Biphasic and bilateral changes in striatal VGLUT1 and 2 protein expression in hemi-Parkinson rats.
ABSTRACT Parkinson's disease is characterized by disturbed glutamatergic neurotransmission in the striatum. Important mediators of extracellular glutamate levels are the vesicular glutamate transporters VGLUT1 and VGLUT2 in respectively corticostriatal and thalamostriatal afferents, next to the high-affinity Na(+)/K(+)-dependent glutamate transporters and the cystine/glutamate antiporter. In the present study, we compared bilateral striatal VGLUT1 and VGLUT2 protein expression as well as VGLUT1 and VGLUT2 transcript levels in the neocortex and parafascicular nucleus of hemi-Parkinson rats at different time intervals post unilateral 6-OHDA injection into the medial forebrain bundle versus controls. Three weeks post-injection we detected increased striatal VGLUT1 expression together with decreased VGLUT2 expression. On the other hand, after twelve weeks, the expression of VGLUT1 was decreased in hemi-Parkinson rats whereas the striatal expression of VGLUT2 was comparable to control rats. No effect could be seen on VGLUT transcript levels in the respective projection areas at any time. In conclusion, we observed a biphasic and bilateral change in the protein expression levels of both VGLUTs in the striatum of hemi-Parkinson rats indicative for a different and time-dependent change in glutamatergic neurotransmission from the two types of striatal afferents.
- SourceAvailable from: Mathieu Favier[Show abstract] [Hide abstract]
ABSTRACT: It has been suggested that glutamatergic system hyperactivity may be related to the pathogenesis of Parkinson's disease (PD). Vesicular glutamate transporters (VGLUT1-3) import glutamate into synaptic vesicles and are key anatomical and functional markers of glutamatergic excitatory transmission. Both VGLUT1 and VGLUT2 have been identified as definitive markers of glutamatergic neurons, but VGLUT 3 is also expressed by non glutamatergic neurons. VGLUT1 and VGLUT2 are thought to be expressed in a complementary manner in the cortex and the thalamus (VL/VM), in glutamatergic neurons involved in different physiological functions. Chronic high-frequency stimulation (HFS) of the subthalamic nucleus (STN) is the neurosurgical therapy of choice for the management of motor deficits in patients with advanced PD. STN-HFS is highly effective, but its mechanisms of action remain unclear. This study examines the effect of STN-HFS on VGLUT1-3 expression in different brain nuclei involved in motor circuits, namely the basal ganglia (BG) network, in normal and 6-hydroxydopamine (6-OHDA) lesioned rats. Here we report that: 1) Dopamine(DA)-depletion did not affect VGLUT1 and VGLUT3 expression but significantly decreased that of VGLUT2 in almost all BG structures studied; 2) STN-HFS did not change VGLUT1-3 expression in the different brain areas of normal rats while, on the contrary, it systematically induced a significant increase of their expression in DA-depleted rats and 3) STN-HFS reversed the decrease in VGLUT2 expression induced by the DA-depletion. These results show for the first time a comparative analysis of changes of expression for the three VGLUTs induced by STN-HFS in the BG network of normal and hemiparkinsonian rats. They provide evidence for the involvement of VGLUT2 in the modulation of BG cicuits and in particular that of thalamostriatal and thalamocortical pathways suggesting their key role in its therapeutic effects for alleviating PD motor symptoms.BMC Neuroscience 12/2013; 14(1):152. · 2.85 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Long term treatment with L-3,4-dihydroxyphenylalanine (l-DOPA) is associated with several motor complications. Clinical improvement of this treatment is therefore needed. Lesions or high frequency stimulation of the hyperactive subthalamic nucleus (STN) in Parkinson's disease (PD), alleviate the motor symptoms and reduce dyskinesia, either directly and/or by allowing the reduction of the l-DOPA dose. N-methyl-d-aspartate (NMDA) receptor antagonists might have similar actions. However it remains elusive how the neurochemistry changes in the STN after a separate or combined administration of l-DOPA and a NMDA receptor antagonist. By means of in vivo microdialysis, the effect of l-DOPA and/or MK 801, on the extracellular dopamine (DA) and glutamate (GLU) levels was investigated for the first time in the STN of sham and 6-hydroxydopamine-lesioned rats. The l-DOPA-induced DA increase in the STN was significantly higher in DA-depleted rats compared to shams. MK 801 did not influence the l-DOPA-induced DA release in shams. However, MK 801 enhanced the l-DOPA-induced DA release in hemi-parkinson rats. Interestingly, the extracellular STN GLU levels remained unchanged after nigral degeneration. Furthermore, administration of MK 801 alone or combined with l-DOPA did not alter the STN GLU levels in both sham and DA-depleted rats. The present study does not support the hypothesis that DA-ergic degeneration influences the STN GLU levels neither that MK 801 alters the GLU levels in lesioned and non-lesioned rats. However, NMDA receptor antagonists could be used as a beneficial adjuvant treatment for PD by enhancing the therapeutic efficacy of l-DOPA at least in part in the STN.Neuropharmacology 10/2014; 85:198–205. · 4.82 Impact Factor
