Comparison Between the Decrease ofDopamine Transporter and that of

L-DOPA Uptake for Detection of Earlyto Advanced Stage of Parkinson’s

Disease in Animal ModelsYASUSHI ITO,1 MASAHIRO FUJITA,1 SHOICHI SHIMADA,3 YOSHIYUKI WATANABE,1

TOMOYA OKADA,1 HIDEO KUSUOKA,1 MASAYA TOHYAMA,2 AND TSUNEHIKO NISHIMURA1*1Division of Tracer Kinetics, Biomedical Research Center, Osaka University Medical School, Osaka, Japan

2Department of Anatomy and Neuroscience, Osaka University Medical School, Osaka, Japan

KEY WORDS [I-125]beta-CIT; Parkinson’s disease; L-DOPA

ABSTRACT Early diagnosis of Parkinson’s disease (PD) is important for thepotential application of neuroprotective therapies. The purpose of this study was toassess the detection of the early changes of PD by either imaging the dopaminetransporter (DAT) or uptake of L-3,4-dihydroxyphenylalanine (L-DOPA). An early toadvanced stage model of PD was induced in rats by stereotaxic injection of 1–10 µg6-hydroxydopamine (6-OHDA) into the substantia nigra pars compacta. Using adjacentsections of the same animals, the binding of [I-125]beta-CIT, which labels DAT and theuptake of [C-14]L-DOPA, were evaluated 4 weeks after induction of the lesion. Anydecrease in dopaminergic neurons was evaluated by in situ hybridization histochemistry(ISH) by detection of DAT mRNA-positive neurons. In addition, the expression levels ofDAT, dopa decarboxylase (DDC), and vesicular monoamine transporter (VMAT2) in eachneuron were studied with ISH. Our results show a decrease in both [I-125]beta-CITbinding and [C-14]L-DOPA uptake in parallel with a decrease in DA neurons from early toadvanced stage models of PD. The decrease in [C-14]L-DOPA uptake was smaller thanthat in [I-125]beta-CIT binding in the same animal (P , 0.0001). Expression levels ofDAT, DDC, and VMAT2 mRNAs were also decreased with the progression of the disease.Although ISH failed to detect the origin of the discrepancy between [I-125]beta-CIT and[C-14]L-DOPA levels, it was concluded that [C-14]L-DOPA levels underestimated thedecrease of dopaminergic neurons and that [I-125]beta-CIT levels more preciselyreflected the decrease. Synapse 31:178–185, 1999. r 1999 Wiley-Liss, Inc.

INTRODUCTION

Parkinson’s disease (PD) is characterized pathologi-cally by the degeneration of pigmented cells and otherbrainstem nuclei, particularly the substantia nigrapars compacta (SNpc), in association with the forma-tion of neuronal eosinophilic Lewy inclusion bodies(Bethlem and Den, 1960). PD becomes clinically mani-fest when patients have lost approximately 50–80% ofdopaminergic (DA) neurons from the SNpc (Jellinger,1987) and the disease progresses gradually due tocontinued degeneration of DA neurons (Gauthier andSourkes, 1982). Specific drugs have recently been triedto slow down the progression of PD, including deprenyl,a monoamine oxidase type B inhibitor (Parkinson StudyGroup, 1989, 1996; Tetrud and Langston, 1989), andamantadine, which is an N-methyl-D-aspartate (NMDA)

receptor antagonist (Uitti et al., 1996; Danysz, 1997). Itis, therefore, important to precisely assess the propor-tion of remaining DA neurons in order to determine thestage of disease and to select an appropriate therapy.

The function of the presynaptic part of the nigrostria-tal DA system is visualized with 6-[F-18]fluoro-L-3,4-dihydroxyphenylalanine ([F-18]FDOPA), which mainlyreflects dopa decarboxylase (DDC) activity (Firnau etal., 1987; Gjedde et al., 1991; Huang et al., 1991;Kuwabara et al., 1993) and dopamine transporter(DAT) ligand levels (Volkow et al., 1996b). Recently,

*Correspondence to: Tsunehiko Nishimura, MD, PhD, Division of TracerKinetics, Biomedical Research Center, Osaka University Medical School, Osaka,Japan. E-mail: nisimura@tracer.med.osaka-u.ac.jp

Received 4 November 1997; accepted 12 June 1998.

