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Experimental Neurology

Astroglial plasticity and glutamate function in a chronic mouse model of

Parkinson’s disease

Adrian G. Dervana, Charles K. Meshulb,c, Mitchell Bealesa, Gethin J. McBeand, Cindy Mooreb,c,

Susan Totterdelle, Ann K. Snydera, Gloria E. Mereditha,*

aDepartment of Cellular and Molecular Pharmacology, Rosalind Franklin University of Medicine and Science, The Chicago Medical School, North Chicago,

IL 60064, USAbResearch Services, V.A. Medical Center, Portland, OR 97239, USA

cDepartment of Behavioral Neuroscience, Oregon Health and Science University, Portland, OR 97239, USAdDepartment of Biochemisty, Conway Institute Biomolecular & Biomedical Research, University College Dublin, Dublin 4, Ireland

eDepartment of Pharmacology, University of Oxford, Oxford OX1 3QT, UK

Received 20 May 2004; revised 25 June 2004; accepted 8 July 2004

Abstract

Astrocytes play a major role in maintaining low levels of synaptically released glutamate, and in many neurodegenerative diseases,

astrocytes become reactive and lose their ability to regulate glutamate levels, through a malfunction of the glial glutamate transporter-1.

However, in Parkinson’s disease, there are few data on these glial cells or their regulation of glutamate transport although glutamate

cytotoxicity has been blamed for the morphological and functional decline of striatal neurons. In the present study, we use a chronic mouse

model of Parkinson’s disease to investigate astrocytes and their relationship to glutamate, its extracellular level, synaptic localization, and

transport. C57/bl mice were treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTP/p). From 4 to 8

weeks after treatment, these mice show a significant loss of dopaminergic terminals in the striatum and a significant increase in the size and

number of GFAP-immunopositive astrocytes. However, no change in extracellular glutamate, its synaptic localization, or transport kinetics

was detected. Nevertheless, the density of transporters per astrocyte is significantly reduced in the MPTP/p-treated mice when compared to

controls. These results support reactive gliosis as a means of striatal compensation for dopamine loss. The reduction in transporter

complement on individual cells, however, suggests that astrocytic function may be compromised. Although reactive astrocytes are important

for maintaining homeostasis, changes in their ability to regulate glutamate and its associated synaptic functions could be important for the

progressive nature of the pathophysiology associated with Parkinson’s disease.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Glial fibrillary acidic protein; MPTP; Astrocytosis; GLT-1; Stereology

Introduction

Astrocytes, which can be detected by glial fibrillary

acidic protein (GFAP) immunoreactivity (Eng and Lee,

1995), are critical for maintaining the homeostatic extrac-

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

doi:10.1016/j.expneurol.2004.07.004

* Corresponding author. Department of Cellular and Molecular

Pharmacology, The Chicago Medical School, Rosalind Franklin University

of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064.

Fax: +1 847 578 3268.

E-mail address: gloria.meredith@finchcms.edu (G.E. Meredith).

ellular environment. They regulate glutamate uptake and

play an important role in synaptogenesis and synaptic

plasticity (Araque et al., 1998; Bowers and Kalivas, 2003;

Slezak and Pfrieger, 2003; Ullian et al., 2001). Pathological

processes in the brain, such as those seen in Alzheimer’s and

Huntington’s disease and in amyotrophic lateral sclerosis,

are associated with a significant astroglial reaction (Eddles-

ton and Mucke, 1993), altered glutamate uptake, and

changes in the expression of glutamate transporters (Beh-

rens et al., 2002; Lin et al., 1998; Rothstein et al., 1992,

1995; Scott et al., 2002). In Parkinson’s disease (PD),

190 (2004) 145–156

A.G. Dervan et al. / Experimental Neurology 190 (2004) 145–156146

however, astrocytosis has not been reported for postmortem

striatum (Forno et al., 1992), and nothing is known of

glutamate transport kinetics.

Under normal conditions, striatal neurons are protected

by an efficient glutamate transport system in neighboring

astrocytes, primarily mediated by the glial glutamate trans-

porter (GLT-1). However, under conditions of oxidative

stress such as occurs in PD and experimental animals treated

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

the functionality of this system might be changed (cf.

Greene and Greenamyre, 1995). When astrocytic cultures

are treated with MPP+, glutamate transporter function is

significantly impaired (Di Monte et al., 1999) and exper-

imental studies in vivo have proposed that striatal neuron

damage arising after dopamine (DA) depleting lesions is

due to increased excitatory activity (Dervan et al., 2003a;

Ingham et al., 1989; McNeill et al., 1998; Meredith et al.,

1995). Certainly, extracellular glutamate is elevated follow-

ing striatal DA loss with 6-hydroxydopamine (6-OHDA) or

acutely administered MPTP (Araki et al., 2001; Meshul et

al., 1999, 2002; Robinson et al., 2003; Sheng et al., 1993).

MPP+, when administered directly into the striatum,

increases the release of glutamate (Carboni et al., 1990),

and glutamatergic receptor blockade seemingly ameliorates

the neurotoxic effects of MPTP (Brouillet and Beal, 1993;

Turski et al., 1991). Nevertheless, less severe treatments

with MPTP, i.e., subacute administration, does not increase

levels of striatal glutamate (Robinson et al., 2003). More-

over, the striatal astroglial reaction to 6-OHDA is unstable

over time, rising, then falling, and rising yet again several

weeks post-lesion (Tripanichkul, 2000). When considered

together, these data indicate that astrocytes and their

regulation of glutamate after DA loss are imperfectly

understood.

