BACKGROUND
it is long recognized that the degeneration of dopaminergic neurons induces an abnormal activation of glutamate systems in the basal ganglia  that is central to the pathophysiology of parkinson’s disease   <cit> .
glutamate mediated mechanisms are also thought to play a role in the development of dyskinesias with long-term administration of l- <dig> -dihydroxyphenylalanine , the most efficient treatment for pd.
many experimental studies also evidence that dopamine denervation induces an increase in corticostriatal glutamate  <cit>  and that l-dopa-induced dyskinesia  are linked to bg network glutamate transmission abnormalities  <cit> .
microdialysis studies have suggested that dopamine lesion may also increase glutamate transmission in the bg output structures, substantia nigra pars reticulata   <cit>  and entopeduncular nucleus  <cit> , presumably as a result of the abnormal activation of the subthalamic nucleus   <cit> .
three subtypes of vesicular glutamate transporters have been identified: vglut <dig>   <dig> and  <dig>  <cit> .
these transporters mediate glutamate uptake inside presynaptic vesicles and are anatomical and functional markers of glutamatergic excitatory transmission  <cit> .
vglut1- <dig> are very similar in structure and function, but are used by different neuronal populations.
vglut <dig> and vglut <dig> are expressed by the cortical and subcortical neurons respectively.
vglut <dig> is expressed by nonglutamatergic neurons, such as cholinergic striatal interneurons, a gabaergic interneuron subpopulation from the cortex and hippocampus and serotoninergic neurons from the dorsal and medial raphe nuclei  <cit> .
since the 1990s, high frequency stimulation  of the stn has become an effective surgical treatment of late-stage parkinson’s disease , improving all motor symptoms in pd patients, particularly in those who experience motor fluctuations  <cit> .
however, the mechanisms underlying the improvement in symptoms remain unclear  <cit> .
beyond its local effect on stn activity, we know that, by activating axons, stn-hfs may generate widespread and heterogeneous distal effects throughout the bg network  <cit> .
indeed, we have already reported in previous studies that in intact or 6-ohda -lesioned rats, stn-hfs increases extracellular glutamate in the striatum, the globus pallidus and the snr  <cit> .
the present study analyzed the effects of da depletion and for the first time those of stn-hfs on vglut1- <dig> expression in several bg nuclei, by using immunoradioautography with affinity-purified rabbit vglut <dig>  vglut <dig> or vglut <dig> antiserum.
we found that da-depletion did not affect vglut <dig> and vglut <dig> expression in almost all bg structures studied while that of vglut <dig> significantly decreased.
interestingly, stn-hfs did not affect vglut1- <dig> expression in normal rats, but systematically increased their expression in most of the bg nuclei studied in da-depleted animals.
according to the changes of vglut1- <dig> expression observed and to their known anatomical localization, we suggest that stn-hfs may achieve its therapeutic effect, at least in part, through normalization of the thalamostriatal and thalamocortical pathways.
methods
animals
adult  male sprague–dawley rats , weighing  <dig> to 270 g, were housed in an animal room on a 12-hour light/dark cycle, with food and water supplied ad libitum.
this study was carried out in strict accordance with the recommendations of the european community council directive of  <dig> november  <dig>  concerning the care of laboratory animals, french ministry of agriculture regulations  and french guidelines for the use of live animals in scientific investigations.
the protocol was approved by the committee on the ethics of animal experiments of the “grenoble institute of neuroscience ethical committee” agreement number  <dig>  all surgery was performed under a mixture of xylazine and ketamaine and all efforts were made to minimize the number of animal used and their suffering.
all operated rats were intraperitoneally treated with rimadyl  to prevent post-surgery suffering.
lesion procedure
forty rats  were anesthetized with a mixture of xylazine  and ketamine  and secured in a kopf stereotaxic apparatus .
all animals received desipramine  pretreatment, to protect noradrenergic neurons.
lesioned animals  received a unilateral injection of 9 μg of 6-hydroxydopamine   dissolved in 3 μl of  <dig> % sterile nacl supplemented with  <dig> % ascorbic acid, administered at a flow rate of  <dig>  μl · min- <dig> to the left snc.
an identical procedure was used for controls  but with the injection of nacl  <dig> %.
the stereotaxic coordinates for the injection site relative to the bregma were as follows: anteroposterior , - <dig>  mm; lateral , + <dig>  mm; dorsoventral , - <dig>  mm, with the incisor bar at  <dig>  mm below the interaural plane, according to the stereotaxic atlas of paxinos and watson  <cit> .