SYNAPSE 31:178–185 (1999)

r 1999 WILEY-LISS, INC.

[I-123]2beta-carbomethoxy-3beta-(4-iodophenyl)tro-pane ([I-123]beta-CIT, RTI-55) has been widely used toimage DAT (Bruck et al., 1993; Innis et al., 1993; Innis,1994; Kuikka et al., 1993, 1995). Moreover, imaging ofDA system presynaptic function has been used for thediagnosis of neurological or psychiatric disorders suchas Parkinson syndrome (Brooks et al., 1990; Brooks,1991; Kuikka et al., 1993; Seibyl et al., 1995), cocaineaddiction (Volkow et al., 1996c), and alcoholism (Thiiho-nen et al., 1995).

Besides these studies, age-related changes in DAneuron distribution have also been rigorously studied(Eidelberg et al., 1993; Martin et al., 1989; Sawle et al.,1990; Van-Dyck et al., 1995; Volkow et al., 1996a). Manypostmortem studies have reported a decrease in DAneurons with age (Fearnley and Lees, 1991; Kish et al.,1992; McGeer et al., 1997). However, results obtainedusing [F-18]FDOPA and DAT imaging results havebeen inconsistent. One reported reduced striatal[F-18]FDOPA uptake with age (Martin et al., 1989),while others have reported no change in uptake (Eidel-berg et al., 1993; Sawle et al., 1990). In contrast, DATimaging has consistently shown a decrease with age(Van-Dyck et al., 1995; Volkow et al., 1996a). Thisdiscrepancy between [F-18]FDOPA uptake and DATbinding may be due to up- or downregulation of DAT,VMAT2, and DDC in the remaining nigrostriatal DAsystem. As for PD, both [I-123]beta-CIT binding (Seibylet al., 1995; Laihinen et al., 1995) and [F-18]FDOPAuptake (Brooks et al., 1990; Brooks, 1991) were corre-lated with the severity of PD symptoms. However, thesuperiority of either the DAT or [F-18]FDOPA tech-niques for measurement of the remaining DA systemfunction in PD remains unclear.

The purpose of this study was to clarify the relativeadvantages of both techniques for evaluation of DAneuron function. Using rat models of early to advancedstage of PD, we compared [I-125]beta-CIT binding and[C-14]L-DOPA uptake in adjacent brain sections fromthe same animals. In addition, to examine the discrep-ancy between [C-14]L-DOPA uptake and [I-125]beta-CITbinding, we examined the expression levels of DAT, DDC,and vesicular monoamine transporter (VMAT2) genes.

MATERIALS AND METHODS

All operational procedures were approved by the localethics committee of Osaka University Medical School.

Animals

Seventeen young female Wistar rats, weighing 80–100 g at the start of the experiment, were used in thisstudy.

6-OHDA lesion

The procedure was essentially the same as previouslyreported (Nishino et al., 1990). Briefly, rats were anes-

thetized with 40 mg/kg, i.p. of sodium pentobarbital(Pitman-Moore) and administered atropine sulfate (0.01mg/kg, i.p.) before being placed on a stereotaxic appara-tus. 6-OHDA (Sigma, St. Louis, MO, USA) solution (2.0mg/ml in 1 ml saline containing 0.5 mg/ml ascorbatesaline) was then injected into the right SNpc (A: 2.0 mmfrom the interaural line, L: 1.6 mm, V: 7.0 mm downfrom the surface of the dura; the nose bar was set 2.4mm below the interaural line) (Konig and Klippel,1963). Rat models of early to advanced stage PD weremade with the injection of 1 µg (n 5 9), 2 µg (n 5 4), 3µg (n 5 3), and 10 µg (n 5 1) of 6-OHDA (2.0 mg/ml).

