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Experimental Neurology 187 (2004) 178–189

Loss of D3 receptors in the zitter mutant rat is not reversed

by L-dopa treatment

Jeffrey N. Joyce,a,* T.C. Der,a,b Lynn Renish,a Tracy Osredkar,a Diane Hagner,a Maria Reploge,a

Shinichi Sakakibara,c and Shuichi Uedac

aThomas H. Christopher Center for Parkinson’s Disease Research, Sun Health Research Institute, Sun City, AZ 85351, USAbDepartment of Anatomy and Neurosciences, Arizona College of Osteopathic Medicine, Midwestern University, Glendale, AZ 85308, USA

cDepartment of Histology and Neurobiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan

Received 11 September 2003; revised 19 November 2003; accepted 13 January 2004

Abstract

In Parkinson’s disease (PD) and animal models of parkinsonism the destruction of nigrostriatal (NSB) system results in a marked loss of

the dopamine D3 receptor and mRNA in the islands of Calleja (ICj) and the nucleus accumbens shell (NAS). In animal models, it has been

reported that both measures are elevated by repeated intermittent administration of L-dopa. However, a large proportion of PD cases are

resistant to L-dopa-induced elevation of D3 receptor number. The zitter mutant (Zi/Zi) rat replicates the slow progressive degeneration of the

NSB observed in PD and also exhibits a loss of D3 receptor number in the NAS or ICj. To test if this could be reversed with subchronic

L-dopa treatment, injections of carbidopa (10 mg/kg ip) were followed an hour later with injection of L-dopa (100 mg/kg ip) twice a day for

10 days. In control Sprague–Dawley (SD) and zitter heterozygote (Zi/�) rats that do not show a loss of D3 receptors with vehicle treatment,

L-dopa produced no change in D3 receptor number or in DA terminal density as measured by dopamine transporter (DAT) binding and

tyrosine hydroxylase immunoautoradiography (TH-IR). There was a marked loss of DAT and TH-IR in caudate–putamen (CPu) and NA, as

well as D3 receptors in NAS and ICj in Zi/Zi rats but no further change with L-dopa treatment. To determine if the resistance to L-dopa-

induced increase in D3 receptor was due to a deficiency in expression of cortical BDNF or its receptor, TrkB, in CPu and NAS, we examined

BDNF mRNA by ISHH in frontal cortex and TrkB mRNA in frontal cortex, CPu, and NA. The loss of the NSB in the Zi/Zi did not alter

levels of BDNF or TrkB mRNA, nor did L-dopa administration alter levels BDNF or TrkB mRNA. Thus, unlike in 6-hydroxydopamine-

treated rats, in Zi/Zi rats administered L-dopa does not reverse the loss of BDNF mRNA or lead to an elevation of D3 receptor number.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Parkinson’s disease; Striatum; Nucleus accumbens; Tyrosine hydroxylase; Substantia nigra; Ventral tegmental area

Introduction to these antiparkinsonian drugs (APDs) and they are inef-

Parkinson’s disease (PD) is a neurodegenerative disorder

with an insidious onset and a prolonged course over many

years. Symptoms are secondary to the death of nigrostriatal

(NSB) DA neurons and the resultant depletion of DA in the

striatum (Hornykiewicz, 1998). Symptomatic treatment

includes levodopa (L-dopa, the most effective antiparkinso-

nian drug), which is converted to DA, and direct-acting DA

agonists (bromocriptine, pergolide, pramipexole, ropinirole)

(Brooks, 2000). However, many patients lose their response

0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.expneurol.2004.01.012

* Corresponding author. Thomas H. Christopher Center for Parkinson’s

Disease Research, Sun Health Research Institute, 10515 West Santa Fe

Drive, Sun City, AZ 85351. Fax: +1-623-876-5695.

E-mail address: Jeff.Joyce@sunhealth.org (J.N. Joyce).

fective for cooccurring dementia and depression (Fabbrini et

al., 1988; Mouradian et al., 1988). Dementia is in fact highly

correlated with the severity of parkinsonism, older age of

onset, and declining response to APDs (Apaydin et al.,

2002; Caparros-Lefebvre et al., 1995; Hughes et al., 2000;

Hurtig et al., 2000; Marder et al., 1995; Portin and Rinne,

1987; Stern et al., 1993). Loss of response, and its correla-

tion with dementia, is differentiated from a complicated

response to APDs or dyskinesias, which can be managed by

titrating the dosage of medication.

Five distinct subtypes of G-protein coupled DA receptors

mediate the actions of DA, three of which, D2, D3, and D4,

belong to the D2 subfamily (Civelli et al., 1993). DA

regulates output pathways of the striatum via interaction

with different DA receptors that are, in turn, differentially

J.N. Joyce et al. / Experimental Neurology 187 (2004) 178–189 179

expressed in these output pathways (Joyce, 2001b). How-

ever, to date, all effective DA agonists stimulate D2-like

receptors, and changes in expression of the D2 receptor have

been hypothesized to underlie alterations in the response to

APDs in PD (Pizzolato et al., 1995). In a recent review

(Joyce, 2001b), we discussed the clinical and preclinical

literature that supports the hypothesis that the initial expres-

sion of parkinsonian symptoms is not only correlated with

the loss of DA innervation and an elevation in receptor

number of the D2 subclass, but also a decrease D3 receptor

number. In animal models of PD, it has been demonstrated

that the reduction of D3 receptor number after the loss of

DA innervation can be reversed by L-dopa in DA denervated

animals (Bordet et al., 1997; Morissette et al., 1998; Quik et

al., 2000). However, we have found in PD that the mainte-

nance of response to APDs (and the absence of PD

dementia) is associated with maintenance or an increase in

D3 receptor number, and conversely that loss of response to

APDs (and presence of PD dementia) is associated with a

decrease in DA D3 receptor number (Joyce et al., 2002;

Ryoo et al., 1998). Interestingly, measurement of DA

terminals with tyrosine hydroxylase (TH) immunoautora-

diography and dopamine transporter (DAT) binding in

striatum, as well as unbiased stereological counts of TH-

IR neurons in the midbrain, showed that damage to the

nigrostriatal and mesolimbic DA system was not different in

PD cases who had loss of response to APDs (and dementia)

compared to PD cases who maintained response to APDs

(and who were free from clinical symptoms of dementia)

(Joyce et al., 2002).

In view of the above findings, it becomes important to

ask why L-dopa does not appear capable of normalizing

deficient D3 receptor expression in PD cases that are

unresponsive to APDs. Unlike that for the D2 receptor,

depletion of DA fibers by 6-hydroxydopamine (6-OHDA)

or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

leads to a reduction of D3 receptor number (Joyce, 1991;

Joyce et al., 1986; Levesque et al., 1995; Morissette et al.,

1998; Wade et al., 2001). The relationship between regional

loss of DA fibers and loss of postsynaptically located D3

receptors has not been clarified, but it is clear that regulation

of D3 receptors is not directed by levels of DA per se (Joyce,

2001a; Joyce et al., 2000; Levesque et al., 1995). Guillin et

al. (2001) have proposed that the D3 receptor is regulated by

brain-derived neurotrophic factor (BDNF). The primary

source of BDNF in striatum is from DA fibers (Altar et

al., 1997) and its reduction following lesions to the nigros-

triatal DA system is thought to correlate with reduced D3

receptor. In fact, it has been proposed that the elevation of

D3 receptor number in L-dopa-treated DA-denervated ani-

mals is regulated by an increased production of BDNF in

corticostriatal fibers (Guillin et al., 2001).

