]fluoro-DOPA in hu-man positron emission tomographic studies.J. Cereb. Blood FlowMetab.11, 898–913.

Huang S.-C., Barrio J. R., Yu D.-C., Chen B., Grafton S., MelegaW. P., Hoffman J. M., Satyamurthy N., Mazziotta J. C., andPhelps M. E. (1991b) Modelling approach for separating bloodtime–activity curves in positron emission tomographic studies.Phys. Med. Biol.36, 749–761.

Huang S.-C., Stout D. B., Yee R. E., Satymurthy N., and Barrio J. R.(1998) Distribution volume of radiolabeled large neutral aminoacid in brain tissue.J. Cereb. Blood Flow Metab.18,1288–1293.

Irwin I., Delanney L. E., Forno L. S., Finnegan K. T., Di Monte D. A.,and Langston J. W. (1990) The evolution of nigrostriatal neuro-chemical changes in the MPTP-treated squirrel monkey.BrainRes.531,242–252.

Ishikawa T., Dhawan V., Chaly T., Margouleff C., Robeson W., DahlJ. R., Mandel F., Spetsieris P., and Eidelberg D. (1996) Clinicalsignificance of striatal DOPA decarboxylase activity in Parkin-son’s disease.J. Nucl. Med.37, 216–221.

Joh T. H., Hwang O., and Abate C. (1986) Phenylalanine hydroxylase,tyrosine hydroxylase, and tryptophan hydroxylase, inNeurometh-ods, Series 1: Neurochemistry, Neurotransmitter Enzymes(Boul-ton A. A., Baker G. B., and Yu P. H., eds), pp. 1–23. HumanaPress, Clifton, New Jersey.

Katz I. R. (1980) Inhibition of 3,4-dihydroxy-L-phenylalanine decar-boxylase in rat striatal synaptosomes by amino acids interactingwith substrate transport.Biochim. Biophys. Acta600,195–204.

Keen R. E., Barrio J. R., Huang S. C., Hawkins R. A., and Phelps M. E.(1989) In vivo cerebral protein synthesis rates with leucyl-transferRNA used as a precursor pool: determination of biochemical



parameters to structure tracer kinetic models for positron emissiontomography.J. Cereb. Blood Flow Metab.9, 429–445.

Keeping E. S. (1995)Introduction to Statistical Inference,pp. 193–197.Dover, New York.

Kilpatrick I. C., Jones M. W., and Phillipson O. T. (1986) A semiau-tomated analysis method for catecholamines, indoleamines, andsome prominent metabolites in microdissected regions of thenervous system: an isocratic HPLC technique employing coulo-metric detection and minimal sample preparation.J. Neurochem.46, 1865–1876.

Kish S. J., Shannak K., and Hornykiewicz O. (1988) Uneven pattern ofdopamine loss in the striatum of patients with idiopathic Parkin-son’s disease.N. Engl. J. Med.318,876–880.

Kish S. J., Zhong X. H., Hornykiewicz O., and Haycock J. W. (1995)Striatal 3,4-dihydroxyphenylalanine in aging: disparity betweenpostmortem and positron emission tomography studies?Ann. Neu-rol. 38, 260–264.

Kozlowski M. R., Sawyer S., and Marshall J. F. (1980) Behaviouraleffects and supersensitivity following nigral dopamine receptorstimulation.Nature287,52–54.

Kuntzman R., Shore P. A., Bogdanshi D., and Brodie D. B. (1961)Microanalytical procedures for fluorometric assay of brain dopa-5HTP decarboxylase, norepinephrine, and serotonin, and a detailedmapping of decarboxylase activity in brain.J. Neurochem.6, 226–232.

Kuwabara H., Cumming P., Yasuhara Y., Le´ger G. C., Guttman M.,Diksic M., Evans A. C., and Gjedde A. (1995) Regional striatalDOPA transport and decarboxylase activity in Parkinson’s dis-ease.J. Nucl. Med.36, 1226–1231.