Rotational behavior

Motor disturbance was assessed by counting fullrotations per minute in a cylindrical container (33 cmdiameter) at 10-min intervals for the first 60 min aftermethamphetamine (3 mg/kg, i.p.) administration(Ungerstedt and Arbuthnott, 1970). These tests wereperformed 1 and 3 weeks after induction of the lesion.

Ex vivo autoradiography with L-DOPA

As the levels of dopamine and dopamine receptorremain stable within 4 weeks after induction of thelesion (Marcotte et al., 1994; Neve et al., 1982), at 4weeks after 6-OHDA injection the rates were injectedi.p. with 25 mg/kg of benserazide (Research Biochemi-cals, Inc., Natick, MA), an inhibitor of peripheral DDC,20 min prior to i.p. injection of [C-14]L-DOPA (92.5MBq/ml, Amersham, Bucks, UK) (370KBq/rat). Onehour after [C-14]L-DOPA injection, the rats were decapi-tated under diethyl ether anesthesia. The brains werequickly removed and frozen in powdered dry-ice. Coro-nal sections of 20 µm thickness including the caudate-putamen (CPu) and 16 µm thickness including theSNpc were cut on a cryostat, thaw-mounted onto silane-coated slides, stored at 240°C and dried rapidly with astream of cool air before being used for autoradiographyand in situ hybridization histochemistry.

Slide-mounted tissue sections and calibrated stan-dards ([C-14]Microscale, Amersham) were placed inX-ray cassettes with an imaging plate (BAS-III, FujiPhoto Film Co., LTD, Kanagawa, Japan).

The level of [C-14]L-DOPA uptake in four sections ineach animal was measured by utilizing imaging soft-ware on a microcomputer system (BAS2000, Fuji). Ineach section, the level of [C-14]L-DOPA uptake wasevaluated as the mean uptake of the entire CPu areatogether with the level of nonspecific uptake in theparieto-occipital cortex of the intact side. The density ofthe uptake was expressed in fmol/mg tissue accordingto the optical density of the calibrated standards. Wecompared the specific uptake of [C-14]L-DOPA in thelesioned CPu with that in the intact CPu.

Percent L-DOPA was expressed by the formula: %L-DOPA 5 [(total uptake of [C-14]L-DOPA in the le-

179PARKINSON’S DISEASE IN ANIMAL MODELS

sioned side 2 nonspecific uptake)/(total uptake of [C-14]L-DOPAin the intact side 2 nonspecific uptake)] 3 100.

In vitro autoradiography with [I-125]beta-CIT

The procedure was the same as previously reported(Fujita et al., 1996). Sections adjacent to those used forautoradiography with [C-14]L-DOPA were used in thisexperiment. Slides were preincubated in buffer (50 mMTris-HCl containing 100 mM NaCl, pH 7.4, at 4°C) for10 sec, then incubated in buffer containing 0.1 nM[I-125]beta-CIT[81.4TBq/mmol] (New England Nuclear,Boston, MA) and 100 nM clomipramine (RBI) for 2 h at4°C to measure total binding, 0.1 nM [I-125]beta-CIT,and 100 nM clomipramine and 100 µM(2)-cocaine todetermine nonspecific binding. Incubation was termi-nated by two consecutive 1-min washes in fresh ice-coldbuffer. After a final dipping in ice-cold distilled water,the slides were rapidly dried with a stream of cool air.Slide-mounted tissue sections and calibrated standards([I-125]Microscale, Amersham) were placed in X-raycassettes along with an imaging film (X-OMAT AR,Kodak).

The level of [I-125]beta-CIT binding in four sectionsfrom each animal was measured by utilizing imagingsoftware on a microcomputer system (MCID ImageAnalysis System, Imaging Research, St. Catharines,Ontario). The level of [I-125]beta-CIT binding in CPuwas measured as the mean level of binding in the wholeCPu. The density of binding was expressed in the sameway as for [C-14]L-DOPA uptake.