Treatments with MPTP administered acutely or 6-OHDA,

either in vivo or in vitro, bear little resemblance to the

progressive nature of PD. Therefore, we questioned whether

a chronic model that is produced by combining MPTP with

probenecid (Lau et al., 1990) to enhance and prolong DA

loss (Petroske et al., 2001) would be more appropriate to

study astrocyte function, glutamate and its uptake. Some of

these results were presented previously (Dervan et al.,

2003b).

Materials and methods

Animals and treatment

One hundred fifty, adult, C57BL/6 male mice (Charles

River Laboratories, Wilmington, MA) at 8 weeks of age and

weighing 20–30 g were housed 2 to the cage. All protocols

were conducted in accordance with the National Institutes of

Health Publications No. 80–23 and were approved by the

Institutional Animal Care and Use Committees at the

Chicago Medical School and Portland VA Medical Center.

Two groups of mice (n = 50 per group) were treated

with 10 doses (2� week; 3.5 days apart) either of MPTP

hydrochloride (25 mg/kg in saline, s.c., Sigma-Aldrich,

St. Louis, MO) and probenecid (250 mg/kg in dimethyl

sulfoxide, i.p., Sigma-Aldrich) (MPTP/p), or probenecid

alone (controls). Previous studies have confirmed that

probenecid administration does not differ from saline

treatment in relation to DA cell number and DA function

(Lau et al., 1990; Petroske et al., 2001). Nevertheless,

results from probenecid-treated mice were compared to

saline-treated controls (n = 24) in some experiments.

Finally, two groups of mice (n = 25/group) were treated

acutely with MPTP (4 � 20 mg/kg in saline i.p. every 2

h). Chronically treated groups were sacrificed 4–8 weeks

after treatment and acutely treated groups, 1 week post-

treatment.

Mice used in transporter assays were killed by cervical

stunning and decapitation. All others were deeply

anesthetized with sodium pentobarbital (130 mg/kg) and

treated as follows: (1) After microdialysis studies and for

glutamate immuno-gold investigations, perfusions con-

sisted of 1000 units/ml of heparin in 0.1 M HEPES

buffer (pH 7.3) followed by 2.5% glutaraldehyde/0.5%

paraformaldehyde in 0.1 M HEPES containing 0.1%

picric acid. For immuno-gold studies, brains were post-

fixed overnight. (2) For other light or electron (EM)

microscopy studies, perfusions consisted of 2.5% sucrose

in 0.1 M phosphate-buffered saline (PBS), followed by

3% paraformaldehyde in 0.1 M PB; brains were post-

fixed for 2 h. For light microscopic investigations, brains

were sunk in 20% sucrose overnight. All brains were

blocked and sectioned at 50-Am thickness in the coronal

plane using a vibrating or freezing microtome.

Immunohistochemistry and electron microscopy

Sections through the striatum were collected in series

(1 in 5) in a uniformly random manner. Adjacent series of

sections were immunoreacted with monoclonal mouse

tyrosine hydroxylase antibodies (TH; Immunochemicals,

Stillwater, MN), rabbit anti-GFAP sera (DAKO, Carpen-

teria, CA), or dually immunolabeled for GLT-1 (rabbit

anti-GLT-1A sera were the kind gift of Dr. David Pow,

University of Queensland, Brisbane, Australia) and GFAP.

These GLT-1 antibodies were previously characterized and

their specificity established (Reye et al., 2002). All treated

and control tissues were immunoreacted at the same time

with the same solutions. Sections reserved for TH and

GFAP immunohistochemistry were incubated for 24 h at

48C in anti-TH sera (1:2000) or rabbit anti-GFAP

(1:2500), then in appropriate biotinylated IgG (1:300),

followed by incubation in avidin–biotin–peroxidase

(ABC) complex. Sections were then reacted in 0.05% 3-

3V diaminobenzidine hydrochloride (DAB) with added

0.01% H2O2, mounted onto slides, dehydrated, and

topped with coverslips.

A.G. Dervan et al. / Experimental Neurology 190 (2004) 145–156 147

The third series of sections were incubated in rabbit anti-

GFAP, as above, followed by donkey anti-rabbit IgG

conjugated to CY2 (Jackson ImmunoResearch, West Grove,

PA), 1:500. After extensive rinsing, sections were incubated

in rabbit anti-GLT-1, 1:20,000, overnight at 48C, followedby 2 h in donkey anti-rabbit IgG conjugated to CY3, 1:500.

Sections were mounted onto subbed slides and topped with

coverslips using a premixed mounting medium compatible

with fluorescent labels.

For EM studies of astrocytes (n = 3/group), sections from

the anterior striatum (+1.0 mm anterior; Franklin and

Paxinos, 1997) were treated with 1% osmium tetroxide

(0.1 M PB) for 30 min, dehydrated, and mounted onto slides

in Durcupan resin (ACM, Fluka, UK). Uranyl acetate (1%)

was added to the 70% ethanol step to increase contrast.