Based on those studies in animal models of PD, it is not

clear why L-dopa administration loses its ability to normal-

ize or elevate D3 receptor number in a subset of PD cases

(Joyce et al., 2002). One possibility is that animals used to

explore regulation of D3 receptor number and response to L-

dopa utilize rapid and massive depletions of DA neurons or

DA fibers. We have explored the regulation of the D3

receptor in a more natural animal model of PD that exhibits

a progressive degeneration of the DAergic and serotonergic

system over a 12-month period, the zitter (Zi/Zi) mutant rat

(Joyce et al., 2000). The Zi/Zi rat is an autosomal recessive

mutant derived from the Sprague–Dawley (SD) rat strain

(Ueda et al., 2002). The Zi/Zi rat exhibits several neurolog-

ical abnormalities that are age progressive. Abnormal me-

tabolism of H202 and catalase leads to an increase in oxygen

species in the brain that is correlated with a significant

reduction in DA and dihydroxyphenylacetic acid (Gomi et

al., 1994; Ueda et al., 2002). In the Zi/Zi rat, there is a

characteristic pattern of DA fiber and neuron degeneration

that is observed initially in the nigrostriatal DA system

followed by a slower degeneration to the mesolimbic DA

system (Ueda et al., 2000). However, even by 3 months of

age when DA levels and fiber density are normal in the

nucleus accumbens (NA) of the Zi/Zi rat, there is a pro-

nounced loss (>70%) of D3 receptor. In the present paper,

we show that the loss of D3 receptors in the Zi/Zi rat is not

normalized or elevated by subchronic L-dopa treatment.

Furthermore, subchronic L-dopa treatment does not produce

an increase in cortical BDNF mRNA, nor are there changes

in TrkB mRNA in striatum of Zi/Zi rats.

Materials and methods

Subjects

Zitter mutant (Zi/Zi), zitter heterozygote (Zi/+) rats were

raised and maintained in the Laboratory Animal Research

Center, Dokkyo University School of Medicine. For Exper-

iment 1, the Sprague–Dawley (SD) controls were also

raised and maintained at the same institution. For Experi-

ment 2, the SD controls were purchased from Charles River

(6 and 10 months old retired breeders) and maintained at

Sun Health Research Institute. All animals were housed in

groups of two or three with continuous access to food and

water and maintained on a 12:12-h light–dark cycle.

Drug treatment

Experiment 1

At 6 or 10 months age, four Zi/Zi and four Zi/+ zitter

male rats were injected with carbidopa (10 mg/kg ip; Sigma,

St Louis MO), a peripheral decarboxylase inhibitor. An hour

later, the animals were given an injection of L-dopa (100

mg/kg ip; Sigma). This procedure was followed twice a day

for 10 successive days. An additional four Zi/Zi, four Zi/+

zitter, and four SD male rats were treated with the vehicle

saline (1 ml/kg ip) twice daily for 10 successive days. At the

end of the 10th day, the rats were overdosed with pento-

barbital and the brain immediately frozen in isopentane

J.N. Joyce et al. / Experimental Neurology 187 (2004) 178–189180

(�30jC) and stored at �80jC and maintained at �45jCduring shipping to Sun Health Research Institute (Sun

City, AZ).

Experiment 2

In Experiment 1, we observed nonsignificant changes in

some analyses that indicated the need for further substan-

tiation of the data, additional analyses, and additional

protocols. An additional study was undertaken with four

female and two male Zi/Zi rats at 6 or 10 months age and

four female and four male Zi/+ zitter male rats. They were

treated with subchronic L-dopa/carbidopa (100:10 mg/kg

ip) twice a day for 10 days in exactly the same manner as

in Experiment 1. An additional four female Zi/Zi and four

female Zi/+ zitter rats were treated with the vehicle saline

(1 ml/kg) two times daily for 10 successive days. To

obtain SD controls, at Sun Health Research Institute, six

SD male rats at 6 months and six at 10 months age were

treated with subchronic L-dopa/carbidopa, (100:10 mg/kg

ip) twice a day for 10 days in exactly the same manner as

in Experiment 1. An additional five SD rats at 6 months

and 6 at 10 months age were treated with the vehicle

saline (1 ml/kg) two times daily for 10 successive days. At

the end of the 10th day, the rats were overdosed, the brains

removed and immediately frozen in isopentane (�30jC)and stored at �80jC. The Animal Welfare Committee of

Dokkyo University School of Medicine and the IACUC

committee of Sun Health Research Institute approved all

procedures.

Autoradiographic procedures

The brains were sectioned at 20 Am thickness in a Leica

CM 1900 cryotome at �20jC, thaw-mounted onto Probe

On Plus slides (Fisher, Pittsburgh, PA), dried on warm plate,

and stored at �70jC until further experiments. Sections

from the striatal complex were processed for D3 receptor

autoradiography, DAT autoradiography, and TH-immunoau-

toradiography per our standard procedures (Joyce et al.,

2002; Murray et al., 1995; Ryoo et al., 1998). Alternate

sections were used for radioligand binding and in situ

hybridization (ISHH) experiments. Sections from the mid-

brain were processed for TH-immunoautoradiography.

[125I](R)-trans-7-hydroxy-2-[N-propyl-N-(3V-iodo-2V-pro-penyl)-amino]tetralin ([125I]trans-7-OH-PIPAT) (NEN, Bos-

ton, MA) binding to D3 receptors was used with conditions

that allow selective labeling of D3 receptor as described

earlier (Gurevich and Joyce, 1999, Gurevich et al., 1999).

The sections were preincubated for 30 min at 30jC in buffer

containing 50 mmol/l Tris–HCl (pH 7.4), 100 mmol/l NaCl.

To each section, 500 Al of incubation buffer containing 50

mmol/l Tris–HCl (pH 7.4), 0.7 nmol/l [125I]trans 7-OH-

PIPAT (Dupont, New England Nuclear, Boston, MA), 40

mmol/l NaCl, and 100 Amol/l GTP was applied for 90 min

at room temperature. Nonspecific binding was defined as

the binding in the presence of 10 Amol/l 7-OH-DPAT. The

sections were then washed three times in the same buffer

without GTP and [125I]trans 7-OH-PIPAT at 4jC for 1 h,

briefly dipped in ice-cold deionized water and dried.

Dopamine transporter sites were labeled with [125I]RTI-

55 (3h-(4-iodophenyl)tropan-2 h-carboxylic acid methyl

ester) (Dupont) (Joyce et al., 2000; Ryoo et al., 1998).

The slide-mounted sections were incubated with 100 pmol/

l [125I] RTI-55 in 50 mmol/l Tris–HCL buffer (pH 7.4)

containing 150 mmol/l NaCl and 5 mmol/l KCl for 2 h at

room temperature. 100 nmol/l of paroxetine was added to

block 5-HT transporter sites. Nonspecific binding was

determined with 4 Amol/l benztropine. Sections were then

washed in the same Tris buffer twice for 20 min at 0jC and

rinsed in cold ddH20 for 30 s before dried on slide warmer.

Sections along with standards were apposed to 3H-Hyper-

film for 18 h.

For tyrosine hydroxylase (TH) immunoautoradiography

(Joyce et al., 2002; Ryoo et al., 1998), sections were cut and

immediately fixed in 4% paraformaldehyde in phosphate-

buffered saline (PBS) for 1 h. Tissues were rinsed three

times in PBS, dehydrated through 70%, 80%, 95%, and

100% ethanol, and stored at �70jC until further use.