Percent beta-CIT was expressed by the formula: %beta-CIT 5 (total binding of [I-125]beta-CIT in thelesioned side 2 nonspecific binding of [I-125]beta-CITin the lesioned side)/(total binding of [I-125]beta-CIT inthe intact side 2 nonspecific binding of [I-125]beta-CITin the intact side) 3 100.

DAT, DDC, and VMAT2 gene expressions

The procedure in situ hybridization histochemistrywas essentially the same as previously reported (Fujitaet al., 1993). In brief, sections were warmed to 22°C,incubated with buffer (4 3 SSC, 50% deionized for-mamide, 0.12 M phosphate buffer (pH 7.2), Denhardt’ssolution, 2.5% tRNA, 10% dextran sulfate) containing[a-35S]dATP (37–55.5TBq/mmol, NEN) labeled hybrid-ization probes (6–9 3 106 dpm/ml, 0.2 ml/slide) for 24 hat 42°C. After hybridization, the sections were rinsedfour times in 1 3 SSC at 55°C for 15 min, dehydratedthrough a graded ethanol series, coated with KodakNTB-2 emulsion diluted 1:1 with water, and exposed for2 weeks in a tightly sealed dark box at 4°C. Sectionswere developed with D-19 developer (Kodak), fixed withphotographic fixer, washed with tap water, counter-stained with a thionine solution, and overlapped with acoverslip.

Oligonucleotide hybridization probes complementaryto bases 87–135 of the rat DAT mRNA (Shimada et al.,1991), 79–117 of the rat DDC (Tanaka et al., 1989), and72–112 of the rat VMAT2 (Erickson et al., 1992) wereused. A computer-assisted homology search for DAT,DDC, VMAT2, mRNA showed a maximal 63.3%, 71%,and 70.7% homology with any sequence in a BLASTSearch, respectively (Release, 26 March, 1996). Thespecificity of hybridization signals was studied in com-petition experiments with an excess of unlabeled probes.Identification of positive cells expressing mRNAs andevaluation of the expression levels were done as previ-ously described (Noguchi et al., 1988). The number ofsilver grains was counted in each 25 µm2 area. Expres-sion of mRNAs was studied in six sections from rostal tocaudal SNpc.

Percent DA neuron was expressed in the formula: %DA neuron 5 (the number of DAT mRNA positiveneurons in the lesioned side)/(the number of DATmRNA positive neurons in the intact side) 3 100.

In addition, percent expression of DAT, DDC, andVMAT2 was expressed by the formula: % expression ofDAT, DDC, VMAT2 5 (the number of silver grains ineach 25 µm2 area in the lesioned side)/(the numberof silver grain in each 25 µm2 area in the intact side) 3100.

Statistical analysis

Comparison of the levels of [C-14]L-DOPA uptakewith that of [I-125]beta-CIT binding was done with thepaired Student’s t-test. One-way ANOVA followed byFisher’s protected least significant difference (PLSD)post-hoc test were used for intergroup comparisons ofthe expression level of DAT, DDC, and VMAT2 mRNAs.Linear regression analysis and the determination ofPearson’s product-moment correlation coefficient (r)were used to test the correlation between percent DAneuron and that of percent L-DOPA or percent beta-CIT.Simple linear regression and the regression throughthe origin were also applied to this analysis. Correla-tion coefficients were compared by Fisher’s Z-transfor-mation. Data were expressed as mean 6 SEM andprobability values less than 0.05 were considered statis-tically significant.

RESULTSLoss of DA neurons

Equivalent numbers of DAT, DDC, and VMAT2mRNA positive neurons were seen at any stage model.Representative results of DAT mRNA positive neurondistribution are shown in Figures 1 2, and 5. Afterinjection of 1–10 µg of 6-OHDA, the proportion of DAneurons ranged from ,2% to .90%. It was 70% or morein five animals, 25–70% in six and 25% or less in sixanimals.