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ABSTRACT: In the striatum, the dendritic tree of the two main populations of projection neurons, called "Medium Spiny Neurons (MSNs)", are covered with spines that receive glutamatergic inputs from the cerebral cortex and thalamus. In Parkinson's disease (PD), striatal MSNs undergo an important loss of dendritic spines, whereas aberrant overgrowth of striatal spines occurs following chronic cocaine exposure. This review examines the possibility that opposite dopamine dysregulation is one of the key factors that underlies these structural changes. In PD, nigrostriatal dopamine degeneration results in a significant loss of dendritic spines in the dorsal striatum, while rodents chronically exposed to cocaine and other psychostimulants, display an increase in the density of "thin and immature" spines in the nucleus accumbens (NAc). In rodent models of PD, there is evidence that D2 dopamine receptor-containing MSNs are preferentially affected, while D1-positive cells are the main targets of increased spine density in models of addiction. However, such specificity remains to be established in primates. Although the link between the extent of striatal spine changes and the behavioral deficits associated with these disorders remains controversial, there is unequivocal evidence that glutamatergic synaptic transmission is significantly altered in both diseased conditions. Recent studies have suggested that opposite calcium-mediated regulation of the transcription factor myocyte enhancer factor 2 (MEF2) function induces these structural defects. In conclusion, there is strong evidence that dopamine is a major, but not the sole, regulator of striatal spine pathology in PD and addiction to psychostimulants. Further studies of the role of glutamate and other genes associated with spine plasticity in mediating these effects are warranted.Neuroscience 07/2013; · 3.33 Impact Factor
Biphasic and bilateral changes in striatal VGLUT1 and 2 protein expression
in hemi-Parkinson rats
Ann Massiea, Anneleen Schalliera, Katia Vermoesena, Lutgarde Arckensb, Yvette Michottea,*
aDepartment of Pharmaceutical Chemistry and Drug Analysis, Research Group Experimental Pharmacology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium
bLaboratory of Neuroplasticity and Neuroproteomics, Katholieke Universiteit Leuven, Naamsestraat 59, Box 2467, 3000 Leuven, Belgium
Parkinson’s disease is a neurodegenerative disorder with a
selective loss of dopaminergic neurons in the substantia nigra pars
between dopaminergic and glutamatergic input in the striatum.
However, until now there is no consensus about the effect of
dopamine deprivation on striatal extracellular glutamate (Linde-
fors and Ungerstedt, 1990; Meshul et al., 1999; Jonkers et al., 2002;
Corsi et al., 2003; Bianchi et al., 2003; Robelet et al., 2004; Walker
et al., 2009).
Extracellular glutamate levels in the striatum are mainly
determined by release of glial cells via the cystine/glutamate
antiporter (Baker et al., 2002), reuptake by glial cells through the
see Danbolt, 2001) and exocytotic release from afferents originat-
ing respectively in the cerebral cortex and the thalamus (Charara
et al., 2002; Smith et al., 2004). Vesicular glutamate transporters
(VGLUTs) located in the membrane of presynaptic glutamatergic
vesicles are responsible for loading glutamate into the vesicles
before they release their content into the synaptic cleft by fusing
with the presynaptic membrane (exocytosis). The driving force of
the VGLUTs is the electrochemical gradient generated by the
V-type H+-ATPase (Takamori, 2006). Since the number of VGLUT
molecules per synaptic vesicle has a major impact on quantal size
in glutamatergic neurons, a variation in VGLUT expression levels
could have significant impact on synaptic transmission (Wojcik
et al., 2004). The expression levels of VGLUT protein are an
indication for the relative synaptic strength of presynaptic
glutamatergic innervation for a given brain region.
Three isoforms of VGLUTs have been characterized (for review
see Takamori, 2006). Whereas VGLUT1 and 2 are specific markers
for glutamatergic neurons, VGLUT3 is not present in glutamatergic
GABA-ergic neurons (Gras et al., 2002; Fremeau et al., 2002). It is
commonly accepted that detection of striatal protein expression of
VGLUT1 and VGLUT2 makes it possible to distinguish between
cortical and thalamic inputs. Whereas cortical neurons innervating
the striatum use VGLUT1, striatal afferents from the thalamic
sources express VGLUT2 (Lacey et al., 2005; Raju and Smith, 2005;
Fujiyama et al., 2006; Raju et al., 2006). However, recent studies in
rat (Barroso-Chinea et al., 2007, 2008) showed that, although
VGLUT2 is the only VGLUT expressed in thalamostriatal projecting
neurons located in the midline and intralaminar nuclei,all neurons
from the ventral thalamic nuclei express both VGLUTs. Neverthe-
less, in the adult central nervous system, VGLUT1 and 2 are still
generally considered to be selective markers of the glutamatergic
corticostriatal and thalamostriatal afferents (Raju et al., 2008).
Neurochemistry International 57 (2010) 111–118
A R T I C L EI N F O
Received 1 March 2010
Received in revised form 15 April 2010
Accepted 23 April 2010
Available online 5 May 2010
Vesicular glutamate transporters
A B S T R A C T
Parkinson’s disease is characterized by disturbed glutamatergic neurotransmission in the striatum.