Regions of interest, selected with the light microscope, were

glued onto preformed resin blocks. Cut serial ultrathin

sections were mounted on Pioloform-coated, single slot

grids, contrasted with lead citrate and photographed in a

Philips 400 EM. Approximately 30 astrocytes were exam-

ined from each group.

Sections prepared for glutamate immuno-gold labeling

came from mice used for microdialysis. However, these

mice were not challenged with l-DOPA before perfusion,

so a comparison could be made between glutamate

immunolabeling density and basal glutamate. Sections

were incubated at room temperature in 1% osmium

tetroxide/1.5% potassium ferricyanide, rinsed and stained

en block with 0.5% uranyl acetate. Tissue was embedded

in Epon/Spurr resin and ultrathin sections cut at 60–90 nm.

Post-embedding immuno-gold EM was performed accord-

ing to Phend et al. (1992), as modified by Meshul et al.

(1994). The glutamate antibody (non-affinity purified,

rabbit polyclonal; Biogenesis, Brentwood, NH), as pre-

viously characterized by Hepler et al. (1988), was diluted

1:400,000 in 0.05 M Tris-buffered saline, pH 7.6.

Aspartate (1 mM) was added to the glutamate antibody

mixture 24 h before incubation to prevent any cross-

reactivity with aspartate within the tissue. The specificity

of glutamate immunolabeling was previously established

(Meshul et al., 1994). Photographs (10 per animal) were

taken randomly through the neuropil at a final magnifica-

tion of �40,000 using a digital camera (AMT, Boston,

MA). The number of gold particles per nerve terminal

associated with an asymmetrical synaptic contact was

counted and the area of the nerve terminal determined

using Image Pro Plus software (Media Cybernetics, Silver

Springs, MD, Version 3.01). The gold particles contacting

the synaptic vesicles within the nerve terminal were

counted and were considered part of the vesicular or

neurotransmitter pool according to previously established

methods (Meshul et al., 1999; Robinson et al., 2001). The

density of gold particles/Am2 of nerve terminal area was

determined for each animal and the mean density for each

treatment group calculated (mean density + SEM). The

differences between treatment groups were analyzed using

the Student’s t test. The total number of synapses for each

of the treatment groups making an asymmetrical synaptic

contact was as follows: MPTP/p group: 234 synapses,

probenecid group: 228 synapses.

Stereological estimates of astrocyte number and size

Striatal borders were demarcated for the stereological

analysis (see Figs. 1A–C; Franklin and Paxinos, 1997) with

the most posterior border set at the level of the anteriormost

amygdala. After randomly selecting a starting point, nine

sections at equally spaced intervals along the rostrocaudal

extent of the striatum were selected for analysis. The

reference volume for the striatum was estimated according

to Cavalieri principles (Coggeshall, 1992) and the total

number (N) and density (Nv) of astrocytes were estimated

with the optical dissector following fractionator rules (West

and Gundersen, 1990) and a semiautomated system (Stereo-

Investigator, version 4.565, Microbrightfield, Williston,

VT).

Video images of GFAP-immunoreactive astrocytes were

acquired with a 100� oil objective (1.35 NA) on a Nikon

E600 microscope equipped with a CCD camera output to a

high-resolution, computer monitor and a Ludl X–Y–Z

motorized stage (Ludl Electronics Products, Hawthorn,

NY). A counting frame of 75 � 75 Am (Fig. 2) and a

disector height of 8 Am with guard zones of 2 Am were

employed. An astrocyte was counted only if it had a clearly

defined nucleus within the disector area, did not intersect

forbidden lines (Fig. 2) and came into focus as the optical

plane moved through the height of the disector. The

precision of each estimate was expressed as the coefficient

of error (CE; Table 1). The somal volume of each astrocyte

cell body was calculated using the nucleator probe (Moller

et al., 1990).

Surgical procedures and in vivo microdialysis

Eight weeks after the last MPTP/p (n = 7) or probenecid

(n = 5) injection, animals were anesthetized (0.1 ml/kg of

2.5% ketamine, 1% xylazine, and 0.5% acepromazine in

normal saline) and placed in a stereotaxic apparatus

(Cartesian Research, Inc., Oregon). A small hole was drilled

in the skull, and the dura was punctured at the following

coordinates from Bregma (Franklin and Paxinos, 1997):

anterior: +1.2 mm; lateral: +1.8 mm. A stainless-steel guide

cannula (8-mm long, 21-gauge; Small Parts, Miami Lakes,

FL) was lowered 0.8 mm from the surface of the skull to the

level of the corpus callosum. The guide cannula was held in

a fixed position by one stainless steel screw attached to the

skull and encompassed by cranioplastic (Plastics One, Inc,

Roanoke, VA).

The dialysis probes, prepared as described by Robinson

and Wishaw (1988) with modifications (Meshul et al.,

1999), were 210 Am in diameter and 2 mm in length. One

day before use, the efficiency of transmitter recovery by the

Fig. 1. (A–C) Light photomicrographs of sections from rostral (A), middle (B), and caudal (C) levels of the striatum in probenecid-treated animals. The lines

drawn in A, B, and C show the boundaries used to delineate the striatum for stereological investigation. (D) In probenecid-treated controls, GFAP-positive

astrocytes are sparsely distributed (arrows) at all levels. (E) In MPTP/p-treated animals, intense GFAP immunostaining is visible (arrows) and corresponds to an

increase in the number of astrocytes and their processes (see Table 1). cc, corpus callosum; Nacc, nucleus accumbens; ac, anterior commissure; gp, globus

pallidus. Scale bars A–C = 500 Am; D and E = 100 Am.