180 ITO ET AL.

Rotational behavior

In general, rats with more than 50% surviving DAneurons showed little or no rotation, while those withless than 50% surviving DA neurons showed precipi-tous rotation. At 3 weeks after induction of lesion, eightof nine rats with less than 50% DA surviving neurons

showed apparent motor disturbance. On the otherhand, only one of eight rats with more than 50% survivingDA neurons showed an apparent motor disturbance (Fig.1A). A similar correlation was noted between percentL-DOPAor percent beta-CIT and the number of metham-phetamine-induced rotations (Fig. 1B,C).

Fig. 1. The relationship between methamphetamine-induced rota-tional behavior at 3 weeks after induction of the lesion and theremaining percentage of the DA neurons (A), percent striatal [C-14]L-DOPA specific uptake (B) and percent striatal [I-125]beta-CIT specificbinding (C). In general, rats with less than half of the DA neuronsshowed apparent motor disturbance (A). On the other hand, ratsretaining more than half of the DA neurons did not show any motordisturbance. A similar correlation was noted between percent L-DOPAuptake (B) or percent beta-CIT binding and the remaining percentageof DA neurons (C).

Fig. 2. Solid and dashed lines show linear regression and 95%confidence interval, respectively. The remaining percentage of DAneurons in SNpc versus percent of striatal [C-14]L-DOPA specificuptake (A) and percent striatal [I-125]beta-CIT specific binding (B).Both percent striatal [C-14]L-DOPA specific uptake and percentstriatal [I-125]beta-CIT specific binding were strongly correlated withthe remaining percentage of dopaminergic neurons in the lesionedsubstantianigra (y 5 1.063 1 12.6, r 5 0.91,P , 0.0001,y 5 1.093 2 2.06,r 5 0.93, P , 0.0001, respectively). In the autoradiography of L-DOPA,nonspecific uptake was determined by the uptake in the parieto-occipital cortex in the intact side. In the autoradiography of beta-CIT,nonspecific binding was evaluated by displacement with cocaine.Binding of beta-CIT to the serotonin transporter was blocked withclomipramine.

181PARKINSON’S DISEASE IN ANIMAL MODELS

Comparison of the decrease of [C-14]L-DOPAuptake with that of [I-125]beta-CIT binding

In general, both percent L-DOPA and percent beta-CIT decreased in parallel with the decrease in DAneurons from early to advanced stage models of PD.Both percent L-DOPA and percent beta-CIT werestrongly correlated with percent DA neuron (Fig. 2;y 5 1.063 1 12.6, r 5 0.91, P , 0.0001, y 5 1.093 2 2.06,r 5 0.93, P , 0.0001, respectively). There was no signifi-cant difference between the regression coefficients forpercent DA neuron versus percent L-DOPA and that forpercent DAneuron versus percent beta-CIT (P 5 0.638).

However, comparison of percent L-DOPA versus per-cent beta-CIT in adjacent brain sections of the sameanimal gave significantly higher values for percentL-DOPA, particularly in moderate to advanced stagemodel of PD (Figs. 3, 4; P , 0.0001).

In addition, while the 95% confidence interval of theregression intercept for percent DA neuron versuspercent beta-CIT was centered around 0 (214.4 to 10.3,Fig. 2B), that for percent DA neuron versus percentL-DOPA was greater than 0 (23.3 to 25.5, Fig. 2A). Theintercept for the regression between percent DA neuronversus percent L-DOPA was significantly higher thanthat for the regression between percent DA neuronversus percent beta-CIT (P , 0.001). Therefore, thevalue of percent beta-CIT was closer to that of percentDA neuron in early to advanced stage models of PD.