Important mediators of extracellular glutamate levels are the vesicular glutamate transporters VGLUT1
and VGLUT2 in respectively corticostriatal and thalamostriatal afferents, next to the high-affinity Na+/
K+-dependent glutamate transporters and the cystine/glutamate antiporter. In the present study, we
compared bilateral striatal VGLUT1 and VGLUT2 protein expression as well as VGLUT1 and VGLUT2
transcript levels in the neocortex and parafascicular nucleus of hemi-Parkinson rats at different time
intervals post unilateral 6-OHDA injection into the medial forebrainbundle versus controls. Three weeks
post-injection we detected increased striatal VGLUT1 expression together with decreased VGLUT2
expression. On the other hand, after twelve weeks, the expression of VGLUT1 was decreased in hemi-
Parkinsonrats whereas the striatalexpression of VGLUT2 wascomparable tocontrol rats.No effectcould
be seen on VGLUT transcript levels in the respective projection areas at any time. In conclusion, we
observed a biphasic and bilateral change in the protein expression levels of both VGLUTs in the striatum
of hemi-Parkinson rats indicative for a different and time-dependent change in glutamatergic
neurotransmission from the two types of striatal afferents.
? 2010 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +32 2 477 47 48; fax: +32 2 477 41 13.
E-mail address: firstname.lastname@example.org (Y. Michotte).
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/neuint
0197-0186/$ – see front matter ? 2010 Elsevier Ltd. All rights reserved.
In Parkinson’s disease patients evidence was found for a
perturbation of both the corticostriatal and the thalamostriatal
glutamatergic systems. The loss of striatal dopamine in patients
(Anglade et al., 1996), as well as in rodent models (Ingham et al.,
1998), is associated with an increased diameter of post-synaptic
densities at corticostriatal synapses. Moreover, a significant cell
loss in the centromedian/parafascicular nuclear complex (CM/Pf),
which is the main source of thalamic inputs to the striatum, was
observed in post-mortem brains of Parkinson patients (Henderson
et al., 2000a,b). Yet, only a few studies addressed the effect of
dopamine depletion on striatal VGLUT protein expression levels in
rodent or primate models for Parkinson’s disease (Robelet et al.,
2004; Chung et al., 2007; Raju et al., 2008). Similar as for the
the results, probably related to the use of different animal models
as well as variations within one model, i.e. site of toxin injection
and survival time after toxin injection.
Within this study, we examined the striatal protein expression
of VGLUT1 and VGLUT2, three to twelve weeks after unilateral
injection of 6-OHDA into the medial forebrain bundle (MFB) using
immunohistochemistry and semi-quantitative Western blotting.
We also used in situ hybridization to study VGLUT transcript
expression levels in those brain regions that represent the major
source of glutamatergic input to the striatum, i.e. neocortex and
parafascicular nucleus (Pf) of the thalamus, corresponding to the
CM/Pf in humans.
1. Materials and methods
Protocols for animal experiments described in this study were
performed according to national guidelines on animal experimen-
tation and were approved by the Ethical Committee for Animal
Experimentation of the Faculty of Medicine and Pharmacy of the
Vrije Universiteit Brussel.
All animals (male albino Wistar rats, Charles Rivers Laborato-
ries) used for this study were housed under standard laboratory
conditions. Some of the animals included in this study were
already used for a previous study (Massie et al., 2008b).
1.2. 6-OHDA lesioning of the medial forebrain bundle
Rats weighing 175–200 g were anaesthetized with a mixture of
ketamine:diazepam (90.5 mg/kg:4.5 mg/kg i.p.) and placed in a
stereotaxic frame. The skull was exposed and a burr hole was
drilled through the skull at the appropriate location. A 6-OHDA
solution (containing 4 mg/ml 6-OHDA (Sigma–Aldrich, St. Louis,
MO, USA) in 0.01% ascorbic acid, pH 5.0) was stereotactically
injected into the MFB at coordinates L ?1.5, AP ?3.2, DV 8.7,
relative to Bregma, according to the atlas of Konig and Klippel. A
total volume of 4 ml 6-OHDA was injected at a flow rate of 1 ml/
min. The syringe was left in place for 2 min and then slowly
removed over a 1–2 min time period. The skin was sutured and the
rats received 4 mg/kg ketofen (Merial, Brussels, Belgium) i.p. to
provide post-operative analgesia. Rats were killed with a lethal
dose of Nembutal (pentobarbital, i.p.) (Sanofi sante, Brussels,
Belgium) three weeks, five weeks or twelve weeks after 6-OHDA
injection. Sham operated rats were injected with vehicle only,
whereas control rats did not undergo any surgical procedure.
Immunohistochemistry was performed as described previously
(Massie et al., 2008a). Briefly, after transcardial perfusion with a
physiological solution followed by freshly depolymerized 4%
paraformaldehyde (Sigma–Aldrich) in 0.15 M phosphate buffered
saline (pH 7.42), brains were removed and post-fixed in the same
fixative overnight, rinsed in tap water for 24 h and stored in
0.015 M phosphate buffered saline at 4 8C. Free floating 50 mm
frontal sections were made with a vibratome and stored in serial
order in 0.015 M phosphate buffered saline at 4 8C.