A.G. Dervan et al. / Experimental Neurology 190 (2004) 145–156148

probe was determined by collecting three 10-min samples

(perfusion flow rate of 2 Al/min) after placing the probe in a

solution of glutamate (200 pg/Al) in artificial cerebrospinal

fluid (aCSF, pH 7.4).

Following the collection of recovery samples and the

day before the actual dialysis procedure, the probe was

lowered into the guide cannula with the entire length of

the dialysis probe in the striatum. The probe was secured

to the guide cannula with epoxy. The artificial aCSF

flowed through the probe overnight at a rate of 0.5 Al/min. The following morning, the pump speed was

increased to 2 Al/min for 1 h and then four samples

were collected every 15 min to determine the basal level

of extracellular glutamate. We have previously verified

that changes in extracellular striatal glutamate are depend-

ent on the presence of calcium within the aCSF (Meshul

et al., 2002). Both the probenecid and MPTP/p-treated

groups were then challenged with 15 mg/kg l-DOPA/12.5

mg/kg benserazide (Sigma) and an additional six 15-min

samples were collected. After the experiment, Vibratome

sections (100 Am) were stained with thionin and the site

of the probe placement within the striatum verified

histologically. Probe placement extended 2 mm along

the lateral quadrant of the striatum. If the placement was

not correct (i.e., outside the striatum), the data from that

animal were discarded. The four baseline data points and

the six l-DOPA-challenged data points were separately

averaged at each time point and a grand mean determined

for either the baseline or l-DOPA-challenged samples.

Values of extracellular striatal glutamate are expressed as

the mean F SEM in pmol/Al. The mean probe recovery

was 10.4 F 1.2%. Data were analyzed using a one-way

ANOVA and significant main effects were further

characterized using Peritz’ f test for comparison of

multiple means.

HPLC detection of dialysate glutamate levels

Glutamate concentration in each dialysate sample was

determined using a Hewlett Packard HPLC 1090 interfaced

with a Hewlett Packard 1046A Programmable Fluorescence

Detector. Dialysates were derivatized with o-phthalaldehyde

and chromatographed according to a modification of the

method of Schuster (1988), as previously reported (Meshul

et al., 1999, 2002; Robinson et al., 2003). Assay sensitivity

was in the subpicomole range.

Glutamate transport assays

To establish whether glutamate transporter function was

impaired, we decapitated mice either 3 days after acute

MPTP or saline treatment or 5 weeks after MPTP/p,

Fig. 2. Light micrograph illustrating the stereological counting frame.

Pictured are two astrocytes from a probenecid-control striatum. Only cells

that lay within the volume (75 � 75 � 8 Am) of the frame and/or touched

the green lines were counted and those that crossed the red lines were

excluded from analysis. Estimates of cell size were generated using the

nucleator probe (white lines). For each cell, eight isotropic lines converged

on the nucleus and intersected the somal boundary. Scale bar = 10 Am.

A.G. Dervan et al. / Experimental Neurology 190 (2004) 145–156 149

probenecid, or saline treatment. Pooled striata (n = 5 per

group) were homogenized in ice-cold medium of 0.32 M

sucrose in 50 mM Tris buffered to pH 7.5. The homogenate

was centrifuged 5 min at 1200 � g at 48C (Sorvall

centrifuge, SS-34 rotor). The supernatant was removed

and centrifuged for 12 min at 17,000 � g at 48C. The pelletwas washed in ice-cold gradient medium, collected by

Table 1

Detailed results from the striatum showing total volume, astrocyte number, densi

Probenecid

controls

Chronic

MPTP/P

V (mm3) CE Astrocytes

counted Q�Disec

P0381 5.681 0.07 258 502

P0382 5.636 0.06 275 500

P0384 5.024 0.05 172 450

P0385 5.829 0.08 214 517

Mean F SE 5.543 F 0.17 0.06 230 492

MP0354 5.103 0.09 572 523

MP0356 5.832 0.07 489 453

MP03266 5.926 0.07 598 527

MP03258 5.430 0.07 547 469

MP03267 6.009 0.06 705 514

Mean F SE 5.660 F 0.18a 0.07 582 497

a P = 0.651 (unpaired Student’s t-test); no significant difference compared to cob P = b0.001 (unpaired Student’s t-test); significantly greater than controls.c P = b0.001 (unpaired Student’s t-test); significantly greater than controls.d P = b0.05 (unpaired Student’s t-test); significantly greater than controls.

centrifugation, and resuspended in a final volume of 750 Alof homogenization medium.

High-affinity Na+-dependent transport was assayed at

substrate concentrations between 4 � 10�6 and 40 �10�6 M essentially as described previously (De Souza et

al., 1999). Briefly, synaptosomes were added to duplicate

microcentrifuge tubes containing Krebs solution, pH 7.4.

