Brief Communication

Diseases

Neurodegenerative Dis 2004;1:109–112
DOI: 10.1159/000080051

Received: April 28, 2003
Accepted after revision: November 4, 2003

Changes in Dopamine D2 Receptors and6-[18F]Fluoro-L-3,4-Dihydroxyphenylalanine
Uptake in the Brain of 6-HydroxydopamineLesioned Rats
Yasushi Ishida a Keiichi Kawai c Yasuhiro Magata b, d Ryuichiro Takeda a
Hiroyuki Hashiguchi a Hiroshi Abe a Takahiro Mukai b Hideo Saji b
a Department of Psychiatry, Miyazaki Medical College, University of Miyazaki, Miyazaki,b Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences,
Kyoto University, Kyoto, c Department of Radiological Technology, School of Health Sciences, Faculty of Medicine,
Kanazawa University, Kanazawa, and d Laboratory of Genome Bio-Photonics, Photon Medical Research Center,
Hamamatsu University School of Medicine, Hamamatsu, Japan

Key Words
6-[18F]Fluoro-L-3,4-dihydroxyphenylalanine W
6-Hydroxydopamine W Cerebral cortex W Dopamine
receptors W Rat W Striatum

Abstract
We studied tracer distributions in positron emission
tomography of ligands for dopamine D1 receptors([11C]SCH23390) and D2 receptors ([11C]raclopride) andthe dopamine precursor analog 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine ([18F]FDOPA), as a measurement ofpresynaptic dopaminergic function, in the brain after 6hydroxydopamine lesioning of the medial forebrain bundle in rats. The unilateral lesions were confirmed behaviorally by methamphetamine-induced rotation 2 weeks
after lesioning, and the brains were analyzed by tissue
dissection following an intravenous bolus of each tracer
3 weeks after lesioning. [11C]Raclopride, but not[11C]SCH23390, showed a higher accumulation in the
striatum on the lesion side compared with that on the
non-lesioned (intact) side. On the other hand, a lower
accumulation of [18F]FDOPA was found in the striatumABC© 2004 S. Karger AG, Basel
1660–2854/04/0013–0109$21.00/0

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and cerebral cortex on the lesion side. Our studies demonstrate upregulation of dopamine D2 receptors in the
striatum and a decrease in FDOPA uptake in both thestriatum and cerebral cortex ipsilateral to the 6-hydroxydopamine lesions. Therefore, the combination of a D2
antagonist and FDOPA may provide a potentially useful
method for assessing the effects of dopamine depletionin Parkinson’s disease.
Copyright © 2004 S. Karger AG, Basel

Introduction

6-[18F]Fluoro-L-3,4-dihydroxyphenylalanine ([18F]
FDOPA) is a positron-emitting analog of the dopamine
precursor L-DOPA and one of the earliest [18F]-labeled
compounds proposed as imaging agents for use in theexternal in vivo examination of the central nervous dopaminergic system [1]. Therefore, [18F]FDOPA has beenused as an imaging agent in the study of dopamine terminals in the living brain using positron emission tomography [2, 3].

Dr. Yasushi Ishida
Department of Psychiatry, Miyazaki Medical College
University of Miyazaki, 5200 Kihara
Kiyotake-cho, Miyazaki 889-1692 (Japan)
Tel. +81 985 85 2969, Fax +81 985 85 5475, E-Mail ishiday@med.miyazaki-u.ac.jp

Experimental animals in which the nigrostriatal pathway is destroyed are considered to be useful models of
Parkinson’s disease. One popular model in rats is produced by the unilateral stereotaxic injection of the neurotoxin 6-hydroxydopamine (6-OHDA) into the medial
forebrain bundle or substantia nigra [4]. In this model,
there is an immediate and almost complete destruction of
the dopamine neurons of the substantia nigra and of theventral tegmental area, resulting in near total depletion(2% of normal) of dopamine in the ipsilateral striatum tothe 6-OHDA injections [5]. Immunohistochemical study
showed there is no evident damage of dopaminergic neurons and fibers in the brain contralateral to the 6-OHDA
injections [5, 6]. There have been many reports of enhanced behavioral (rotational behavior) effects of directly
acting dopamine agonists such as apomorphine after unilateral 6-OHDA lesioning. The rotational behavior induced by dopamine agonists has been attributed to postsynaptic dopamine receptor supersensitivity in the striatum that occurs following the unilateral destruction ofmesostriatal dopamine fibers [7]. From these observations, it is conceivable that the 6-OHDA-lesioned rat
model may be useful for evaluating brain functions in
Parkinson’s disease. However, little is known aboutchanges in dopamine receptors and FDOPA uptake in thebrain following 6-OHDA lesioning under the same experimental conditions.
In the present study, therefore, we studied the tracer distributions of ligands for dopamine D1 receptors([11C]SCH23390), D2 receptors ([11C]raclopride) and of[18F]FDOPA in the brain after 6-OHDA lesioning of themedial forebrain bundle in rats.