All rinsing steps and incubations of the staining procedure were
performed in Tris–saline (0.01 M, 0.1% Triton X-100 (Sigma–
Aldrich), pH 7.4) at room temperature, and under gentle agitation.
The sections underwent a permeabilizing treatment consisting of
incubation in 0.1% trypsin (Fluka, Buchs, Switzerland) for 1 h at
37 8C prior to a blocking step with normal goat serum (diluted 1/5,
45 min; Millipore, Temecula, CA, USA). Thereafter the sections
were incubated overnight with the immunoaffinity purified
polyclonal VGLUT1 (1/10,000; raised in Guinea pig, AB5905,
Millipore) or VGLUT2 antibodies (1/10,000; raised in Guinea pig,
AB5907, Millipore). The next day, sections were processed by the
avidin–biotin method using a Vectastain ABC kit (Vector Labora-
tories, Burlingame, CA, USA) and immunoreactivity was visualized,
after a final rinsing step with acetate buffer, using the glucose
oxidase-diaminobenzidine–nickel method (Shu et al., 1988).
Immunohistochemical stainings at the level of the striatum, using
anti-tyrosine hydroxylase (TH) antibodies (raised in rabbit, diluted
1/2000, AB152, Millipore), were performed as a control for correct
lesioning. The protocol for the TH staining is identical to the
protocol described above. Photomicrographs were made of the
stained sections and processed using Adobe Photoshop 7.0.
1.4. Western blotting
After decapitating the rats, brains were removed, snap-frozen
and 100 mm cryosections were made at the level of the striatum.
For protein extraction, striatal tissue was collected from 12
cryosections, located between Bregma +1.6 and ?0.4, and
homogenized in 300 ml of extraction buffer (2% sodium dodecyl
sulphate (SDS), 60 mM Tris base, pH 6.8, 100 mM dithiothreitol
(DTT) and 1 mM Na2EDTA). After homogenization, samples were
incubated for 30 min at 37 8C. Next, samples were processed 4
times through 20 gauge needles, once through a 26 gauge needle
and spun at 10,000 g at 4 8C (Massie et al., 2003). Supernatants
were stored at ?20 8C. Protein concentrations were determined
concentrations of protein were loaded on the gel.
Proteins were separated by SDS-polyacrylamide gel electro-
phoresis (PAGE) (4–12% gel; Invitrogen, Groningen, The Nether-
nitrocellulose membrane using an iBlot system (Invitrogen).
Non-specific binding was blocked by incubating the membrane
for 1 h at room temperature in 5% ECL Membrane Blocking Agent
(GE Healthcare, Roosendaal, The Netherlands). Blots were incu-
bated overnight at 4 8C with the immunoaffinity purified
polyclonal antibodies against VGLUT1 (diluted 1/10,000, raised
in Guinea pig, AB5905, Millipore), VGLUT2 (diluted 1/5000, raised
in mouse, MAB5504, Millipore) or TH (raised in rabbit, 1/4000,
AB152, Millipore). The next day, after a 30 min incubation with
horseradish-peroxidase-conjugated anti-Guinea pig (1/20,000,
Millipore), anti-mouse or anti-rabbit antiserum (1/4000; Dako,
Glostrup, Denmark), immunoreactive proteins were visualized
using enhanced chemiluminescence (ECL) (ECL Plus kit, GE
Healthcare). After visualizing the immunoreactive signal, mem-
branes were rinsed thoroughly and stripped by incubating them
for 45 min in a buffer containing 2% SDS, 1.5% DTT, 62.5 mM Tris,
pH 6.7, at 50 8C. After thorough rinsing, aspecific binding was
blocked again for 1 h before the membrane was incubated
overnight with primary antibodies against b-tubuline (raised in
A. Massie et al./Neurochemistry International 57 (2010) 111–118
rabbit for the VGLUT1 and 2 blots and raised in mouse for the TH
blots; 1/250; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).
Next the membrane was incubated with horseradish-peroxidase-
conjugated anti-rabbit or anti-mouse antiserum (1/4000, 30 min;
Dako) and the immunoreactive signal revealed using the ECL+kit
(GE Healthcare). All washing and dilution steps were performed
with Tris–saline (0.01 M, pH 7.4). The MultiMark Multi-Colored
Standard (Novex, Invitrogen) was used as molecular weight
standard. Negative controls included the omission of the immu-
noaffinity purified antibody and the secondary antibody, respec-
tively. Densitometric analysis of the immunoreactive bands was
performed using the ImageJ software (National Institute of Health,
USA). Densities of glutamate transporter immunoreactive bands
are normalized to densities of b-tubuline immunoreactive bands,
detected on the same membrane. All samples that were being
compared were always loaded on one gel and experiments were
repeated 3–5 times for each survival time.
An arbitrary chosen sample of the control group was set as
reference (100%) and relative expression levels of the glutamate
transporters in the control group as well as the lesioned groups are
expressed as a percentage of this reference. Expression levels are
expressed as means ? standard error of the mean (SEM). Statistical
analysis of data was performed using an unpaired Student’s t-test
(comparison young (n = 4) and old (n = 4) control rats) or one-way
ANOVA followed by a Tukey’s test for multiple comparison
(comparison control rats (n = 8) and 6-OHDA lesioned rats (n = 4))
(a = 0.05).