Following protein determination, the synaptosomes were

incubated at 378C with 15 nCi of d-[2,3-3H]-aspartic acid

(New England Nuclear, Downers Grove, IL). The

reaction was stopped with 200 Al of ice cold 1 mM d-

aspartate, followed by microcentrifugation at 15,700 � g

at 48C. The pellets were washed twice without resus-

pending using sodium-free Krebs medium, microcentri-

fuged as described and dissolved by overnight incubation

in 200 Al of 2% sodium dodecyl sulfate and the next day

suspended in 5 ml of Ecoscint A (National Diagnostics,

Atlanta, GA). The quantity of radioactivity was deter-

mined by liquid scintillation spectroscopy and the rate of

transport was plotted as a function of substrate concen-

tration, using nonlinear regression analysis.

Quantitative analysis of GLT and GFAP immunoreactive

profiles

Although high-affinity, Na+-dependent transport assays

give an indication of the number of functional trans-

porters present in synaptosomes prepared from the

striatum, they provide no information about the location

or density of transporters on individual astrocytes.

Therefore, we quantified the density of GLT-1 on

GFAP-immunolabeled cells. To minimize the variability

of immunohistochemical processing, all sections were

processed simultaneously using buffers, antibodies, and

solutions made at the same time. Single images (1024 �1024 pixels) were acquired digitally from an Olympus

Fluoview Series 43 confocal laser-scanning microscope

(CLSM; oil objective 60� and �2 magnification) without

ty, and somal volume

tors, P CE Total number

N (�105)

Astrocyte density

Nv (�103/mm3)

Somal

volume (Am3)

0.06 2.38 4.18 289.0

0.06 2.51 4.45 326.7

0.08 1.54 3.04 301.5

0.06 1.75 2.99 259.2

0.06 2.045 F 0.24 3.67F 0.38 294.1 F 14.0

0.04 4.89 9.59 330.4

0.04 4.24 7.27 319.6

0.04 4.52 7.62 342.6

0.04 4.10 7.55 320.9

0.03 5.21 8.67 343.7

0.04 4.592 F 0.20b 8.14 F 0.43c 331.6 F 5.1d

ntrols.

A.G. Dervan et al. / Experimental Neurology 190 (2004) 145–156150

saturation of pixel intensities and under constant con-

ditions by adjusting the CLSM parameters: pinhole size,

laser power, and detector voltage. Four mice from each

group and a single section through the striatum (mid-

rostral level from Bregma: anterior +1.2 mm; same as in

Fig. 3) for each mouse were selected for study.

Essentially all cells, i.e., approximately 20 per section

(each a merged image of a GFAP-immunoreactive

astrocyte and accompanying GLT-1 immunolabeling)

from the dorsomedial quadrant of the striatum from each

coded section, were captured by an investigator blind to

the animal treatment. The images were imported into the

Image J 1.29 program (NIMH), where the gray level of

each was inverted (no signal = 0 and maximum level =

255, corresponding to eight bits). The gray levels of the

merged images were measured by placing four open

rectangles, each with a diameter of 4 pixels, across the

cell body and proximal astrocytic processes of each cell

(see Results for further detail). Optical density within each

rectangle was recorded and measurements were pooled. The

mean F standard error of the mean (SEM) was established

for each group; group means were compared with a

Student’s t test.

Results

Effect of MPTP/p treatment on striatum

At 3 weeks post-MPTP/p treatment, mice show a

significant behavioral impairment and reduction in DA

neurons and striatal DA function (Dervan et al., 2003a;

Fig. 3. Representative sections of the rostral striatum from probenecid control (A) a

striatum contains numerous TH-immunoreactive fibers. (B) The extent of loss of T

Note the relative sparing in the ventral striatum and along a thin band dorsolater

Meredith et al., 2002; Petroske et al., 2001). In the

present study, mouse striata from MPTP/p-treated mice

that were immunoreacted for TH show a significant

reduction in TH-positive fibers, except for an outer rim

of spared fibers in the dorsolateral striatum and a

relatively spared ventral striatum (compare Fig. 3B with

A). This pattern of TH immunoreactivity is consistent

with that described in our earlier work (Meredith et al.,

2002; Petroske et al., 2001).

Astrocyte distribution, number, and size

In control animals, GFAP immunoreactivity is moderate to

light in the striatum and cortex (Figs. 1A–C); GFAP-

immunopositive cells are typically grouped into small

dislandsT that follow the course of blood vessels. At the

cellular level, each astrocyte in control mice (probenecid- or

saline-treated) has a tightly configured set of fibrous

processes that are initially thick as they emerge from the cell

body but rapidly become fine and wispy distally (Fig. 4A). In

contrast, MPTP/p treatment is associated with an increase in

GFAP-immunoreactive profiles in the striatum (compare Fig.

1E with D), and immunopositive astrocytes display a reactive

morphology with thickened processes (Fig. 4B). The reactive

cells are often located close to the ventricle and along the

corpus callosum. Ultrastructurally, most astrocytes from

MPTP/p-treated, but not control mice, have nuclear inden-

tations and clumped chromatin (Figs. 4C–D).