after injections, animals were kept warm and allowed to recover from the anesthetic before being returned to the animal house for three weeks until the stimulation experiments.
this time interval was left to allow the da system degeneration induced by the neurotoxin to stabilize.
implantation of the stimulation electrode
rats from the two experimental groups  were first anesthetized by the inhalation  of a mixture of 3% isoflurane in air  and mounted in a stereotaxic frame .
the dorsal skull was exposed and holes were drilled for the implantation of the stimulation electrode into the left stn.
during the implantation and stimulation procedure, anesthesia was maintained with an inhaled mixture of 1% isoflurane in air  and body temperature was maintained at 37°c with a feedback-controlled heating pad .
stereotaxic coordinates were chosen according to the atlas of paxinos and watson  <cit>  and were as follows relative to the bregma: ap, - <dig>  mm; l, + <dig>  mm; and dv, - <dig>  mm as previously described  <cit> .
electrical stimulation
for electrical stimulation, we used a concentric stimulating bipolar electrode , with an outer diameter of 250 μm and a distance between the poles of 1 mm.
stimuli were delivered under anesthesia during 4 hours with a world precision instrument  acupulser and stimulus isolation units giving a rectangular pulse.
this duration of stimulation  was chosen to be sure that the proteic expression of vgluts can be detected and stabilized and almost corresponds to that used in previous studies analyzing mrna levels of different target proteins of basal ganglia circuits  <cit> .
as previously reported, the stimulation parameters  matched those routinely used in parkinsonian patients  <cit> .
at the end of each experiment, an electrical lesion was created in the stn so that the position of the electrode could be checked post-mortem.
in control rats  the stimulation was never switched “on”.
histology
at the end of the electrical stimulation, all animals were perfused transcardially with  <dig> % saline, under chloral hydrate anesthesia.
brains were rapidly removed and frozen in cooled  isopentane, then stored at −20°c.
serial frontal sections  were cut with a cryostat , collected on microscopic slides and stored at −20°c.
tissue sections from different bg nuclei and related structures , nucleus accumbens, motor and somatosensory cortices, thalamus , subthalamic nucleus, globus pallidus and substantia nigra pars reticulata ) were selected to analyze changes in vglut expression.
the correct location of the stimulation electrode was checked by collecting several subthalamic tissue sections   and counterstaining with cresyl violet.
the tip of the electrode was systematically implanted directly in the stn at the top of its dorsal part.
these histological controls were systematically carried out for all the animals in each experimental group.
all animals with incorrectly positioned stimulation electrodes were excluded .
th-immunohistochemistry
we assessed the extent of the dopaminergic denervation induced by nigral 6-ohda injection by th immunostaining on striatal and nigral sections from the fixed brains of lesioned animals.
th immunostaining was carried out as previously described  <cit> .
briefly, striatal and nigral tissue sections from 6-ohda-lesioned rats were mounted on silane-coated microscope slides.
tissue sections were postfixed in 4% paraformaldehyde, thoroughly washed with tris buffered-saline  and incubated for 1 hour in  <dig> % triton x- <dig> in tbs  and 3% normal goat serum .
they were then incubated with primary antisera diluted in tbst supplemented with 1% normal goat serum  for 24 h, at 4°c.
the antiserum was diluted 1: <dig> for th staining .
antibody binding was detected with avidin-biotin-peroxidase conjugate , with  <dig>  3’-diaminobenzidine as the chromagen.
the detection reaction was allowed to proceed for one to three minutes, as previously described.
sections were dehydrated in a series of graded ethanol solutions, cleared in xylene, mounted in dpx  and covered with a coverslip for microscopy.
vglut 1– <dig> immunoradioautography
tissue sections were air-dried, post-fixed by immersion in fixative , and then washed in pbs.
nonspecific binding sites were saturated by incubation with 3% bovine serum albumin  in pbs, 1% ngs and 2 mm nai .
sections were incubated overnight at 4°c in buffer a supplemented with affinity-purified rabbit vglut <dig>  vglut <dig> or vglut <dig> antiserum , and then for 1 hour with an affinity-purified goat anti-rabbit  igg  in buffer a supplemented with  <dig> % sodium azide.
the sections were rinsed in water, dried and placed against x-ray films  for  <dig> to 11 days.
the specificity of all antisera used in this study have been previously validated by our group .
for each labeled section, a background value was estimated by measuring optical density in the corpus callosum, since this structure is devoided of specific staining for vglut 1– <dig> antibodies.