1.5. In situ hybridization
After decapitating the rats, brains were removed, snap-frozen
and 25 mm cryosections were made throughout the brain.
Prior to in situ hybridization analysis, correct lesioning was
verified in each animal using immunohistochemical staining for
TH at the level of the striatum as well as substantia nigra. Briefly,
frozen sections were post-fixed in 4% paraformaldehyde and
immunohistochemistry was performed on the glass slide as
described above for free floating sections. No trypsin treatment
was performed. A methanolic H2O2treatment for 20 min preceded
the blocking step in normal goat serum.
In situ hybridization was performed on sections with an
interval of 200 mm according to a protocol described before
(Arckens et al., 1995). Synthetic oligonucleotide probes (VGLUT1:
Eurogentec, Seraing, Belgium) were end-labeled with33P-dATP
(NEN, Zaventem, Belgium) using terminal deoxynucleotidyl
transferase (Invitrogen). The radioactively labelled probe, sepa-
rated from the unincorporated nucleotides by running the
mixture over a miniQuick Spin column (Roche Diagnostics,
Brussels, Belgium), was mixed with a hybridization cocktail (50%
formamide, 4? standard saline citrate (SSC), 1? Denhardt’s
solution, 10% dextran sulphate, 100 mg/ml salmon sperm DNA,
250 mg/ml tRNA, 60 mM DTT, 1% N-lauryl-sarcosine, 26 mM
NaHPO4, pH 7.4) and applied to the dehydrated sections for an
overnight incubation at 37 8C. The next day, sections were rinsed
in 1? standard saline citrate buffer at 42 8C and radioactive signal
detected using an autoradiographic film (Kodak, Zaventem,
Belgium). After developing the films, autoradiographic images
were scanned and quantitative analysis was done using the
ImageJ software. A calibration curve of relative optical density
(ROD; log265/mean gray value) versus radioactivity concentra-
tion of the [14C] microscales (GE Healthcare) was constructed by
the program. Autoradiographic brain images were compared
with their counterstained Nissl sections. Then, contours were
drawn bilaterally over the cortex and the Pf and optical densities
were measured. Since brain sections were collected at 200 mm
intervals, the data gathered was from 16 sections per brain for
determined by subtracting the non-specific hybridization signal
obtained from the film in the vicinity of each section (background)
from the values obtained with the oligoprobes. The average mean
gray value was determined for duplicate measurements for each
by comparing RODs of the [14C] microscales.
2.1. Striatal TH immunoreactivity
All hemi-Parkinson rats used in this study showed a near
complete loss of TH immunoreactivity in the ipsilateral striatum
(Fig. 1A, 2C0, 3A) and substantia nigra pars compacta (Fig. 1B), as
assessed with immunohistochemistry (Figs. 1 and 2C0) or Western
blotting (Fig. 3A).
2.2. Striatal VGLUT distribution
VGLUT1 immunoreactivity was seen in the neuropil of the
striatum whereas the fiber bundles and neuronal cell bodies were
devoid of staining (Fig. 2A). The distribution of VGLUT2 immuno-
reactivity in the striatum was similar to VGLUT1 (Fig. 2E).
Immunohistochemical stainings for VGLUT1 and 2 did not reveal
any effect of 6-OHDA injection into the MFB on the striatal
distribution of the respective transporters. A homogeneous
neuropil staining could be observed for VGLUT1 as well as VGLUT2
in the left and right striatum of control rats (Fig. 2A, B, E, F) as well
as all hemi-Parkinson rats, independent of post-lesion time
(Fig. 2C, D, G, H).
2.3. Striatal VGLUT protein expression levels
In first instance, the effect of a small difference in age on the
striatal expression of the VGLUTs was studied. We compared the
expression levels of VGLUT1 and 2 in striatum of control rats with
an age corresponding to hemi-Parkinson rats three weeks post-
lesion (‘‘young rats’’) to control rats with an age corresponding to
hemi-Parkinson rats twelve weeks post-lesion (‘‘mature rats’’). No
age-related changes in expression level could be observed for
VGLUT2 (young: 106.0 ? 9.8%; mature: 102.0 ? 7.9%) (Fig. 3D). On
the other hand, for VGLUT1, we detected increased expression levels
in mature control animals (206.8 ? 23.8%) compared to young ones
(91.8 ? 8.6%) (Fig. 3D). Therefore, we always compared expression
levels of the VGLUTs in lesioned animals to their age-matched
controls. A general and very important finding was that we could not
detect any significant imbalance between the expression levels of the
VGLUTs between the dopamine depleted and intact striatum of the
Fig. 1. To confirm correct lesioning of the nigrostriatal pathway of the hemi-
Parkinson rats used in the in situ hybridization experiments, tyrosine hydroxylase
(TH) stainings were performed on 25mm frozen sections. In the ipsilateral striatum
(Str), a near complete loss of TH-positive afferents could be observed in hemi-
Parkinson rats as soon as three weeks after 6-OHDA lesioning (A). Also in the
ipsilateral substantia nigra pars compacta (SNc), neurodegeneration was almost
complete as no TH-immunoreactive neurons could be visualized (B). Scale bar
2 mm (A), 1 mm (B) (SNr, substantia nigra pars reticulate).