Sections were first incubated in the primary antibody, mouse

anti-TH (Chemicon, Temecula, CA) at 1:500 dilution for 24

h in PBS with 0.1% bovine serum albumin (BSA) and 0.3%

Triton X-100. The following day, sections were rinsed three

times in 0.1% BSA-PBS solution and then incubated with

the secondary antibody, [125I] F(ab)2 sheep anti-mouse

immunoglobulin G (Amersham, Arlington Heights, Ill) at

1:200 dilution in 0.1% BSA-PBS for 1 h. Sections were then

washed three times in PBS, three times in deionized water,

and dried on warm plate. Sections along with standards

were apposed to 3H-Hyperfilm for 24 h.

In situ hybridization histochemistry

Adjacent sections were processed for TrkB and BDNF

ISHH per our routine procedures (Gurevich et al., 1999).

Antisense BDNF mRNAwas generated from the rat recom-

binant plasmid pR1112-8 with T3 RNA polymerase after

digestion with BamHI. This cRNA probe contains 384 bases

complementary to the mRNA encoding mature rat BDNF

protein (Conner et al., 1997; Isackson et al., 1991). The

cRNA probe for TrkB contains 483 bases complementary to

extracellular domain of the mouse TrkB protein (Klein et al.,

1990). Digestion was completed with HindIII and transcrip-

tion with T7. Sense probes for both BDNF and TrkB were

also produced. The probes were labeled in standard in vitro

transcription reaction with 33P-UTP (Perkin Elmer, Boston,

MA). Before hybridization, sections were treated with

proteinase K (10 min at 37jC, 1 Ag/ml), rinsed briefly in

water, and incubated in 0.1 M triethanolamine buffer con-

taining 0.25% acetic anhydride for 15 min at room temper-

ature. After brief wash in 2 � SSC, sections were

dehydrated in ethanols, dried, and approximately 1–2 �106 cpm of the probe in 25 Al of hybridization buffer

J.N. Joyce et al. / Experimental Neurology 187 (2004) 178–189 181

containing 75% formamide, 50 mM Tris–HCl, 2.5 mM

EDTA, 3 � SSC, 10% dextran sulfate, 25 mM DTT, 1 �Denhardt’s solution, 0.5 mg/ml tRNA, and 0.2 mg/ml

Salmon testes DNAwere applied per section. Sections were

coverslipped and placed in hybridization oven at 55jC for

20 h. Upon completion of hybridization, sections were

subjected to RNAse digestion and washes of increased

stringency culminating in 1 h wash in 0.1 � SSC at

60jC. Following dehydration in ethanols, sections were

exposed to Kodak Biomax MR film (Amersham) along

with standards for 10 days. Film was developed with Kodak

GBX developer and fixer. The sections were developed in

Kodak D-19 developer, fixed in 30% sodium thiosulfate,

washed extensively in water, dehydrated, counterstained

with Nuclear Fast Red, and mounted from xylene in

Permount. Four sections for antisense probe and two sec-

tions for sense probe hybridization were used for each

animal. Labeling of the sense probe used as control for

specificity of hybridization signal was negligible as was the

background labeling of the antisense probe (in such areas as

white matter). In addition, we utilized pre-proenkephalin

cRNA probe as a control for stringency of ISHH protocols.

Data analysis

For Experiment 1, the autoradiographs were analyzed

using a Macintosh computer-based image analysis system

(BRAIN version 2.0, Drexel University), which converts

gray values to the amount of radioligand bound in fmol/mg

of protein (Artymyshyn et al., 1990). For Experiment 2,

autoradiographs were analyzed using a PC computer-based

image analysis system (AIS, Imaging Research Inc., Ontario

Canada) that converts transmitted optical density to the

amount of radioligand bound in fmol/mg of protein. The

optical density values derived from the autoradiographs of

[125I]radioligand binding were converted to fmol/mg protein

using standards calibrated for 125I. For ISHH autoradiogra-

phy, optical density values were converted to dpm/cm2

using 14C standards calibrated for 33P and then converted

into femtomole of probe per gram of wet weight using

specific activity of the probe in each experiment. For

analysis of the autoradiographs, four sections for total and

Table 1

Effects of strain and drug treatment on the dopamine system

Dopamine transporter

CPu NAc NAs Olf Tub

SD with saline 45.7 F 4.7 28.9 F 3.1 25.8 F 5.8 27.9 F 3

Zi/+ with saline 45.3 F 5.3# 23.7 F 5.7 19.8 F 5.5 25.9 F 3

Zi/Zi with saline 33.1 F 3.1* 29.7 F 4.4 15.8 F 4.1* 18.6 F 4

Zi/+ with L-dopa 46.1 F 5.4y 24.7 F 4.0 13.8 F 4.4* 22.2 F 3

Zi/Zi with L-dopa 25.5 F 2.0* 21.5 F 3.7 10.4 F 3.9* 13.6 F 9

*Significant at P < 0.05 vs. SD with saline.# Significant at P < 0.05, Zi/+ with saline vs. Zi/Zi with saline.y Significant at P < 0.05, Zi/+ with L-dopa vs. Zi/Zi L-dopa.£ Significant at P < 0.05, Zi/+ with saline vs. Zi/+ with L-dopa.

two sections for nonspecific binding from each animal were

utilized. Optical densities in defined brain areas were

measured bilaterally, allowing for eight measurements for

total and four measurements for nonspecific binding. For

each animal, the total and nonspecific binding were aver-

aged and nonspecific binding was subtracted from total

binding to yield values for specific binding for each region

of each animal. For the present study, the striatum was

divided into subdivisions: caudate–putamen (CPu), nucleus

accumbens shell (NAs), nucleus accumbens core (NAc),

islands of Calleja (ICj), and olfactory tubercle (Olf Tub).

For ISHH autoradiography, optical density values were

converted to ACi/g using 14C standards (American Radio-

labeled Chemicals, Inc., St. Louis MO) utilizing the cali-

bration scale provided with each standard (Gurevich et al.,

1999). For analysis of the autoradiographs of the sections

processed for ISHH, four sections for the antisense probe

and two sections for the sense probe from each animal were

utilized. Optical densities in defined brain areas were

measured bilaterally, allowing for eight measurements for

the antisense probe and four measurements for the sense

probe. For each animal, the readings from the sense probe-

labeled sections were subtracted from antisense probe-la-

beled sections to yield values for specific labeling for each

region of each animal. For the ISHH analysis, readings were

taken from the cingulate cortex (CG), frontal cortex (FC),

and nucleus accumbens (NA) (Paxinos and Watson,1986).

For statistical analysis, a two-way ANOVA, with group and

region as main factors, and pairwise comparisons performed

using post hoc Dunnett with Bonferroni correction. For

Experiment 2, data from the zitter groups were analyzed

separately from the SD groups because the autoradiographic

procedures were completed independently.