[11C]SCH23390 was synthesized by the [11C]methylation reaction of SCH24518 in an automated synthesis apparatus (CUPID
C-100, Sumitomo Heavy Industries, Tokyo, Japan), according to themethod reported by Halldin et al. [9], with a slight modification. The
radiochemical purity of [11C]SCH23390 was 1 97.4%. The specificactivity of the product was 38.5–81.4 GBq/Ìmol. [11C]Raclopride
was synthesized by [11C]methylation of O-desmethylraclopride in the
CUPID C-100. The radiochemical purity of [11C]raclopride was
1 98.4%. The specific activity of the product was 32.6–88.9 GBq/
Ìmol. [18F]FDOPA was synthesized in a multipurpose synthetic system [10], according to the method reported by Namavari et al. [11],with a slight modification. The radiochemical purity of [18F]FDOPA
was 1 98.5%. The specific activity of the product was 33.0–33.7
MBq/Ìmol.
The regional distribution of radioactivity in the rat brain was
examined after intravenous injection of [11C]SCH23390, [11C]raclopride or [18F]FDOPA 3 weeks after the 6-OHDA lesions. In previous
studies, many investigators reported that both degeneration of nigral
dopaminergic neurons and dopamine receptor supersensitivity in the
denervated striatum have been established at this time point after
6-OHDA lesioning [12, 13]. One hour ([11C]SCH23390 and [11C]raclopride) or 1.5 h ([18F]FDOPA) after the intravenous injection, therats were killed by decapitation, and the brains (each side) were dissected on ice into the different brain areas (striatum, cerebral cortex
and cerebellum) as described by Glowinski and Iversen [14] for the
rat brain. Brain samples were weighed, and radioactivity accumulation of each tracer was determined for each brain region bilaterally. Radioactivity was measured with a scintillation counter(model 5003, Packard, Downers Grove, Ill., USA). Firstly, results
were expressed as radioactivity per unit wet tissue weight (cpm/g).
Then, the biodistribution of [11C]SCH23390, [11C]raclopride or[18F]FDOPA was computed as a ratio of the lesion side to the intact
side of each brain region of the 6-OHDA rats.
Data of the lesion side/intact side ratio of each brain region wereanalyzed nonparametrically using the Wilcoxon signed rank test.
p values ! 0.05 were regarded as significant.

Results
Materials and Methods

Table 1 shows the biodistribution of [11C]SCH23390,[11C]raclopride and [18F]FDOPA in three brain regions

The subjects were male Wistar rats (Kiwa Laboratory Animals,
Kaisou, Japan) weighing 120–130 g at the beginning of the experiment. The experimental protocols used in this study were approvedby the ethical committees of animal experimentation at the Miyazaki
Medical College and Kyoto University. Rats were anesthetized withpentobarbital (40 mg/kg, i.p.), and unilateral lesions of the left medial
forebrain bundle were made by injecting 8 Ìg of 6-OHDA hydrobromide (Sigma, St. Louis, Mo., USA) in 4 Ìl of sterile saline containing
0.01% ascorbic acid. Stereotaxic coordinates for the lesions were as
follows: anteroposterior 3.2 mm rostral to the interaural line, 1.3 mm
to the left of the midline, and 6.7 mm ventral to the dural surface,with the incisor bar set 2.4 mm below the level of the ear bars [8].
Two weeks after the 6-OHDA treatments, the relative completenessof the lesions was confirmed by testing for rotation following intraperitoneal injection of methamphetamine (3 mg/kg) [5, 6]. Animals
accomplishing no less than 7 turns/min on the methamphetamine
challenge were included in the study.

110

Neurodegenerative Dis 2004;1:109–112(cerebral cortex, striatum, and cerebellum) on the lesionside of the 6-OHDA rats, represented as the ratio to theintact side of the brain. In the striatum on the lesion side,
the biodistribution of [11C]raclopride was significantly
higher (p ! 0.01; 191% of the intact side) than that of thestriatum on the intact side of the 6-OHDA rats. On theother hand, the biodistribution of [18F]FDOPA was lower
in both the striatum (p ! 0.05; 80% of intact side) andcerebral cortex (p ! 0.05; 89% of intact side) on the lesionside compared with the intact side. Regarding the three
regions, there was no significant difference in the biodistribution of [11C]SCH23390 between the lesion and intact
sides in any of the three regions of the brain.