A. Massie et al./Neurochemistry International 57 (2010) 111–118
lesioned rats (Fig. 3B, C, E, F). Three weeks after injecting6-OHDAinto
the left MFB, we detected a significant bilateral upregulation of the
221.0 ? 29.9%; Fig. 3B and E) and a significant bilateral decrease in
the expression of VGLUT2 (ipsi: 61.3 ? 4.9%; contra: 73.5 ? 4.7%;
Fig. 3C and F). Five weeks post-lesion, the expression of VGLUT1 was
(ipsi:215.7 ? 33.9%;contra:
190.0 ? 27.9%), although not significant at the contralateral side.
At this survival time, the expression of VGLUT2 was unaltered
compared to control rats (ipsi: 98.3 ? 7.2%, contra: 76.0 ? 7.9%)
(Fig. 3F). Twelve weeks post-lesion, wedetected a significantbilateral
decrease in the expression of VGLUT1 (control: 93.8 ? 14.4%; ipsi:
bilaterally upregulated (ipsi:257.0 ? 39.1%;contra:
Fig. 2. Immunohistochemical stainings for VGLUT1 (A–D) and VGLUT2 (E–H) in frontal sections, at the level of the striatum, of a control rat (A, B, E, F, Ctrl) and a hemi-
Parkinsonratthreeweeksafter6-OHDAinjectionintothemedial forebrainbundle (C,D, G,H).The*inAandEshows thearea thatisenlarged inthehigher-powerviewinB/F.
C/G represents the same area of a contralateral and D/H of an ipsilateral striatum of a lesioned rat. C0shows the loss of tyrosine hydroxylase (TH)-immunoreactivity in the
striatum ipsilateral to 6-OHDA injection. Scale bar E (applies to A as well) 1 mm, H (applies to B–D, F–G) 100 mm, C03.5 mm.
Fig. 3. Striatal protein expression levels of VGLUT1 (B, D, E) and VGLUT2 (C, D, F) in ‘‘mature’’ control rats (n = 4) relative to ‘‘young’’ control rats (n = 4) (D) and in hemi-
Parkinson as well as sham rats (n = 4) relative to age-matched control rats (B, C, E, F). Tyrosine hydroxylase (TH)-immunoreactivity was measured as a control for correct
lesioning (A). A representative example of a Western blotting experiment on samples of a hemi-Parkinson rat with a post-lesion time of three weeks is shown (2
concentrations of each sample were loaded. Upper bands correspond to TH (?60 kDa), VGLUT1 (?50 kDa) or VGLUT2 (?55 kDa), lower bands tob-tubuline) (A–C). In hemi-
Parkinson rats, expression levels were examined in the ipsi- and contralateral striatum three (n = 4), five (n = 4) and twelve (n = 4) weeks after injecting 6-OHDA into the left
medial forebrain bundle (E, F). Student’s t-test (D) or one-way ANOVA followed by Tukey’s multiple comparison test (E, F), *p < 0.05, **p < 0.01, ***p < 0.001.
A. Massie et al./Neurochemistry International 57 (2010) 111–118
44.8 ? 11.7%; contra: 35.0 ? 8.7%; Fig. 3E) whereas VGLUT2 expres-
sionwasstillunaffectedatthispost-lesiontime(control:89.5 ? 7.4%;
ipsi: 78.0 ? 4.8%; contra: 87.5 ? 7.8%; Fig. 3F). Sham lesioning
induced a small, though not significant, decrease in VGLUT2
expression in the ipsilateral striatum and did not have any effect
on striatal expression level of VGLUT1 (Fig. 3E and F).
2.4. In situ hybridization
VGLUT1 mRNA expression levels were measured in neocortex
between Bregma +2.7 and Bregma ?2.6 (Fig. 4A–C), whereas the
VGLUT2 mRNA expression levels were evaluated in the Pf (lateral
and medial) of the thalamus (Fig. 4D–I) three, five and twelve
weekspost 6-OHDAinjectionintotheMFBaswell asincontrol and
sham operated rats. For none of the vesicular glutamate
transporters and none of the conditions, we could detect a
significant change in mRNA expression level (Fig. 5). Yet, for
VGLUT2 mRNA there was a tendency towards a decreased
expression level in the Pf of the ipsilateral hemisphere (Fig. 5B).
This decrease could be observed in the sham operated rats as well,
on mRNA (Fig. 5B) and protein level (Fig. 3F), and is most probably
the result of the fact that the injection needle passes thisnucleus in
its way towards the MFB.