Results

Experiment 1

The results of Experiment 1 (shown in Table 1) confirm

our previous results that the zitter homozygotic rats (Zi/Zi)

have significantly lower concentrations of DAT labeling of

D3 receptor binding

CPu NAc NAs ICj

.3 3.0 F 1.9 9.17 F 1.8 13.31 F 3.2 22.96 F 4.1

.5 5.6 F 3.3 13.37 F 2.9# 18.97 F 2.9*,# 23.75 F 3.6#

.1* 2.3 F 2.5 2.85 F 1.5* 3.25 F 2.5* 9.63 F 2.2*

.0y 4.0 F 1.5 8.21 F 1.6 8.83 F 2.1£ 17.69 F 5.1

.1* 1.1 F 3.6 2.04 F 2.1*,y 2.66 F 2.7*,y 13.14 F 5.0*

J.N. Joyce et al. / Experimental Neurology 187 (2004) 178–189182

DA fibers, a marker for the integrity of the DA fibers (Joyce

et al., 2002; Murray et al., 1995; Ryoo et al., 1998), as

compared to SD control rats (Joyce et al., 2000), and

intermittent L-dopa treatment nonsignificantly exacerbated

that effect. Analysis of the effects of group (SD, Zi/+ with

saline, Zi/Zi with saline, Zi/+ with L-dopa, and Zi/Zi with L-

dopa) and regions demonstrated group (P < 0.001, F =

21.42), region (P < 0.001, F = 83.68), and interaction

effects (P = 0.0012, F = 3.68) for DAT binding. The

concentrations of DAT binding sites were not different in

zitter heterozygotes (Zi/+) treated with saline or L-dopa as

compared to the SD controls (Table 1). Zi/Zi rats did have

significantly lower concentrations of DAT in the CPu

(�28%), NAs (�39%), and Olf Tub (�34%) as compared

to SD rats. Treatment of Zi/Zi rats with L-dopa nonsignif-

icantly enhanced the loss of DAT sites in the CPu (�45% of

Fig. 1. Low-power photomicrograph of the original autoradiograph of the cauda

binding from individual subjects of four groups: (A) Zi/Zi with saline, (B) Zi/Zi wit

saline, and (D) Zi/+ with L-dopa/carbidopa. Note the marked depletion of DAT site

The ROI used for analysis is depicted by dashed lines in panel D. Abbreviation

accumbens core; Olf Tub, olfactory tubercle.

SD), NAs (�60% of SD), and Olf Tub (�45% of SD) as

compared to the Zi/Zi treated with saline.

As reported previously (Joyce et al., 2000), Zi/Zi rats had

significantly lower concentrations of D3 receptors in striatal

regions as compared with SD rats, but intermittent L-dopa

treatment did not further alter the concentrations of D3

receptors in the Zi/Zi rats. Analysis of the effects of group

(SD, Zi/+ with saline, Zi/Zi with saline, Zi/+ with L-dopa, Zi/

Zi with L-dopa) and regions demonstrated group (P < 0.001,

F = 39.91), region (P < 0.001, F = 80.50), and interaction

effects (P = 0.0019, F = 3.094) for concentrations of D3

receptors. There were no differences in the concentration of

D3 receptors in the CPu, NAc, or ICj between SD and Zi/+

rats treated with saline or L-dopa, although the Zi/+ rats

treated with saline did have a somewhat greater concentra-

tion of D3 receptors in the NAs as compared to the SD rats.

te–putamen and nucleus accumbens of tissue sections processed for DAT

h L-dopa/carbidopa (100:10 mg/kg ip) twice a day for 10 days, (C) Zi/+ with

s from the caudate–putamen in Zi/Zi and lack of affect of L-dopa treatment.

s: CPu, caudate–putamen; NAs, nucleus accumbens shell; NAc, nucleus

Fig. 2. Effects of zitter homozygosity and drug treatment with L-dopa on

DAT binding (A) andD3 receptor number (B) in regions of the striatum. Zi/Zi

and Zi/+ rats were treated with L-dopa/carbidopa (100:10 mg/kg ip) twice a

day for 10 days or saline. Histogram A is for [125I]RTI-55 labeling of DAT

sites and B is for [125I]trans-7-OH-PIPAT labeling of D3 receptors. Data are

means F standard deviation of the mean from four to eight samples per

group. Significant differences are represented by: *P < 0.01 for between

regions within group; #P < 0.01 for between groups within region.

Abbreviations: CPu, caudate–putamen; ICj, Islands of Calleja; NAc,

nucleus accumbens core; NAs, nucleus accumbens shell; OLF, olfactory

tubercle.

Fig. 3. Effects of zitter homozygosity and drug treatment with L-dopa on

TH-IR in striatum (A) and midbrain (B). Zi/Zi and Zi/+ rats were treated

with L-dopa/carbidopa (100:10 mg/kg ip) twice a day for 10 days or saline.

Histogram A is for TH-IR in regions of the striatum and B is for the SN and

VTA. Data are means F standard deviation of the mean from four to eight

samples per group. #P < 0.01 for between groups within region.

Abbreviations: CPu, caudate–putamen; NA, nucleus accumbens; OLF,

olfactory tubercle; SN, substantia nigra; VTA, ventral tegmental area.

J.N. Joyce et al. / Experimental Neurology 187 (2004) 178–189 183

Zi/Zi rats did have significantly lower concentrations of D3

receptors in the NAc (�69%), NAs (�76%), and ICj

(�59%) as compared with SD rats. Zi/Zi rats treated with

L-dopa also demonstrated significantly lower levels of D3

receptors in the NAc (�78% of SD), NAs (�80% of SD),

and Olf Tub (�43% of SD). Treatment of Zi/Zi rats with L-

dopa did not alter the loss of D3 receptors in NAc and NAs as

compared to the Zi/Zi treated with saline (Table 1).

Experiment 2

Analysis in Zi/Zi and Zi/+ rats

As shown in Fig. 1, concentrations of DAT sites were

heterogeneous in the striatum of the Zi/+ rats. Similar to that

of SD control rats, there were higher concentrations of sites

in the CPu than NAs, NAc, or Olf Tub. The concentrations

of DAT sites were considerably lower in the CPu of the Zi/

Zi rats as compared to the Zi/+ rats regardless of whether

they were administered saline or L-dopa. There was no

effect of L-dopa on the concentration of DAT sites. Statis-

tical analysis confirmed these observations as there were

significant group (P < 0.001, F = 20.96), region (P < 0.001,

F = 161.2), and interaction (P < 0.001, F = 11.62) effects

for the analysis of DAT binding (Fig. 2A). The Zi/Zi with

saline group had significantly lower levels of DAT than the

Zi/+ with saline group for the CPu, and the Zi/Zi with L-

dopa group had significantly lower levels of DAT than the

Zi/+ with L-dopa group for the CPu. There were no

significant differences between Zi/+ with saline and Zi/+

with L-dopa or between Zi/Zi with saline and Zi/Zi with L-

dopa groups. The CPu contained higher levels of DAT than

any other region (P < 0.001) for all groups, and within the

Zi/+ with saline and Zi/+ with L-dopa groups, the NAc

contained higher levels than the NAs and Olf Tub.

We also analyzed TH-IR levels in the CPu, nucleus

accumbens (NAs and NAc combined), and Olf Tub, an

indicator of the integrity of DA fibers (Joyce et al., 2002;

Murray et al., 1995; Ryoo et al., 1998), and also in the SN

and VTA, an indicator of the integrity of DA cell bodies

(Joyce et al., 2003,2004; Murray et al., 1995). For the

J.N. Joyce et al. / Experimental Neurology 187 (2004) 178–189184

striatum, there were significant group (P = 0.0002, F =

7.73) but no region or interaction effects (Fig. 3A). TH-IR

was significantly lower in the Zi/Zi with saline group as

compared to the Zi/+ with saline group, and the Zi/Zi with

L-dopa group was significantly lower than the Zi/+ with L-

dopa group. There were no significant differences between

Zi/+ with saline and Zi/+ with L-dopa groups or between Zi/

Zi with saline and Zi/Zi with L-dopa groups. Analysis of

TH-IR in the SN/VTA of the zitter groups revealed signif-

icant group (P < 0.001, F = 18.67), region (P < 0.05, F =

4.297), and interaction (P < 0.05, F = 3.669) effects (Fig.