DAT, DDC, and VMAT gene expression

To clarify this discrepancy between [C-14]L-DOPAuptake and [I-125]beta-CIT binding, we examined theexpression of the DAT, DDC, and VMAT2 genes. Thelevel of expression of each of these genes in each DAneuron in the lesioned SNpc was compared with that inthe intact SNpc in the same section. Specificity ofhybridization signal was confirmed by competition ex-periments with excess unlabeled probe. With the de-crease in DA neurons, the expression of all three genesdecreased similarly, with no apparent difference in thepattern of decrease (Fig. 5). In the advanced stagemodels of PD, the expression of all three genes in thelesioned SNpc was lower than that in the intact side. Incontrast, in the early stage model the expression of allthree genes was equivalent between the lesioned sideand the intact side of the SNpc. The expression of allthree genes in moderate stage model of PD (percent DAneuron: 70% or more) was significantly lower than thatin early stage model of PD (percent DA neuron: 70% ormore) (P , 0.01), but significantly higher than that inthe advanced stage model of PD (percent DA neuron:25% or less) (P , 0.05) (Fig. 5).

DISCUSSION

The loss of DA neurons is the cause of PD and thisdecrease has been studied mainly with two types of

radiolabeled tracers in positoron emission tomography(PET) or single photon emission tomography (SPECT).One is [F-18]FDOPA, an analogue of L-DOPA (Brooks etal., 1990; Brooks, 1991), and the others are DA trans-porter ligands such as [I-123]beta-CIT (Laihinen et al.,1995; Seibyl et al., 1995) and other ligands (Eidelberg etal., 1993) in SPECT. Although the decreased uptake of[I-123]beta-CIT-FP and [F-18]FDOPA was reported tobe corrected in a small group of PD patients (Ishikawaet al., 1996), few combined studies using both types oftracers have been published and it remains unclearwhich technique is the most accurate for the detectionof DA neuron decrease. Neuroprotective therapy, anintervention which may slow the progression of degen-eration in residual neurons (Parkinson Study Group,1989, 1996; Tetrud and Langston, 1989), has under-lined the importance of measurement of the remainingnumber of DA neurons. Using a rat model of early toadvanced stage PD, we compared [I-125]beta-CIT bind-ing and [C-14]L-DOPA uptake in adjacent sections fromthe same animals.

Both [I-125]beta-CIT binding and [C-14]L-DOPA up-take decreased in parallel with the decrease of DAneurons from early to advanced stage models of PD(Fig. 2). However, percent L-DOPA uptake was signifi-cantly greater than percent beta-CIT binding (Fig. 3),particularly in moderate stage models of PD(P , 0.0001) and the regression intercept between per-cent DA neuron versus percent L-DOPA did not inter-sect with the origin and was significantly greater thanthat between percent DA neuron versus percent beta-CIT (Fig. 2). These results are consistent with reports ofunchanged uptake of [F-18]FDOPA and decreased DATbinding with age (Eidelberg et al., 1993; Sawle et al.,

Fig. 3. Percent striatal [C-14]L-DOPA specific uptake versus per-cent striatal [I-125]beta-CIT specific binding. Percent striatal [C-14]L-DOPA specific uptake was significantly greater than percent striatal[I-125]beta-CIT specific binding. (P , 0.0001) (paired Student’s t-test).

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1990; Van-Dyck et al., 1995; Volkow et al., 1996a).These findings suggest the superiority of the [I-125]beta-CIT binding assay as an indicator of DA neuron loss.

A certain level of dopaminergic degeneration (50–80%) must be reached for symptoms to become appar-ent (Gauthier and Sourkes, 1982; Jellinger, 1987), atwhich point neuroprotective therapy should be started.In our study, the discrepancy between L-DOPA uptakeand beta-CIT binding reached its highest level inmoderate stage models of PD (Figs. 2, 3), which furthersuggests that the [I-125]beta-CIT binding is a superiorindicator of DA neuron loss.