this background value was then systematically subtracted from the optical density values obtained for each corresponding section.
quantification and statistical analysis
for the evaluation of the extent of da-denervation, striatal and nigral th immunostained sections were directly processed by using the calopix software of the computerized image analysis system .
six th-immunostained sections from each structure  and for each rat were used for quantification.
the loss of th immunostaining in the snc or in the striatum was evaluated by comparing the total surface of both structures, as revealed by the th immunolabelling, in normal and lesioned animals.
for quantification of vglut1- <dig> contents, four ap levels ) were choosen.
for each rat, three stained sections of the same ap level were used for quantification and the triplicate od values obtained for each structure analyzed were averaged.
immunoradioautograms obtained from x-ray films were analyzed with autoradio v <dig>  software .
values of optical densities measured from each structure analyzed are expressed as a mean ± standard error  in table  <dig>  histograms presented in figures show the mean ± standard error of the mean  of optical densities expressed as a percentage of control values.
data were analyzed for each brain structure by kruskal-wallis tests with sigmastat  <dig>  software.
post-hoc analyses were carried out with the dunn’s method.
a, modifications of vglut1- <dig> expression induced by unilateral 6-ohda-lesion of snc .
*p< <dig> , vs control group.
b, modifications of vglut1- <dig> expression induced by unilateral stn-hfs in 6-ohda lesioned rats .
*p< <dig> , vs non stimulated 6-ohda-lesioned group.
values of optical densities measurements are expressed as a mean ± standard error .
data were analyzed for each brain structure by kruskal-wallis tests with sigmastat  <dig>  software.
post-hoc analyses were carried out with the dunn’s method.
bold numbers correspond to significant differences.
ipsilateral side: related to the lesion and/or stimulation side; controlateral side: related to the lesion and/or stimulation side; acb, accumbens nucleus; cpu, caudate putamen ; gp, globus pallidus; pm cx, premotor cortex; snr, substantia nigra pars reticulata; ss cx, somatosensory cortex; stn, subthalamic nucleus; thalamus .
RESULTS
histological controls of the extent of the dopamine lesion and of electrode location
three weeks after the unilateral injection of 6-ohda, all lesioned animals presented a substantial loss of th immunostaining in the ipsilateral snc and the striatum , as shown by comparison with the contralateral side  or with control animals.
an analysis of densitometric measurements of th immunostaining showed an absence of statistical difference between the two lesioned groups .
in da-depleted animals, the loss of snc th + neurons was evaluated by comparing the total snc surface on the intact side with the homologous area on the lesioned side.
a loss of 92 ± 5%  of th immunolabeled surface was measured.
in the striatum of the same rats, the loss of da nerve terminals, as revealed by th immunostaining mainly affected the dorsal part of the striatum .
this loss affected around 83 ± 4% of the striatal surface as compared to the total striatal surface of the control side.
in this denervated striatal area, th immunolabeling, as evaluated by a mean of densitometric values, was decreased by 85 ± 5%  when compared to the controlateral intact side.
the correct implantation of the stimulation electrode in the stn is illustrated in figure 1c-e. figure 1e shows, at a higher magnification, the small electrical lesion  created at the end of the experiment, indicating the point stimulated.
regional distribution of vglut1- <dig> in control rats
vglut1- <dig> expression was qualitatively analyzed in control rats that had been neither lesioned nor stimulated, to ensure the validity and specificity of the immunoradioautographical staining.
immunoradioautograms from the different sections showed a distribution of vglut1- <dig> similar to that previously reported  <cit> , confirming the validity of our vglut1- <dig> staining procedure and the lack of cross-reactivity between the antibodies used.
vglut <dig> immunostaining was dense in almost all the structures studied, including, especially, the striatum, nucleus accumbens, cortex, the motor part of the thalamus  and hippocampus.
by contrast, no vglut <dig> labeling was found in the globus pallidus, the substantia nigra and in most of the brainstem .
vglut <dig> proteins were detected in almost the same set of structures as vglut <dig> although the density of vglut <dig> immunostaining was slightly lower than that for vglut <dig> in striatal, cortical and thalamic areas, whereas the opposite was observed in many sub-cortical structures.
these data are consistent with the well-described complementary pattern of expression of vglut <dig> and vglut <dig> in the rat brain.
vglut <dig> staining, unlike that for vglut <dig>  was detectable in the substantia nigra pars reticulata, hypothalamic nuclei and midbrain, which displayed widespread staining.