In this study we describe time-dependent and bilateral changes
in the striatal protein expression level of VGLUT1 as well as
VGLUT2 upon dopamine depletion by unilateral injection of 6-
OHDA into the MFB. Although no change in distribution pattern
could be observed using immunohistochemistry, we detected a
bilateral upregulation of VGLUT1 and a downregulation of VGLUT2
at a post-lesion time of three weeks. Twelve weeks after dopamine
depletion, VGLUT1 was bilaterally downregulated. We were
unable to correlate these changes in striatal protein expression
to changes in mRNA levels of the respective VGLUTs in the
Fig. 4. Representative autoradiograms illustrating the absence of an effect of 6-OHDA lesioning of the nigrostriatal tract on VGLUT1 mRNA levels in neocortex (A–C, Cx) and
VGLUT2 mRNA levels in the parafascicular nucleus (D–F, Pf) at a post-lesion time of three, five and twelve weeks. A cresylviolet staining of the boxed area in D is shown in
panel G–H and shows the outline of the Pf. The Pf in the rat consists of medial and lateral parts surrounding the fasciculus retroflexus (fr). Scale bar 2 mm (A–F), 0.5 mm (G–I).
Fig. 5. Bar diagram showing the relative optical density (ROD) of the hybridization signal for VGLUT1 (A)and VGLUT2 (B) in respectively neocortex and parafascicular nucleus
three, five and twelve weeks after 6-OHDA injection into the medial forebrain bundle. Results are expressed as mean ? SEM of n = 6 animals per group.
A. Massie et al./Neurochemistry International 57 (2010) 111–118
corresponding projection areas. Both VGLUT1 and VGLUT2
transcripts in respectively neocortex and Pf remained stable
during the whole time-window included in this study.
Although a consensus exists that changes in striatal glutama-
tergic neurotransmission are linked to the pathogenesis of
Parkinson’s disease, there is controversy as to the effect of
nigrostriatal damage on striatal extracellular glutamate concen-
trations. Whereas some groups reported increased extracellular
glutamate concentrations (Lindefors
Meshul et al., 1999; Jonkers et al., 2002; Walker et al., 2009),
others do not detect any change in extracellular glutamate after 6-
OHDA lesioning of the nigrostriatal pathway (Corsi et al., 2003;
Bianchi et al., 2003; Robelet et al., 2004). Most probably these
contradictory data are the result of different lesioning protocols as
well as different post-lesion times. Since Meshul et al. (1999)
described biphasic changes in striatal glutamate after 6-OHDA
injection into the MFB, with an increase at three weeks and a
decrease at twelve weeks, we investigated VGLUT transcript and
protein expression levels at these time intervals. Moreover, we
included a post-lesion time of five weeks, since it was reported by
Robelet et al. (2004) that neither changes in VGLUT expression nor
extracellular glutamate could be observed at this post-lesion time.
and Ungerstedt, 1990;
3.1. Bilateral effect on striatal VGLUT expression after unilateral 6-
Similar as for the high-affinity glutamate reuptake transporters
(Massie et al., submitted for publication) as well as for the
extracellular glutamate concentrations (Lindefors and Ungerstedt,
1990), we observed bilateral changes in the striatal protein
expression levels of both VGLUTs after unilateral injection of 6-
OHDA. Crosstalk between the two hemispheres caninvolve several
routes. Although basal ganglia connectivity is largely sustained by
ipsilateral projections, several decussations have been described
(Kerkerian-Le Goff et al., 2006). Not only a small part of the
nigrostriatal pathway is crossing over to the contralateral striatum
(Fass and Butcher, 1981) but also crossed thalamic efferents are a
source of descending bilateral projections (Marini and Tredici,
1995; Marini et al., 1999; Castle et al., 2005). Moreover, at the level
of the cortex, interhemispheric communication is common
through cortical loops and projections via the corpus callosum.
3.2. Biphasic changes in VGLUT1 protein expression in the striatum of
the hemi-Parkinson rat
The increase in striatal VGLUT1 protein expression three weeks
by Raju et al. (2008) in striatum of the MPTP-treated monkey as
well as by Kashani et al. (2007) in putamen of parkinsonian
humans. Although it was postulated by Baker et al. (2002) that in
the nucleus accumbens extracellular glutamate concentrations are
mainly determined by glutamate release via the cystine/glutamate
antiporter, an increase in glutamate filling of the synaptic vesicles
due to increased expression of VGLUT1 may result in an increase of
quantal size (Wojcik et al., 2004) and could possibly also result in
strengthening of corticostriatal transmission. Interestingly, all
studies in which increased extracellular glutamate has been
reported in the dopamine depleted striatum, have been conducted
in rats at three to four weeks post 6-OHDA injection into the
nigrostriatal pathway (Lindefors and Ungerstedt, 1990; Meshul
et al., 1999; Jonkers et al., 2002). Most likely, the increased
expression of VGLUT1, together with the increase in the cystine/
glutamate antiporter expression that was observed before (Massie
et al., 2008b), will contribute to such an increase in extracellular
glutamate. At five weeks post-lesion, we observed a similar
bilateral increase. This is in contrast to the findings of Robelet et al.
(2004) who described no effect of striatal dopamine deprivationon
VGLUT1 expression. Possibly, this discrepancy is related to
technical details. Whereas we inject 6-OHDA into the MFB, in
the former study the injection was placed in the substantia nigra.
The decreased expression of VGLUT1 that we observed twelve
weeks after 6-OHDA lesioning, correlates to the decreased
extracellular glutamate concentrations as described by Meshul
et al. (1999).