3B). As would be expected from the previous study (Ueda et

al., 2000), TH-IR was significantly lower in the Zi/Zi with

saline group as compared to the Zi/+ with saline group for

the SN, and the Zi/Zi with L-dopa group was significantly

lower than the Zi/+ with L-dopa group for both the SN and

VTA. There were no significant differences between Zi/+

Fig. 4. Low-power photomicrograph of the original autoradiograph of the caud

receptor binding from individual subjects of four groups: (A) Zi/Zi with saline, (B)

Zi/+ with saline, and (D) Zi/+ with L-dopa/carbidopa. Note the marked reduction of

dopa treatment. The ROI used for analysis is depicted by dashed lines in panel D

with saline and Zi/+ with L-dopa groups or between Zi/Zi

with saline and Zi/Zi with L-dopa groups. Thus, as with

analysis of DAT binding, degeneration of DA fibers as

measured with striatal TH-IR and of TH-IR cell bodies in

SN/VTA was evident in the Zi/Zi rats but not enhanced by

treatment with L-dopa.

As shown in Fig. 4, the concentrations of D3 receptors

were heterogeneously distributed in the striatum of the Zi/+

rats, and similar to SD controls, there were higher concen-

trations of sites in the ICj than NAs, NAc, or the CPu. The

concentrations of D3 receptors were considerably lower in

the CPu of the Zi/Zi rats administered saline or L-dopa as

compared with the Zi/+ rats administered saline or L-dopa.

There was no effect of L-dopa on the concentration of D3

receptors. Statistical analysis confirmed these observations,

as there were significant group (P < 0.001, F = 154.9),

region (P < 0.001, F = 181.1), and interaction (P < 0.001, F

ate–putamen and nucleus accumbens of tissue sections processed for D3

Zi/Zi with L-dopa/carbidopa (100:10 mg/kg ip) twice a day for 10 days, (C)

D3 receptor sites from the caudate–putamen in Zi/Zi and lack of affect of L-

. Abbreviations as in Fig. 1.

J.N. Joyce et al. / Experimental Neurology 187 (2004) 178–189 185

= 27.12) effects for the analysis of D3 receptor binding (Fig.

2B). The Zi/Zi with saline group had significantly lower

levels of D3 receptors than the Zi/+ with saline group for the

NAc, NAs, and ICj; and the Zi/Zi with L-dopa group had

significantly lower concentrations of D3 receptors than the

Zi/+ with L-dopa group for the NAc, NAs, and ICj. There

were no significant differences between Zi/+ with saline and

Zi/+ with L-dopa groups or between Zi/Zi with saline and

Zi/Zi with L-dopa groups. The ICj contained higher con-

centrations of D3 receptors than any other region for all

groups, and within the Zi/+ with saline and Zi/+ with L-dopa

groups, the NAs contained higher levels than the NAc and

was lowest in the CPu.

Analysis in SD control rats

Analysis of DAT binding in the SD groups also demon-

strated significant group (P < 0.05, F = 6.701), region (P <

Fig. 5. Low-power photomicrograph of the original autoradiograph of the forebrai

individual subjects of four groups: (A) Zi/Zi with saline, (B) Zi/Zi with L-dopa/car

(D) Zi/+ with L-dopa/carbidopa. Methods for labeling of the tissue sections with th

used for analysis is depicted by dashed lines in panel A. Abbreviations: CG, cing

0.001, F = 157.5), and interaction (P < 0.05, F = 2.978)

effects (data not shown). The SD with L-dopa group had a

slightly greater level of DAT in the CPu as compared to the

SD with saline group, and the CPu contained higher levels

of DAT than any other region for both groups (P < 0.001).

Analysis of D3 receptor binding in the SD groups also

demonstrated significant region (P < 0.001, F = 121.3) but

not group or interaction effects (Fig. 5B). For both the SD

with saline and SD with L-dopa groups, the ICj contained

higher concentrations of D3 receptors than any other region

and the NAs contained higher levels than the NAc and

binding was lowest in the CPu.

BDNF and TrkB ISHH

Representative photomicrographs of sections for rat

forebrain of Zi/Zi and SD rats treated with L-dopa or saline

and prepared for ISHH for BDNF and TrkB are shown in

n of tissue sections processed for ISHH for antisense to BDNF mRNA from

bidopa (100:10 mg/kg ip) twice a day for 10 days, (C) Zi/+ with saline, and

e 33P-labeled riboprobe is described in the Materials and methods. The ROI

ulate cortex; FR, frontal cortex.

J.N. Joyce et al. / Experimental Neurology 187 (2004) 178–189186

Figs. 5 and 6. Analysis of BDNF ISHH was limited to the

CG and deep layers of the FC and for TrkB, the CG, FC,

CPu, and NA (NAs and NAc combined), as these were

found to be regions with significant alterations in 6-OHDA

lesioned and L-dopa-treated rats (Guillin et al., 2001). While

the levels of BDNF mRNA (Fig. 5) were higher in the CG

as compared to the FR (P < 0.001, F = 18.50), there were

not any differences between the four groups analyzed (Zi/Zi

saline, Zi/Zi L-dopa, SD saline, and SD L-dopa). Analysis of

the TrkB ISHH (Fig. 6) revealed significant group (P <

0.001, F = 10.13) and region (P < 0.001, F = 18.50) but

not interaction effects. There was a consistent pattern of

labeling with the CG higher than the FR, which was higher

than NA and the NA was higher than the CPu. The Zi/Zi

saline group had higher levels of TrkB labeling than the

other groups for the CG, but no other group differences

existed.

Fig. 6. Low-power photomicrograph of the original autoradiograph of the forebra

individual subjects of four groups: (A) Zi/Zi with saline, (B) Zi/Zi with L-dopa/car

(D) Zi/+ with L-dopa/carbidopa. Methods for labeling of the tissue sections with th

used for analysis is depicted by dashed lines in panel A. Abbreviations: CG, cing

Discussion

The results of Experiments 1 and 2 were largely in accord

that reduction of D3 receptors in Zi/Zi rats could not be

reversed with subchronic treatment with L-dopa. We utilized

a regimen of L-dopa treatment that in 6-OHDA-treated rats

does lead to the reversal of the loss and an elevation of D3

receptor binding (Bordet et al., 1997; Guillin et al., 2001).

One of the possible explanations we considered was that the

L-dopa treatment in the Zi/Zi rats might lead to further

damage to the presynaptic DA system, unlike in 6-OHDA-

treated rats, as L-dopa can elevate production of reactive

oxygen species (Blunt et al., 1993; Lai and Yu, 1997;

Naudin et al., 1995; Spencer et al., 1995), and the zitter

mutant rat has abnormal metabolism of oxygen species in

the brain (Gomi et al., 1994; Ueda et al., 2002). In fact, in

Experiment 1, we observed a nonsignificantly lower level of

in of tissue sections processed for ISHH for antisense to TrkB mRNA from

bidopa (100:10 mg/kg ip) twice a day for 10 days, (C) Zi/+ with saline, and

e 33P-labeled riboprobe is described in the Materials and methods. The ROI

ulate cortex; FR, frontal cortex; NA, nucleus accumbens.

J.N. Joyce et al. / Experimental Neurology 187 (2004) 178–189 187

DAT in the Zi/Zi rats treated with L-dopa as compared with

Zi/Zi rats treated with saline. To examine this in more detail,

an additional group of Zi/Zi, Zi/+, and SD rats were

analyzed for the effects of subchronic L-dopa on the pre-

synaptic DA system by measuring both DAT and TH-IR in

the striatum and TH in the midbrain. Those results were

confirmatory, as we did not identify any differences in the

degree of loss of DAT or TH-IR in the striatal regions of the

Zi/Zi rats treated with L-dopa as compared with Zi/Zi with

saline rats. The pattern of loss of DAT in the Zi/Zi rat

striatum observed here is very similar to that described for

degenerative changes in TH-IR fibers in histologically well-

preserved tissue (Ueda et al., 2000). These findings along

with our observations of the reduction of striatal levels of

TH-IR and TH-IR in the SN of the Zi/Zi rats support our

belief that we are accurately measuring any possible impact

of L-dopa on the presynaptic DA system in the Zi/Zi rats.