different, complementary patterns of immunostaining for vglut <dig> and vglut <dig> were observed in the hippocampus.
the density of vglut <dig> proteins was highest in layers iv and vi of the cortex and in the hypothalamus, the central gray matter and the superior colliculus .
vglut <dig> staining was weaker than that for vglut <dig> and vglut <dig>  but was also observed in many different areas.
vglut <dig> levels were moderate in the striatum, but high in the hippocampus, with a complementary distribution for vglut <dig> and vglut <dig> .
effects of 6-ohda-lesion and/or stn-hfs on vglut1- <dig> expression
da lesion and stn stimulation were unilaterally performed in this study.
however, we found similar changes of vgluts expression on both sides, as revealed by the optical density measurements of immunoreactive signals for vglut1- <dig> in all structures examined .
in order to simplify the presentation of our data, we decided to only show the results obtained from the ipsilateral side  on figures  <dig> and  <dig>  as precised in materials and methods, changes in vgluts expression induced by 6-ohda lesion and stn stimulation were first analyzed by kruskal-wallis tests with sigmastat  <dig>  software.
results of these tests for each brain structure are presented in legends of figures  <dig> and  <dig>  post-hoc analyses were then carried out with the dunn’s method.
effect of the 6-ohda-lesion on vglut1- <dig> expression
da depletion did not affect vglut <dig> and vglut <dig> expression whatever the brain structure analyzed.
slight changes were observed in the striatum, nucleus accumbens, somatosensory cortex and thalamus , but they were not statistically significant .
by contrast, 6-ohda lesioning induced a significant decrease in vglut <dig> expression of nearly 50% with respect to that in sham-operated rats .
this decrease was particularly strong in the stn, thalamus  , and cortical areas  which displayed decreases of 55%, 48% and 42% with respect to control  rats, respectively .
effect of stn-hfs on vglut1- <dig> expression in sham-operated control  rats and in 6-ohda-lesioned animals
no significant change in vglut1- <dig> expression was detected after four hours of stn-hfs in sham-operated control rats  rats.
levels of vglut1- <dig> expression were similar between the two experimental groups  in all different structures studied, as show in figures  <dig> and  <dig>
on the contrary, stn-hfs induced a marked increase in vglut1- <dig> expression in 6-ohda-lesioned rats.
this increase affected almost all the brain structures studied .
vglut <dig> levels were always higher for all brain areas studied in stimulated 6-ohda rats when compared to non-stimulated 6-ohda-rats except for the nucleus accumbens  and stn .
the largest differences were found in the somatosensory cortex  and the motor part of the thalamus   .
for many brain structures analyzed, vglut <dig> levels measured in stimulated 6-ohda-rats were comparable to those detected in control  rats.
interestingly, for the striatum and the thalamus , vglut <dig> levels remained moderately overexpressed versus controls  .
stn-hfs induced a significant increase in vglut <dig> expression in all structures studied in 6-ohda-lesioned rats when compared to non-stimulated 6-ohda-rats.
however this increase did not affect the globus pallidus.
.
thus, stn-hfs more or less completely reversed the decrease in vglut <dig> expression induced by the da-depletion in all structures analyzed.
stn-hfs induced a strong upregulation of vglut <dig> expression in 6-ohda-lesioned rats .
interestingly, this effect was particularly marked in the thalamus   and the somatosensorial cortex .
discussion
the key findings of this study were: 1) da depletion decreased vglut <dig>  in all brain structures studied; 2) stn-hfs did not affect vglut1- <dig> expression in control  rats whatever the brain structure analyzed; 3) stn-hfs increased vglut1- <dig> expression in 6-ohda-lesioned rats in almost all structures analyzed.
thereby stn-hfs: i) normalized vglut <dig> levels after the decrease induced by da depletion, and ii) significantly increased vglut <dig> levels above those detected in control animals.
this was also true for vglut <dig> levels but only for the striatum.
this remodeling suggests that the mode of action of stn-hfs results from a global effect on basal ganglia network and related structures and that its therapeutic efficacy may to be linked, at least in part, to the normalization of thalamostriatal and thalamocortical neurotransmissions.
bilateral effects of unilateral da lesioning and stn-hfs on vglut expression
as stated above, unilateral da lesioning and stn-hfs caused similar changes in vglut expression on both sides of the brain.