Stereological estimates showed that the striatum of

MPTP/p-treated animals had significantly more GFAP-

immunoreactive cells than does that of controls. However,

the reference volume of the striatum is not different between

nd MPTP/p (B) treated animals immunoreacted for TH. In controls (A), the

H-immunoreactivity can be seen in the striatum of MPTP/p-treated animals.

ally. Scale bars = 500 Am.

Fig. 5. In vivo microdialysis of the lateral striatum from MPTP/p (n = 7) or

probenecid (n = 5) treated animals, 8 weeks after final administration.

Baseline samples were collected from each group and then both groups

were injected with a single dose of l-DOPA (15 mg/kg, l-DOPA + 12.5

mg/kg, benserazide) and additional samples collected. There was no

difference in the basal extracellular levels of striatal glutamate between the

two groups [Control (baseline) vs. MPTP/p (baseline)]. Following the

injection of l-DOPA, there was a significant increase in the extracellular

levels of glutamate in both groups compared to their respective baseline

levels. Values are means F SEM (pmol/Al). *P b 0.05 and **P b 0.05

compared to control (baseline) or MPTP/p (baseline) groups using Peritz’ f

test for comparison of multiple means.

Fig. 4. Light photomicrographs of GFAP-immunopositive astrocytes of probenecid-treated (A) and MPTP/p-treated (B) animals. Note the increased thickness

of processes on cells after MPTP/p treatment (B). (C) Representative electron micrograph of a striatal astrocyte of a probenecid-treated animal. Note the

electron-lucent cytoplasm and nucleus. In astrocytes from MPTP/p-treated animals (D), the cells are electron-dense and contain an indented nucleus with

clumped chromatin (arrows). Scale bars in A and B = 10 Am and in C and D = 2 Am.

A.G. Dervan et al. / Experimental Neurology 190 (2004) 145–156 151

groups (Table 1). In addition, cell bodies immunopositive

for GFAP are significantly increased in size following

MPTP/p treatment when compared to controls (Table 1).

Extracellular glutamate levels

Eight weeks following administration of MPTP/p or

probenecid (i.e., a total of 13 weeks after the first dose of

MPTP/P), in vivo microdialysis of the lateral striatum

revealed no difference in basal extracellular glutamate

between the two groups (Fig. 5). To determine if the

MPTP/p group is differentially sensitive to the effects of l-

DOPA, used to restore brain levels of DA, both groups

received a single dose of the drug following the collection of

baseline samples. This resulted in a significant increase in

the extracellular levels of striatal glutamate in both groups

compared to baseline (Fig. 5). Although the increase in the

control (probenecid) group (21.4%) is greater than that of

the MPTP/p group (12.3%), this difference is not statisti-

cally significant.

Nerve terminal glutamate immuno-gold labeling

In a separate group of MPTP/p- or probenecid-treated

animals, quantitative immuno-gold EM was carried out to

determine the immunolabeling associated with the synaptic

vesicle (i.e., neurotransmitter) pool in endings making an

asymmetrical synaptic contact (Figs. 6A–B). There is no

difference in the density of nerve terminal glutamate

labeling between MPTP/p-treated and control groups (Fig.

Fig. 7. The effect of chronic MPTP/p (A) and acute MPTP (B) treatment on

the kinetics of d-[3H]aspartate transport. Synaptosomes were incubated at

358C for 4 min in Krebs’ medium and the uptake of d-[3H]aspartate at the

specified concentrations was measured. The results from each treatment are

plotted as a mean of five independent experiments, performed in duplicate

and expressed as the mean F SEM.

Fig. 6. Nerve terminal glutamate immuno-gold labeling in the dorsolateral

striatum, 8 weeks after MPTP/p or probenecid (n = 8/group). (A)

Representative electron micrograph from the probenecid (control) group.

Note the number of gold particles (arrowhead) associated with synaptic

vesicles and within three nerve terminals making an asymmetrical synaptic

contact (arrows) with an underlying dendritic spine. (B) Glutamate nerve

terminal immunolabeling from the MPTP/p group: There are four terminals

making an asymmetrical synaptic contact (arrows) with an underlying

dendritic spine. (C) The density of nerve terminal glutamate (gold particles/

Am2) in asymmetrical (excitatory) synapses was determined. There was no

change in the density of gold particles, the number of gold particles, or in

the area of the terminals between the two groups. Values are meansF SEM.

Scale bar = 0.5 Am.

A.G. Dervan et al. / Experimental Neurology 190 (2004) 145–156152

6C). Previous work has shown that 97% of the immuno-

gold labeling is associated with the synaptic vesicle pool

and only 3% with the cytoplasmic pool (Meshul et al.,

1999). This cytoplasmic pool does not change following a

6-OHDA lesion (Meshul et al., 1999). In previous studies,

the mitochondrial (metabolic) pool was analyzed separately

as compared to the vesicular pool. There appears to be no

change in the mitochondrial pool of glutamate after a 6-

OHDA lesion (Meshul and Allen, 2000; Robinson et al.,

2001). In the present study, the gold particles associated

with the mitochondrial pool were not included in the density

measurements of nerve terminal glutamate.