Not surprisingly, the subchronic treatment with L-dopa in

the SD rats did not lower DAT binding, and L-dopa

treatment has not been observed to alter levels of TH-IR

labeling of DA fibers in SD rats treated for longer periods

with L-dopa (Murer et al., 1998).

We believe our data demonstrate that the failure to

elevate D3 receptor number with L-dopa treatment in zitter

mutant rats does not occur because of further damage to

the DA innervation to the striatum since we did not find

that L-dopa treatment caused a further reduction of DAT

and TH in the striatum nor of TH-IR in SN/VTA. We also

eliminated the possibility that zitter mutation had effects

other than through the DA system, as the zitter hetero-

zygotes had normal levels of D3 receptors and DAT

binding to DA fibers. Another group has examined ex-

pression of a number of neurotrophic factors in the zitter

mutant rat (Muto et al., 1999). Quantification of mRNA

levels for NGF, BDNF, NT-3, ciliary neurotrophic factor,

and GDNF showed that only BDNF was down-regulated

in the cerebrum and brainstem of the adult Zi/Zi rat.

Furthermore, there was an age-related decrease in the

cerebrum that was evident at an early postnatal period in

the brainstem. The timing of this reduction of BDNF in

brainstem is consistent with the slowly progressive degen-

eration of the DA system in the Zi/Zi rat (Ueda et al.,

2000, 2002). Given the evidence for the direct effect of

BDNF on control of expression of the D3 receptor (Guillin

et al., 2001), we hypothesized that the failure for proper

adult expression of the D3 receptor in the Zi/Zi rat is due

to the combination of the lowered BDNF and degeneration

of DA innervation. Furthermore, we hypothesized that the

failure of L-dopa treatment to up-regulate D3 receptor

might be due to deficient cortical expression of BDNF.

In fact, in another study, we have confirmed the findings of

Guillin et al. (2001) that BNDF plays an important role in

the proper development of the D3 receptor as BDNF

heterozygote mice with 1/2 the levels of BDNF demon-

strate significantly lower levels of D3 receptors in forebrain

of adult mice (Joyce et al., 2004). While in this study we

did not observe lower cortical levels of BDNF mRNA in

the Zi/Zi rats as compared with the SD rats, treatment with

L-dopa failed to increase BDNF mRNA in the Zi/Zi or SD

rats. In the study by Guillin et al. (2001), it was also

demonstrated that 6-OHDA lesioned and L-dopa-treated

rats exhibited an elevation of TrkB receptor mRNA, and

L-dopa-induced up-regulation of D3 receptor mRNA was

antagonized by blocking the TrkB receptor with IgG-TrkB.

In this study, there was no difference in TrkB receptor

mRNA in the CPu or NA of the Zi/Zi rats as compared

with the SD rats, and treatment with L-dopa did not alter

TrkB receptor mRNA levels in the Zi/Zi or SD rats. Thus,

the absence of alterations in FR BDNF mRNA and striatal

TrkB receptor mRNA with L-dopa in the Zi/Zi rats could

underlie the resistance to the impact of L-dopa in regulating

expression of the D3 receptor in the Zi/Zi rats.

Interestingly, unlike what has been observed by another

group (Muto et al., 1999), we did not observe lower levels

of BDNF mRNA in FR of Zi/Zi rats. This could suggest that

the lowered D3 receptor number in the Zi/Zi rats is not due

to reduced BDNF availability. In another test of the role of

BDNF and DA fiber loss in the control of expression of D3

receptors in forebrain, we have examined the impact of

methamphetamine toxicity to DA fibers in combination with

lowered BDNF (BDNF +/� mice) on D3 receptors in

forebrain of adult mice (Joyce et al., 2004). We found that

in BDNF +/� mice, with 1/2 levels of BDNF, given a

methamphetamine regimen that produces 50% loss of DA

innervation, there was no greater reduction in the number of

D3 receptors than in the BDNF +/� mice treated with the

vehicle. In MPTP-treated mice with significantly greater

damage to the DA system than achieved with methamphet-

amine treatment, there is a reduction in striatal D3 receptor

number (Gross et al., 2003). In concert, these results would

suggest that reduced BDNF and reduced DA innervation are

not additive in the control of expression of D3 receptors. In

other models of DA denervation with 6-OHDA (Bordet et

al., 1997; Guillin et al., 2001) and MPTP (Morissette et al.,

1998; Wade et al., 2001) that lead to reduced expression of

forebrain D3 receptors, there is massive loss of DA fibers

that is not matched by the zitter mutation (Ueda et al., 2000,

2002). Thus, our current evidence does not support our

hypothesis that BDNF expression plays a predominant role

in regulating expression of the D3 receptor in the Zi/Zi rats.

We believe that there are other factors than the control of

BDNF release by DA fibers that regulate expression of the

D3 receptor. Perhaps additional specific cortical pathology

(Kondo et al., 1995), and/or loss of serotonergic afferents

(Ueda et al., 1998), or loss of cortical DA is required for

deficient control of expression of the D3 receptor in the

zitter rat.

The results of the current study are particularly relevant

for our studies in PD (Joyce et al., 2002; Ryoo et al., 1998),

where we find that a population of PD patients fail to show

normalization of D3 receptors with chronic L-dopa treatment

and this is related to loss of response to APDs and presence

J.N. Joyce et al. / Experimental Neurology 187 (2004) 178–189188

of dementia. Certainly, there is widespread changes in the

cortex of PD cases with dementia (Apaydin et al., 2002;

Hughes et al., 2000; Hurtig et al., 2000; Mattila et al., 2000).

While we believe that the loss of DA fibers and abnormal

morphology of the remaining DA fibers alters axonal flow

of a number of factors that regulate expression of the D3

receptor, other pathological substrates might play a contrib-

utory role. Perhaps additional specific cortical pathology

(Apaydin et al., 2002; Hurtig et al., 2000), and/or loss of

serotonergic afferents (Hornykiewicz, 1998; Jellinger,

1991), or loss of cortical DA (McRitchie et al., 1997) is

required for deficient control of expression of the D3

receptor in PD. Since all of these changes have been

observed to occur in the zitter mutant rat, it further sub-

stantiates the value of the ZI/Zi rat as a pathological model

of PD. Certainly, other models of PD than that caused by

rapid and extensive DA fiber or cell loss induced by 6-

OHDA and MPTP must be considered for exploring the

regulation of the D3 receptor.