Langston J. W., Forno L. S., Rebert C. S., and Irwin I. (1984) Selectivenigral toxicity after systematic administration of 1-methyl-4-phe-nyl-1,2,5,6-tetrahydropyridine (MPTP) in the squirrel monkey.Brain Res.292,390–394.

Leenders K. L., Salmon E. P., Tyrrell P., Perani D., Brooks D. J., SagerH., Jones T., Marsden D., and Frackowick S. J. (1990) Thenigrostriatal dopaminergic system assessed in vivo by positronemission tomography in healthy volunteer subjects and patientswith Parkinson’s disease.Arch. Neurol.47, 1290–1298.

Lin K.-P., Huang S.-C., Baxter L. R., and Phelps M. E. (1994) Ageneral technique for interstudy registration of multifunction andmultimodality images.IEEE Trans. Nucl. Sci.40, 2850–2855.

Lin K.-P., Huang S.-C., Yu D.-C., Melega W. P., Barrio J. R., andPhelps M. E. (1996) Automated image registration for FDOPAPET studies.Phys. Med. Biol.41, 2775–2788.

Lloyd K. G. and Hornykiewicz O. (1972) Occurrence and distributionof aromaticL-amino acid (L-DOPA) decarboxylase in the humanbrain.J. Neurochem.19, 1549–1559.

Mackay A. V. P., Davies P., Dewar A. J., and Yates C. M. (1978)Regional distribution of enzymes associated with neurotransmis-sion by monoamines, acetylcholine, and GABA in the humanbrain.J. Neurochem.30, 827–839.

McCaman R. E., McCaman M. W., Hunt J. M., and Smith M. S. (1965)Microdetermination of monoamine oxidase and 5HTP decarbox-ylase activity in nervous tissue.J. Neurochem.12, 15–23.

Melamed E., Hefti F., Pettibone D. J., Liebman J., and Wurtman R. J.(1981) AromaticL-amino acid decarboxylase in rat corpus stria-tum: implications for actions ofL-DOPA in parkinsonism.Neu-rology 31, 651–655.

Melega W. P., Hoffman J. M., Schneider J. S., Phelps M. E., and BarrioJ. R. (1991a) 6-[18F]Fluoro-L-DOPA metabolism in MPTP-treatedmonkeys: assessment of tracer methodologies for positron emis-sion tomography.Brain Res.543,271–276.

Melega W. P., Grafton S. T., Huang S.-C., Satyamurthy N., PhelpsM. E., and Barrio J. R. (1991b) L-6-[18F]Fluoro-DOPA metabo-lism in monkeys and humans: biochemical parameters for theformulation of tracer kinetic models with positron tomography.J. Cereb. Blood Flow Metab.11, 890–897.

Muenter M. D. and Tyce G. M. (1971)L-Dopa therapy of Parkinson’sdisease: plasmaL-dopa concentration, therapeutic response, andside-effects.Mayo Clin. Proc.46, 231–239.

Mura A., Jackson D., Manley M. S., Young S. J., and Groves P. M.(1995) Aromatic L-amino acid decarboxylase immunoreactivecells in the rat striatum: a possible site for the conversion ofexogenousL-DOPA to dopamine.Brain Res.704,51–60.

Murakami Y., Tanahashi N., Tanaka K., Omura S., and Hayashi S.(1996) Proteasome pathway operates for the degradation of orni-thine decarboxylase in intact cells.Biochem. J.317,77–80.

Mytilineou C., Cohen G., and Heikkila R. E. (1985) 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine is toxic to mesencephalic DAneurons in culture.Neurosci. Lett.57, 19–24.

Nahmias C. and Wahl L. (1996) Modeling fluorine-18-6-fluoro-L-DOPA in humans.J. Nucl. Med.37, 432–437.