Effect of MPTP treatment on high-affinity, Na+-dependent

transport

We studied the rate of transport of d-[3H] aspartate

into crude synaptosomes from mouse striatum over a

concentration range of 0.4–40 AM and compared high-

affinity transport in chronically (MPTP/p) and acutely

(MPTP) treated mice and their respective probenecid or

saline controls. Fig. 7A shows that Vmax for chronically

MPTP/p-treated mice (2.48 F 0.04 nmol mg protein�1

min�1) does not differ significantly from that of the

probenecid controls (2.36 F 0.12 nmol mg protein�1

min�1; P = 0.616). The values for Km are not

significantly different between groups (7.75 F 1.1 AMfor probenecid vs. 8.69 F 0.43 AM for MPTP/p treated

animals; P = 0.377). Fig. 7B shows that the Vmax for

animals treated acutely with MPTP (2.68 F 0.08 nmol

mg protein�1 min�1) also does not differ significantly

from saline-treated controls (2.71 F 0.07 nmol mg

protein�1 min�1; P = 0.772) and the values for Km also

A.G. Dervan et al. / Experimental Neurology 190 (2004) 145–156 153

did not differ significantly between groups (8.97 F 0.61

AM for saline vs. 9.81 F 0.74 AM for MPTP treated

mice; P = 0.914).

Comparisons between acute and chronic treatments

show no significant difference in Vmax (ANOVA, F =

0.497; P = 0.689) or Km (F = 0.463; P = 0.711) between

groups. Furthermore, there is no difference between the

probenecid and saline control groups (Vmax, P = 0.299;

Km, P = 0.465).

GLT-1 labeling on GFAP-immunolabeled astrocytes

The GLT-1 transporter is the glutamate transporter

commonly found on striatal astrocytes. As our transport

studies showed that the number of functional transporters in

the striata of MPTP/p-treated mice did not differ from that

of controls (Vmax was unchanged), but our stereological

analysis revealed a doubling in the number of astrocytes

after MPTP/p treatment, we hypothesized that the density of

transporters per astrocyte would be lower in the toxin-

treated group compared to controls. To test this possibility,

we employed dual-label confocal microscopy under con-

trolled conditions (Figs. 8A–F). Qualitatively, sections from

MPTP/p-treated mice showed more GFAP-labeled pro-

cesses in each field. GLT-1 immunoreactivity on the cell

soma and proximal processes of GFAP-immunopositive

cells appeared less obvious when compared to controls

(compare Fig. 8C with F). We quantified the density of

GLT-1 immunoreactivity per GFAP-immunopositive astro-

cyte by capturing merged images and averaging the pixel

densities after inversion of each image (see Materials and

methods). After pooling the data for each group and

comparing groups with a Student’s t test, we found that

the mean optical density for the MPTP/p-treated group

(104.6 F 17 SEM) was significantly lower (P b 0.05) than

that in controls (174.6 F 25 SEM). This result can also be

interpreted as a 40% reduction in GLT-1 density per

Fig. 8. Confocal images showing the cellular localization of GFAP (A, D) and GL

month after final treatment. Four animals from the MPTP/p group and four probe

localization on astrocytes from MPTP/p treated animals (F) appears to be less (y

astrocyte after MPTP/p treatment compared to controls

(compare Fig. 8C with F).

Discussion

Many of the classic symptoms of Parkinson’s disease

are thought to arise by elevated glutamate levels follow-

ing the loss of DA (Fornai et al., 1997). Experimental

studies have shown increased striatal neuron activity after

complete 6-OHDA lesions (Schultz and Ungerstedt, 1978)

and elevated glutamate 1 month after acute MPTP

(Robinson et al., 2003). Although glutamatergic pathways

to the striatum could potentially show sustained over-

activity after DA loss (Fornai et al., 1997), there are data

to suggest that the striatum compensates remarkably well

for DA reductions, especially if the loss is progressive

(Zigmond et al., 1990). In the present study, we show for

the first time that extracellular glutamate and its transport

kinetics are not changed 13 weeks after the start of

chronic MPTP/p treatment, although greater than 90% of

the striatal DA function is gone (Petroske et al., 2001).

The question arises as to the mechanism responsible for

such results. Our study indicates that it is due to a

significant increase in reactive astrocytes, which through

their large numbers maintain striatal homeostasis. Never-

theless, each GFAP-immunopositive cell has a reduced

complement of GLT-1 transporters, a change that may

eventually contribute to disease pathophysiology.

Changes in extracellular glutamate could be reflected in

the time course of DA loss. Acute MPTP treatment, which

down-regulates TH levels within 24 h (Mandel et al., 2002),

is associated with significant elevations in extracellular

glutamate after 4 weeks (Robinson et al., 2003). In the

chronic model, DA levels decline progressively (Petroske et

al., 2001), and although earlier time points have not been

studied, striatal glutamate probably rises rapidly after each

T-1 (B, E) in probenecid-treated (A–C) and MPTP/p-treated mice (D–F) 1

necid controls were used in this study. In merged images (C, F), the GLT-1

ellow) compared to the probenecid controls (C). Scale bar = 10 Am.

A.G. Dervan et al. / Experimental Neurology 190 (2004) 145–156154

dose of MPTP but then subsides in parallel with astrocytic

proliferation. Certainly, the striatal, astroglial reaction to 6-

OHDA rises and falls with time after the lesion (Tripanich-

kul, 2000).