Conclusions

The principle finding of the present study is that the zitter

mutant rat model of PD pathology, which demonstrates a

reduction of striatal D3 receptors as in the 6-OHDA and

MPTP models of PD, fails to exhibit a normalization of D3

receptor to L-dopa. Furthermore, we provide evidence that

this may be the result of the failure of L-dopa to regulate

forebrain BDNF and TrkB receptor mRNAs. This is differ-

ent from what is observed in the 6-OHDA and MPTP

models of PD but is comparable to what occurs in PD. In

a recent review (Joyce, 2001b), we discussed the clinical

and preclinical literature that supports the hypothesis that

the initial expression of parkinsonian symptoms is not only

correlated with the loss of DA innervation and an elevation

in receptor number of the D2 subclass, but also a decrease

D3 receptor number. Furthermore, a subgroup of PD cases

that become resistant to the clinical benefits of antiparkin-

sonian mediation also exhibit a sustained loss of D3 recep-

tors (Joyce et al., 2002; Ryoo et al., 1998). The role of the

D3 receptor in the therapeutic response to antiparkinsonian

drugs in PD remains unproven, but recent data support the

hypothesis that it plays an important contributory role as a

selective D3 receptor antagonist blocks the ability of L-dopa

to reverse the parkinsonian symptoms in MPTP-treated

monkeys (Bezard et al., 2003). Hence, identifying factors

that regulate the expression of the D3 receptor may be

important for developing therapeutic interventions for treat-

ing the clinical symptoms of PD.

Acknowledgments

Funded by Federal Grant NS40669. The technical

assistance of Han Ryoo is greatly appreciated.

References

Altar, C.A., Cai, N., Bliven, T., Juhasz, M., Conner, J.M., Acheson, A.L.,

Lindsay, R.M., Wiegand, S.J., 1997. Anterograde transport of brain-

derived neurotrophic factor and its role in the brain. Nature 389,

856–860.

Apaydin, H., Ahlskog, J.E., Parisi, J.E., Boeve, B.F., Dickson, D.W., 2002.

Parkinson disease neuropathology: later-developing dementia and loss

of the levodopa response. Arch. Neurol. 59, 102–112.

Artymyshyn, R., Smith, A., Wolfe, B.B., 1990. The use of 3H standards in

125I autoradiography. J. Neurosci. Methods 32, 185–192.

Bezard, E., Ferry, S., Mach, U., Stark, H., Leriche, L., Boraud, T.,

Gross, C., Sokoloff, P., 2003. Attenuation of levodopa-induced dys-

kinesia by normalizing dopamine D3 receptor function. Nat. Med. 9,

762–767.

Blunt, S.B., Jenner, P., Marsden, C.D., 1993. Suppressive effect of L-dopa

on dopamine cells remaining in the ventral tegmental area of rats pre-

viously exposed to the neurotoxin 6-hydroxydopamine. Mov. Disord. 8,

129–133.

Bordet, R., Ridray, S., Carboni, S., Diaz, J., Sokoloff, P., Schwartz, J.C.,

1997. Induction of dopamine D3 receptor expression as a mechanism of

behavioral sensitization to levodopa. Proc. Natl. Acad. Sci. U.S.A. 94,

3363–3367.

Brooks, D.J., 2000. Dopamine agonists: their role in the treatment of Par-

kinson’s disease. J. Neurol. Neurosurg. Psychiatry 68, 685–689.

Caparros-Lefebvre, D., Pecheux, N., Petit, V., Duhamel, A., Petit, H., 1995.

Which factors predict cognitive decline in Parkinson’s disease?. J. Neu-

rol. Neurosurg. Psychiatry 58, 51–55.

Civelli, O., Bunzow, J.R., Grandy, D.K., 1993. Molecular diversity of the

dopamine receptors. Annu. Rev. Pharmacol. Toxicol. 33, 281–307.

Conner, J.M., Lauterborn, J.C., Yan, Q., Gall, C.M., Varon, S., 1997.

Distribution of brain-derived neurotrophic factor (BDNF) protein and

mRNA in the normal adult rat CNS: evidence for anterograde axonal

transport. J. Neurosci. 17, 2295–2313.

Fabbrini, G., Mouradian, M.M., Juncos, J.L., Schlegel, J., Mohr, E., Chase,

T.N., 1988. Motor fluctuations in Parkinson’s disease: central patho-

physiological mechanisms: Part I. Ann. Neurol. 24, 366–371.

Gomi, H., Ueno, I., Yamanouchi, K., 1994. Antioxidant enzymes in the

brain of zitter rats: abnormal metabolism of oxygen species and its

relevance to pathogenic changes in the brain of zitter rats with genetic

spongiform encephalopathy. Brain Res. 653, 66–72.

Gross, C.E., Ravenscroft, P., Dovero, S., Jaber, M., Bioulac, B., Bezard, E.,

2003. Pattern of levodopa-induced striatal changes is different in normal

and MPTP-lesioned mice. J. Neurochem. 84, 1246–1255.

Guillin, O., Diaz, J., Carroll, P., Griffon, N., Schwartz, J.C., Sokoloff, P.,

2001. BDNF controls dopamine D3 receptor expression and triggers

behavioural sensitization. Nature 411, 86–89.

Gurevich, E.V., Joyce, J.N., 1999. Distribution of dopamine D3 receptor

expressing neurons in the human forebrain: comparison with D2 recep-

tor expressing neurons. Neuropsychopharmacology 20, 60–80.

Gurevich, E.V., Himes, J.W., Joyce, J.N., 1999. Developmental regulation

of expression of the D3 dopamine receptor in rat nucleus accumbens

and islands of Calleja. J. Pharmacol. Exp. Ther. 289, 587–598.

Hornykiewicz, O., 1998. Biochemical aspects of Parkinson’s disease. Neu-

rology 51, S2–S9.

Hughes, T.A., Ross, H.F., Musa, S., Bhattacherjee, S., Nathan, R.N., Mind-

ham, R.H., Spokes, E.G., 2000. A 10-year study of the incidence of and

factors predicting dementia in Parkinson’s disease. Neurology 54,

1596–1602.

Hurtig, H.I., Trojanowski, J.Q., Galvin, J., Ewbank, D., Schmidt, M.L.,

Lee, V.M., Clark, C.M., Glosser, G., Stern, M.B., Gollomp, S.M.,

Arnold, S.E., 2000. Alpha-synuclein cortical Lewy bodies correlate

with dementia in Parkinson’s disease. Neurology 54, 1916–1921.

Isackson, P.J., Huntsman, M.M., Murray, K.D., Gall, C.M., 1991. BDNF

mRNA expression is increased in adult rat forebrain after limbic seiz-

ures: temporal patterns of induction distinct from NGF. Neuron 6,

937–948.

J.N. Joyce et al. / Experimental Neurology 187 (2004) 178–189 189

Jellinger, K.A., 1991. Pathology of Parkinson’s disease. Changes other than

the nigrostriatal pathway. Mol. Chem. Neuropathol. 14, 153–197.

Joyce, J.N., 1991. Differential response of striatal dopamine and muscarinic

cholinergic receptor subtypes to the loss of dopamine: I. Effects of

intranigral or intracerebroventricular 6-hydroxydopamine lesions of

the mesostriatal dopamine system. Exp. Neurol. 113, 261–276.

Joyce, J.N., 2001a. D2 but not D3 receptors are elevated after 9 or 11

months chronic haloperidol treatment: influence of withdrawal period.

Synapse 40, 137–144.

Joyce, J.N., 2001b. Dopamine D3 receptor as a therapeutic target for anti-

psychotic and antiparkinsonian drugs. Pharmacol. Ther. 90, 231–259.

Joyce, J.N., Marshall, J.F., Bankiewicz, K.S., Kopin, I.J., Jacobowitz,

D.M., 1986. Hemiparkinsonism in a monkey after unilateral internal

carotid artery infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

(MPTP) is associated with regional ipsilateral changes in striatal dopa-

mine D-2 receptor density. Brain Res. 382, 360–364.

Joyce, J.N., Yoshimoto, K., Ueda, S., 2000. The zitter mutant rat exhibits

loss of D3 receptors with degeneration of the dopamine system. Neuro-

Report 11, 2173–2175.