Ishida/Kawai/Magata/Takeda/Hashiguchi/
Abe/Mukai/SajiTable 1. Biodistribution of each tracer in three brain regions of the
6-OHDA rats

TracerRegionLesion side/intactside, cpm/g

e[11C]SCH23390(n = 7)

cerebral cortex
striatumcerebellum

1.04B0.03
1.11B0.02
1.00B0.00[11C]Raclopride(n = 9)cerebral cortex
striatumcerebellum

1.14B0.02
1.91B0.04**
1.01B0.01[18F]FDOPA(n = 9)cerebral cortex
striatumcerebellum

0.89B0.02*
0.80B0.01*
0.99B0.00* p ! 0.05, ** p ! 0.01, vs. corresponding values of the intact side
(Wilcoxon signed rank tests).

Discussion

In the present study, the biodistribution of[11C]SCH23390 did not change in any brain region of the
6-OHDA animals. An upregulation of dopamine D1 receptors after lesioning of the medial forebrain bundle has
been demonstrated in receptor autoradiographic studies[15, 16]. Furthermore, supersensitivity of dopamine D1
receptors has been reported in striatal tissue homogenates
after medial forebrain bundle lesioning with 6-OHDA[17, 18]. In contrast, there are other contradicting results
showing no change or a decrease in the density of striatal
dopamine D1 receptors after denervation of the nigrostriatal dopaminergic pathway [19, 20]. The precise reason for these discrepancies is unknown. However, the discrepancies may be related to different methods (e.g. radioligand binding assay, autoradiography) used and/or
different time points after 6-OHDA lesioning in the earlier studies.
On the other hand, denervation of the nigrostriatal
pathway can cause the postsynaptic population of dopamine D2 receptors to become supersensitive [15, 16].
These observations are consistent with our present findings. Furthermore, consistent with the present findings,
Araki et al. also observed that the upregulation in D2
receptors was more pronounced than that in D1 receptors
in the striatum after 6-OHDA lesioning [21, 22].
The present study also showed that [18F]FDOPA uptake significantly decreased in both the striatum and cerebral cortex ipsilaterally after unilateral 6-OHDA lesion-

Cerebral [11C]Raclopride and [18F]FDOPA
Uptake in 6-OHDA Rats

ing. The degree of the decrease in the cerebral cortex,
however, was relatively slight compared with that in the
striatum. A lowered presynaptic dopamine function was
found not only in the striatum but also in the cerebral cortex on the lesion side. The [18F]FDOPA uptake wasreported to increase [23] or decrease [24] in the cerebral
cortex in patients with Parkinson’s disease. Taken together with the evidence that the degree of dopamine denervation in the 6-OHDA animals was almost complete [5], thepresent findings suggest that the [18F]FDOPA uptake may
decrease in the cerebral cortex in parkinsonian patients inan advanced stage. Further studies on the tracer distributions in rats with partial 6-OHDA lesions and/or in theacute stage (e.g. days 1–7) following the complete 6OHDA lesioning may clarify the issue.
FDOPA is an exogenous substrate of DOPA decarboxylase which is the enzyme directly responsible for the synthesis of dopamine and serotonin, and indirectly of noradrenaline. In the central nervous system, DOPA decarboxylase is localized in both monoaminergic neurons [25]and a subset of non-monoaminergic neurons [26]. In the
cerebral cortex, afferent fibers of serotonin and noradrenaline are distributed in most areas [27]. Previous cerebral
dialysis and immunohistochemical studies showed aslight decrease in serotonin overflow and immunoreactivity, respectively, in the denervated striatum in 6-OHDA
rats [5]. Thus at least some of the cortical FDOPA uptake
could be accounted for serotonergic and/or noradrenergic
neuron innervation.
In conclusion, the present study demonstrates upregulation in dopamine D2 receptors in the striatum anddecrease in FDOPA uptake in both the striatum and cerebral cortex ipsilateral to the 6-hydroxydopamine lesions. Therefore, the combination of a D2 antagonist and
FDOPA may provide a potentially useful method forassessing the effects of dopamine depletion in Parkinson’s
disease. However, we must be careful not to interpret theresults illogically. In spite of many similarities, the characteristics of acute 6-OHDA lesions may be rather different
from the slowly progressive course of Parkinson’s diseasein humans.

Acknowledgments
This research was supported by grants-in-aid for scientific research (11470193, 14370273, 14570935) and a 21st century COE
program at the Miyazaki Medical College from the Japan Society for
the Promotion of Science. We are grateful to Miss Okitsu (Kanazawa
University) for technical assistance.

Neurodegenerative Dis 2004;1:109–112

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Ishida/Kawai/Magata/Takeda/Hashiguchi/
Abe/Mukai/Saji