We were unable to link these striatal protein changes to
changes in cortical VGLUT1 transcript levels. Most probably, the
changes in VGLUT1 mRNA expression in cortical neurons project-
ing to the striatum are camouflaged by VGLUT1 expressing
intracortical projecting neurons. Indeed, only a subpopulation of
the cortical neurons projects to the striatum. Moreover, as
described by Barroso-Chinea et al. (2008), VGLUT1 cannot be
considered as a definitive marker for glutamatergic striatal
afferents from cortical sources since thalamostriatal afferents
arising from the ventral thalamic nuclei also express VGLUT1
transcript. However, VGLUT1 hybridization signal was faint in the
ventral thalamic nuclei compared to the neocortex, making
quantification rather difficult and inaccurate. Yet, at first sight,
no strong increase in signal could be seen in 6-OHDA lesioned rats
with a short post-lesion time (personal observation). Furthermore,
the striatal VGLUT1 protein changes might also depend on post-
3.4. Decreased striatal VGLUT2 expression in the hemi-Parkinson rat
three weeks after 6-OHDA lesioning
Although the existence of the thalamostriatal system has long
been known to contribute to the basal ganglia circuitry, from a
functional point of view it remains poorly characterized (Barroso-
Chinea et al., 2008). In the current study, we detected a bilateral
decrease in striatal VGLUT2 protein expression at three weeks
whereas at five and twelve weeks we did not detect any significant
change, in line with the observations of Robelet et al. (2004). A
decrease in VGLUT2 expression, a marker for thalamostriatal
afferents, might reflect the neuronal loss that has been found in the
CM/Pf of patients with Parkinson’s disease (Henderson et al.,
2000a,b). Though, we do not detect any significant loss of VGLUT2
transcript in Pf at any post-lesion time. However, neuronal loss
might be masked by an increase in VGLUT2 transcript per cell as
the loss of Pf neurons innervating the dopamine depleted striatum
can be accompanied by a marked increase in the metabolic activity
of the remaining thalamostriatal projecting neurons (Aymerich
et al., 2006; Kashani et al., 2007). Possibly, this increase in
metabolic activity is already apparent at the mRNA level at three
weeks after lesioning and will result in a restoration of striatal
VGLUT2 protein from five weeks on. Another possible explanation
for the discrepancy between striatal protein expression and Pf
transcript levels could be linked to the fact that, although the main
sources of thalamostriatal projections are the intralaminar
thalamic nuclei, they also arise from midline and specific relay
thalamic nuclei (Smith et al., 2004).
3.5. No effect of 6-OHDA lesioning on VGLUT2 transcript levels in the
In rodents, some controversy exists as to the effect of striatal
dopamine depletion on Pf neurodegeneration and VGLUT2 mRNA
expression. The lack of effect of striatal dopamine depletion on
VGLUT2 transcript in the Pf is in contradiction to the observations
of Sedaghat et al. (2009) who described an overall reduction of
A. Massie et al./Neurochemistry International 57 (2010) 111–118
VGLUT2 hybridization density, linked to a reduced density of
neurons, in Pf ipsilateral to intranigral 6-OHDA injection at a post-
lesion time of one and five months. Also, one month after 6-OHDA
injection into the MFB more than 50% loss of Pf neurons projecting
to the striatum was observed (Aymerich et al., 2006). However, as
described above, in the latter study degeneration of thalamic
neurons was accompanied by hyperactivity and increased VGLUT2
mRNA expression in surviving neurons. Systemic MPTP adminis-
tration in mice (Freyaldenhoven et al., 1997) as well as striatal
injection of MPP+in rats (Ghorayeb et al., 2002) induced significant
loss of neurons in midline and intralaminar thalamic nuclei,
whereas in the striatum of the dopamine depleted monkey no
significant change in relative prevalence of VGLUT2 thalamos-
triatal terminals was observed (Raju et al., 2008). Here again, it is
obvious that the discrepancy that can be observed in the available
reports is most probably the result of the use of different animal
The data presented in this paper, together with our previous
findings for the glial high-affinity Na+/K+-dependent glutamate
transporters and xCT, the specific subunit of the cystine/glutamate
antiporter, demonstrate a clear, time-dependent effect of dopa-
mine deprivation on striatal glutamate transporter expression
(Massie et al., 2008b; Massie et al., submitted for publication) and
support the idea of a biphasic change in striatal glutamatergic
neurotransmission in the hemi-Parkinson rat model (Meshul et al.,
In conclusion, changes in VGLUT expression will certainly
contribute to the aberrant glutamatergic neurotransmission in the
hemi-Parkinson rat model. Moreover, in post-mortem tissue of
Parkinson patients identical results were obtained for VGLUT1 as
in this study at three to five weeks post-lesion (Kashani et al.,
2007). This strongly supports the idea that VGLUT1 might be an
interesting target in the search for new therapeutic strategies for
the treatment of Parkinson’s disease and should encourage the
search for specific in vivo ligands of the VGLUTs.
This work was supported by grants of the Brussels Capital-
Wetenschappelijk Onderzoek-Flanders and the Vrije Universiteit
Brussel. AS is a research assistant of the FWO-Flanders and KV is a
research assistant of the IWT-Flanders. The authors wish to
acknowledge Mr. G. De Smet, Ms. R. Vanlaer and Ms. R. Berckmans
for excellent technical assistance.
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