Nahmias C., Wahl L., Chirakal R., Firnau G., and Garnett S. (1995) Aprobe for intracerebral aromatic amino acid decarboxylase activ-ity: distribution and kinetics of [18F]6-fluoro-L-m-tyrosine in thehuman brain.Mov. Disord.3, 298–304.

Neff N. H. and Hadjiconstantinou M. (1995) AromaticL-amino aciddecarboxylase modulation and Parkinson’s disease.Prog. BrainRes.106,91–97.

Nyberg P., Nordberg A., Wester P., and Winblad B. (1983) Dopami-nergic deficiency is more pronounced in putamen than in nucleuscaudatus in Parkinson’s disease.Neurochem. Pathol.1, 193–202.

Pate B. D., Kawamata T., Yamada T., McGeer E. G., Hewitt K. A.,Snow B. J., Ruth T., and Calne D. B. (1993) Correlation of striatalfluorodopa uptake in MPTP monkey with dopaminergic indices.Ann. Neurol.34, 331–338.

Pifl C. H., Schingnitz G., and Hornykiewicz O. (1991) Effect of1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on the regional dis-tribution of brain monoamines in the rhesus monkey.Neuro-science44, 591–605.

Rahman M. K., Nagatsu T., and Kato T. (1981) AromaticL-amino aciddecarboxylase activity in central and peripheral tissues and serumof rats with DOPA and 5HTP as substrates.Biochem. Pharmacol.30, 645–649.

Reith J., Dvye S., Kuwabara H., Guttman M., Diksic M., and Gjedde A.(1990) Blood–brain transfer and metabolism of 6-[18F]fluoro-L-DOPA in rat.J. Cereb. Blood Flow Metab.10, 707–719.

Reubi J.-C., Iversen L. L., and Jessell T. M. (1977) Dopamine selec-tively increases3H-GABA release from slices of rat substantianigra in vitro.Nature268,652–654.

Robertson G. S., Damsma G., and Fibiger H. C. (1991) Characteriza-tion of dopamine release in the substantia nigra by in vivo micro-dialysis in freely moving rats.J. Neurosci.11, 2209–2216.

Rossor M. N., Watkins J., Brown M. J., Reid J. L., and Dollery C. T.(1980) Plasma levodopa, dopamine, and therapeutic response fol-lowing levodopa therapy of Parkinson’s patient.J. Neurol. Sci.46,385–392.

Scherman D., Desnos C., Darchen F., Pollak P., Javoy-Agid F., andAgid Y. (1989) Striatal dopamine deficiency in Parkinson’s dis-ease: role of aging.Ann. Neurol.26, 551–557.

Stout D. B. (1999) Small monkeys as clinical models for the study ofthe central dopaminergic system using positron emission tomog-raphy. Ph.D. thesis, University of California, Los Angeles.

Stout D. B., Huang S.-C., Melega W. P., Raleigh M. J., Phelps M. E.,and Barrio J. R. (1998) Effects of large neutral amino acid con-centrations on 6-[F-18]fluoro-L-DOPA kinetics.J. Cereb. BloodFlow Metab.18, 43–51.

Tsukada H., Lindner K.-J., Hartvig P., Tani Y., Valtysson J., BjurlingP., Kihlberg T., Werterburg G., Watanabe Y., and Långstro¨m B.(1996) Effects of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin and in-fusion of L-tyrosine on the in vivoL-[b-11C]DOPA disposition inthe monkey brain.Brain Res.713,92–98.

Yee R. E., Huang S.-C., and Barrio J. R. (1998) Neuronal membranetransport and compartmental modeling of 6-[18F]fluoro-L-Dopa(FDOPA) striatal kinetics. (Abstr.)Neuroimage7, A14.



Nigrostriatal Reduction of Aromatic L-Amino Acid Decarboxylase Activity in MPTP-Treated Squirrel Monkeys: In Vivo and In Vitro Investigations

Documents