Joyce, J.N., Ryoo, H.L., Beach, T.B., Caviness, J.N., Stacy, M., Gurevich,

E.V., Reiser, M., Adler, C.H., 2002. Loss of response to levodopa in

Parkinson’s disease and co-occurrence with dementia: role of D3 and

not D2 receptors. Brain Res. 955, 138–152.

Joyce, J.N., Presgraves, S., Renish, L., Borwege, S., Hagner, D., Osred-

kar, T., Replogle, M., Paz Soldan, M.M., Millan, M.J., 2003. Neuro-

protective effects of the novel D3/D2 receptor agonist and anitparkinson

agent S32504, in vitro against 1-methyl-4-phenylpyridinium (MPP+)

and in vivo against 1-methyl- 4-phenyl-1,2,2,6-tetrahydropyridine

(MPTP): a comparison to ropinirole. Exp. Neurol. 184, 393–407.

Joyce, J.N., Renish, L., Osredkar, T., Walro, J.M., Kucera, J., Dluzen, D.E.,

2004. Methamphetamine induced loss of striatal dopamine innervation

in BNDF heterozygote mice does not further alter D3 receptor concen-

trations. Synapse 52, 11–19.

Klein, R., Conway, D., Parada, L.F., Barbacid, M., 1990. The trkB tyrosine

protein kinase gene codes for a second neurogenic receptor that lacks

the catalytic kinase domain. Cell 61, 647–656.

Kondo, A., Sendoh, S., Miyata, K., Takamatsu, J., 1995. Spongy degener-

ation in the zitter rat: ultrastructural and immunohistochemical studies.

J. Neurocytol. 24, 533–544.

Lai, C.T., Yu, P.H., 1997. Dopamine- and L-beta-3,4-dihydroxyphenylala-

nine hydrochloride (L-dopa)- induced cytotoxicity towards catechol-

aminergic neuroblastoma SH-SY5Y cells. Effects of oxidative stress

and antioxidative factors. Biochem. Pharmacol. 53, 363–372.

Levesque, D., Martres, M.P., Diaz, J., Griffon, N., Lammers, C.H., Sokol-

off, P., Schwartz, J.C., 1995. A paradoxical regulation of the dopamine

D3 receptor expression suggests the involvement of an anterograde

factor from dopamine neurons. Proc. Natl. Acad. Sci. U.S.A. 92,

1719–1723.

Marder, K., Tang, M.X., Cote, L., Stern, Y., Mayeux, R., 1995. The fre-

quency and associated risk factors for dementia in patients with Parkin-

son’s disease. Arch. Neurol. 52, 695–701.

Mattila, P.M., Rinne, J.O., Helenius, H., Dickson, D.W., Roytta, M., 2000.

Alpha-synuclein-immunoreactive cortical Lewy bodies are associated

with cognitive impairment in Parkinson’s disease. Acta Neuropathol.

(Berl.) 100, 285–290.

McRitchie, D.A., Cartwright, H.R., Halliday, G.M., 1997. Specific A10

dopaminergic nuclei in the midbrain degenerate in Parkinson’s disease.

Exp. Neurol. 144, 202–213.

Morissette, M., Goulet, M., Grondin, R., Blanchet, P., Bedard, P.J., Di

Paolo, T., Levesque, D., 1998. Associative and limbic regions of mon-

key striatum express high levels of dopamine D3 receptors: effects of

MPTP and dopamine agonist replacement therapies. Eur. J. Neurosci.

10, 2565–2573.

Mouradian, M.M., Juncos, J.L., Fabbrini, G., Schlegel, J., Bartko, J.J.,

Chase, T.N., 1988. Motor fluctuations in Parkinson’s disease: central

pathophysiological mechanisms, Part II. Ann. Neurol. 24, 372–378.

Murer, M.G., Dziewczapolski, G., Menalled, L.B., Garcia, M.C., Agid, Y.,

Gershanik, O., Raisman-Vozari, R., 1998. Chronic levodopa is not toxic

for remaining dopamine neurons, but instead promotes their recovery, in

rats with moderate nigrostriatal lesions. Ann. Neurol. 43, 561–575.

Murray, A.M., Weihmueller, F.B., Marshall, J.F., Hurtig, H.I., Gottleib,

G.L., Joyce, J.N., 1995. Damage to dopamine systems differs between

Parkinson’s disease and Alzheimer’s disease with parkinsonism. Ann.

Neurol. 37, 300–312.

Muto, Y., Hayashi, T., Higashi, Y., Endo, T., Yamamoto, T., Sato, K., 1999.

Age-related decrease in brain-derived neurotrophic factor gene expres-

sion in the brain of the zitter rat with genetic spongiform encephalop-

athy. Neurosci. Lett. 271, 69–72.

Naudin, B., Bonnet, J.J., Costentin, J., 1995. Acute L-DOPA pretreatment

potentiates 6-hydroxydopamine-induced toxic effects on nigro-striatal

dopamine neurons in mice. Brain Res. 701, 151–157.

Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates.

Academic Press, Sydney Australia.

Pizzolato, G., Chierichetti, F., Rossato, A., Cagnin, A., Fabbri, M., Dam, M.,

Ferlin, G., Battistin, L., 1995. Alterations of striatal dopamine D2 recep-

tors contribute to deteriorated response to L-dopa in Parkinson’s disease:

a [123I]-IBZM SPET study. J. Neural. Transm., Suppl. 45, 113–122.

Portin, R., Rinne, U.K., 1987. Predictive factors for cognitive deterioration

and dementia in Parkinson’s disease. Adv. Neurol. 45, 413–416.

Quik, M., Police, S., He, L., Di Monte, D.A., Langston, J.W., 2000. Ex-

pression of D3 receptor messenger RNA and binding sites in monkey

striatum and substantia nigra after nigrostriatal degeneration: effect of

levodopa treatment. Neuroscience 98, 263–273.

Ryoo, H.L., Pierrotti, D., Joyce, J.N., 1998. Dopamine D3 receptor is

decreased and D2 receptor is elevated in the striatum of Parkinson’s

disease. Mov. Disord. 13, 788–797.

Spencer, J.P., Jenner, P., Halliwell, B., 1995. Superoxide-dependent deple-

tion of reduced glutathione by L-DOPA and dopamine. Relevance to

Parkinson’s disease. NeuroReport 6, 1480–1484.

Stern, Y., Marder, K., Tang, M.X., Mayeux, R., 1993. Antecedent clinical

features associated with dementia in Parkinson’s disease. Neurology 43,

1690–1692.

Ueda, S., Aikawa, M., Ishizuya-Oka, A., Koibuchi, N., Yamaoka, S., Yosh-

imoto, K., 1998. Age-related degeneration of the serotoninergic fibers

in the zitter rat brain. Synapse 30, 62–70.

Ueda, S., Aikawa, M., Ishizuya-Oka, A., Yamaoka, S., Koibuchi, N.,

Yoshimoto, K., 2000. Age-related dopamine deficiency in the meso-

striatal dopamine system of zitter mutant rats: regional fiber vulnera-

bility in the striatum and the olfactory tubercle. Neuroscience 95,

389–398.

Ueda, S., Sakakibara, S., Watanabe, E., Yoshimoto, K., Koibuchi, N., 2002.

Vulnerability of monoaminergic neurons in the brainstem of the zitter

rat in oxidative stress. Prog. Brain Res. 136, 293–302.

Wade, T.V., Rothblat, D.S., Schneider, J.S., 2001. Changes in striatal do-

pamine D3 receptor regulation during expression of and recovery from

MPTP-induced parkinsonism. Brain Res. 905, 111–119.