In the present study, we decapitated rats 1 h after i.p.injection of [C-14]L-DOPA and then evaluated [C-14]L-DOPA uptake as the mean of the entire area of CPu. Weadministered [C-14]L-DOPA i.p. because preliminaryexperiments with i.v. injection showed a low signal-to-noise ratio. Since a number of representative[F-18]FDOPA PET studies selected a reference regionfor nonspecific uptake in the parieto-occipital cortex(Eidelberg et al., 1993; Martin et al., 1989; Tetrud andLangston, 1989), we chose the same region. The signal-to-noise ratio in our work was similar to that of the PETstudy (Martin et al., 1989) and percent L-DOPA de-creased together with percent DA neuron. Therefore,[C-14]L-DOPAuptake in this study reflected the remain-ing DA system activity. On the other hand, the signal-to-noise ratio of [C-14]L-DOPA uptake was lower than thatof [I-125]beta-CIT binding. However, the SEM of [C-14]L-

DOPA uptake in each section was equivalent to that of[I-125]beta-CIT binding (data not shown). Thus, thelow signal-to-noise ratio is not the source of the discrep-ancy between [C-14]L-DOPA uptake and [I-125]beta-CIT binding in the present study. [I-125]beta-CITbinding was measured with in vitro autoradiographybecause of the ease with which values parallel to Bmax

could be obtained. Although the peak uptake time of[F-18]FDOPA in the striatum was not different betweenhealthy subjects and PD patients (Martin et al., 1989;Brooks et al., 1990), that of [I-123]beta-CIT was appar-ently different between these two groups (Laruelle etal., 1994).

The discrepancy between L-DOPA uptake and beta-CIT binding may be due to up- or downregulation ofDAT, DDC, or VMAT2 gene expression; one reason whywe examined the expression level of three genes with insitu hybridization histochemistry was in order to clarifythis discrepancy. However, in situ hybridization histo-chemistry failed to detect any difference in levels ofexpression of these genes with different levels of[I-125]beta-CIT binding and [C-14]L-DOPA uptake (Fig.5), indicating that the regulation of DAT, DDC, orVMAT2 activity might occur at the protein level but notat the mRNA level. On the other hand, Bannon et al.(1992) showed that the postmortem DAT transcriptlevels in the substantia nigrae of 18–57-year-old sub-jects were relatively constant, but in older subjects aprecipitous decline (.95%) in DAT transcripts was

Fig. 4. In situ hybridizationhistochemistry detecting the do-pamine transporter mRNA-posi-tive neurons, ex vivo autoradiog-raphy with L-DOPA and in vitroautoradiography with beta-CITin advanced and moderate stagemodels. The lesioned side is onthe right side of the figure. Inthe advanced stage model, thedecrease in L-DOPA uptake wasalmost equal to that of beta-CITbinding. However, in the moder-ate model the decrease of beta-CIT binding was more promi-nent than that of L-DOPAuptake.

183PARKINSON’S DISEASE IN ANIMAL MODELS

evident in the substantia nigra. In contrast, tyrosinehydrpxylse (TH) gene expression in the same samplesdeclined in a linear manner with increasing age (Ban-non et al., 1992). The discrepancy between our resultsand those of the above-mentioned work may be due to amethodological difference.

Alternatively, there may be a discrepancy betweenthe dopamine transporter and L-DOPA uptake system.If glial cells or serotonergic and noradrenergic nerveterminals also have an L-DOPA uptake system but lacka dopamine transporter, this difference may be the

origin of the discrepancy. Recent reports have sug-gested that besides DA neurons, DDC exists in glialcells (Li et al., 1992), serotonergic neurons (Melamed etal., 1981; Tinson et al., 1992), and noradrenargic neu-rons (Bertler et al., 1996).

In conclusion, both beta-CIT binding and L-DOPAuptake decreased in parallel with a decrease in DAneurons from the early to advanced stage models of PD.However, L-DOPA uptake was greater than beta-CITbinding in the moderate to advanced stage model of PDand the y intercept of the regression line for L-DOPA didnot reach the origin. Taken together, these resultsindicate that beta-CIT may be superior in reflecting thedecrease in DA neurons.

ACKNOWLEDGMENTS

We thank Prof. Osamu Inoue (School of Allied HealthScience, Osaka University Faculty of Medicine) forhelpful discussions and Prof. Hitoo Nishino (Depart-ment of Phisiology, Nagoya City University MedicalSchool) for his support in making PD model rats.

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