The rise in striatal extracellular glutamate following the

single dose of l-DOPA in MPTP/p-treated mice is

consistent with results after acute MPTP treatment (Rob-

inson et al., 2003) and presumably reflects changes in basal

ganglia function (Albin et al., 1989). DA stimulation, either

indirectly via l-DOPA to restore brain levels of DA, or via

DA D-1 or D-2 receptors in the striatum, would lead to a

decrease in the GABAergic output from the basal ganglia to

the motor thalamus, resulting in increased activity of the

corticostriatal pathway.

The striatum appears to adapt to glutamate changes by up-

regulating astrocytic function through an increase in GFAP-

immunoreactive cells. The ability of glia to regulate

glutamate levels is well known, and early elevations in

glutamate could be counteracted by a rapid astrocytic reaction

that fades later in a manner similar to that described in models

of CNS injury (see Ridet et al., 1997). Acute MPTP treatment

does trigger a reactive gliosis, but it is short-lived (Muramatsu

et al., 2003), which may explain the lack of change in

transporter kinetics (present results) at 1 week post treatment,

and the high level of extracellular glutamate 1 month later

(Robinson et al., 2003). Thus, reduced levels of GFAP

immunoreactivity found in postmortem PD striatum (Forno et

al., 1992) could be due to the late stage of the disease and not

to a lack of a glial response, which may have peaked earlier.

Moreover, subacute l-DOPA treatment significantly elevates

GLT-1 mRNA (Lievens et al., 2001), a change that may

enhance glutamate recycling and limit gliosis.

We have measured high-affinity uptake using synaptoso-

mal preparations, which are generally considered to be

neuronal in nature. Nevertheless, there is good evidence that

uptake activities by these particles reflect glial-mediated

transport (see Danbolt, 2001), presumably because glial cell

fragments contaminate the preparation (Nakamura et al.,

1993) and because mice deficient in the GLT-1 transporter

show greatly reduced glutamate uptake from synaptosomes

compared to controls (Tanaka et al., 1997).

Our results show that the kinetics of glutamate are

unchanged after chronic MPTP. Although single application

of MPP+ to astrocytic cultures is accompanied by impaired

glutamate uptake (Di Monte et al., 1999), the lack of change

seen in our study may not be surprising because the animals

were analyzed 5 weeks after an extended exposure to MPTP.

Other in vivo studies have shown that nigrostriatal denerva-

tion has no effect on glutamate transporter mRNA (Lievens et

al., 2001).

As the basis of MPTP toxicity is negotiated via astrocytic

uptake and conversion (Markey et al., 1984), it is unclear

whether MPP+ affects host astrocyte function. When

compared to their mesencephalic counterparts, striatal

astrocytes seem to have a stronger intrinsic facility for

dealing with oxidative stress, especially in the presence of

MPTP (Wong et al., 1999). Striatal astrocytes can increase

levels of superoxide dismutase, which affords them

increased protection by absorbing reactive oxygen species

(Wong et al., 1999). Therefore, it seems unlikely that the

glial changes seen in the present study are strictly due to the

inability of striatal astrocytes to cope with the pyridinium

ion, MPP+, but see Di Monte et al. (1999). Once reactive,

astrocytes show a reduced ability to carry out normal

support functions (Behrens et al., 2002; Ginsberg et al.,

1995; Krum et al., 2002; Masliah et al., 1996; Ridet et al.,

1997). Therefore, their activation could conceivably con-

tribute to progressive brain pathology (Blandini et al., 1996;

Olanow and Tatton, 1999).

Glutamate rapidly diffuses out of the cleft of excitatory

synapses (Clements, 1996) and is quickly cleared by

glutamate transporters, as measured in hippocampal cultures

(Diamond and Jahr, 1997; Tong and Jahr, 1994). Although

reuptake blockers have essentially failed to prolong AMPA

receptor-mediated synaptic currents at some synapses (Hes-

trin et al., 1990; Isaacson and Nicoll, 1993; Sarantis et al.,

1993), glutamate transporters contribute to synaptic efficacy

if AMPA receptor desensitization is blocked (Mennerick

and Zorumski, 1994). Glutamate transporters affect both the

duration and amplitude of excitatory postsynaptic currents

and ultimately influence the induction of synaptic plasticity

(Bowers and Kalivas, 2003; Diamond and Jahr, 1997;

Mennerick and Zorumski, 1994; Turecek and Trussell,

2000). Moreover, any reduction in uptake sites for

glutamate, as seen in the present results, could increase

the vulnerability of striatal neurons to toxic insults (McBean

and Roberts, 1985). Maintaining the normal complement of

glutamate transporters appears to be critical for modulating

synaptic strength, and altered synaptic efficacy, rather than

elevated glutamate, could therefore be a strong contributing

factor to progressive pathogenesis in PD.

Acknowledgments

This research was supported by a USPHS NS41799

(GEM), a Wellcome Trust Biomedical Collaboration Grant

(GEM and ST), and the Department of Veterans Affairs Merit

Review Program (CKM). We thank Dr. David Pow,

University of Queensland, Brisbane, Australia, for his

generous gift of the GLT1 antibodies and Prof. B.L. Roberts

for his comments on the manuscript. We also thank Heather

Milligan and Julie Frederickson for their technical assistance.

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