bilateral effects of unilateral da lesioning have already been reported in the striatum for tissue concentrations of glutamate  <cit> , extracellular glutamate content assessed by microdialysis or voltammetry  <cit> , glutamate receptor mrna  <cit>  and the glial glutamate transporter glt- <dig>  <cit> .
similarly, unilateral stn-hfs has been reported to induce bilateral increases in striatal and nigral glutamate content  <cit> .
these bilateral effects may result from crossed glutamatergic projections from the cortex or the thalamus  innervating the bg on the contralateral side, consistent with cross-talk in cortico-bg-cortical loops  <cit> .
effect of 6-ohda-snc-lesioning on vglut expression
we found here that 6-ohda-lesions had no effect on vglut <dig> or vglut <dig> levels in any of the structures studied.
by contrast, vglut <dig> levels decreased significantly three weeks after lesioning.
at first glance, our observations contrast with previous reported data showing that dopamine depletion is associated with an increase in synaptic glutamate release  <cit>  and with high striatal extracellular glutamate levels and glutamatergic activity  <cit>  and greater thalamostriatal activity  <cit> , two to four weeks after lesioning of nigral dopaminergic neurons.
however, other studies have reported an absence of change in glutamate levels  <cit> .
these differences may be accounted for by differences in the extent of the dopamine lesion, lesion sites, methodologies and time courses.
we cannot exclude the possibility that different cellular mechanisms underlie presynaptic glutamate processes and extracellular glutamate release after lesioning.
indeed, striatal extracellular glutamate levels have been reported to depend on a complex balance between vesicular release and non vesicular release via glutamate transporters on both neurons and glia and the cysteine-glutamine antiporter  <cit> .
dopamine depletion leads to complex, biphasic changes in striatal glutamatergic transmission over the first few weeks, possibly stabilizing over three months.
contradictory data have been reported, for the cysteine-glutamate antiporter  <cit>  and glial transporters  <cit>  for example.
furthermore, the changes in vgluts expression induced by 6-ohda lesions are also complex.
vglut <dig> levels increase in the three weeks following the injection but then decrease, whereas vglut <dig> levels decrease and then normalize  <cit> .
in monkeys, mptp treatment increases vglut <dig> expression but does not affect vglut <dig> levels  <cit> .
in postmortem samples of parkinsonian patients vglut <dig> and vglut <dig> levels are increased in the putamen while vglut <dig> levels is lowered in the prefrontal and temporal cortex  <cit> .
however, the decrease in vglut <dig> levels observed here in all the brain structures of 6-ohda rats closely parallels the thalamic hypoactivity induced by the strengthening of gabaergic inputs from the snr and ep/gpi by the stn overactivity observed in da-depleted bg networks  <cit> .
furthermore, neuronal degeneration has been observed postmortem in the thalamic nuclei of pd patients  <cit>  and in the parafascicular nucleus in 6-ohda-lesioned rats  <cit> .
vglut <dig> is massively expressed by thalamic nuclei  <cit>  and have be postulated as selective marker of thalamo-striatal activity  <cit> .
this observation support the notion of a potential decreased glutamatergic afferences from the thalamus .
effect of stn-hfs on vglut1- <dig> expression in control and 6-ohda-lesioned rats
stn-hfs had no effect on vglut1- <dig> expression in any of the brain structures studied in control rats.
this suggests that in the absence of dopamine depletion, stn-hfs did not affect vglut1- <dig> expression.
these data are rather surprising since we reported in previous microdialysis study that in intact rats stn-hfs increases extracellular glutamate in the striatum, the globus pallidus and the snr  <cit> .
however, the duration of stimulation used here was longer than that used in our previous studies.
thus, we can speculate that increase of extracellular glutamate levels induced by stn-hfs in physiological conditions mainly involves non vesicular release.
by contrast, after 6-ohda lesions, stn-hfs induced an increase in vglut1- <dig> expression in almost all the structures analyzed.
in these da depleted conditions, we cannot exclude the possibility that the balance between non vesicular and vesicular release of glutamate is disturbed, involving more glutamate transporters on both neurons and glia  <cit> .
indeed, it is well documented that following da nigrostriatal lesion, there is an increase in the number of glial cells, including astrocytes and microglia.
therefore, the non vesicular release may be due to an increase in membrane transporters, such as glial glutamate transporters  and the neuronal glutamate transporter eeac <dig>  <cit> .
thus, our previous findings concerning increased extracellular glutamate levels in the striatum, globus pallidus and snr of 6-ohda-lesioned rats  <cit>  are consistent and confirm that stn-hfs affects not only its direct targets, but also more distant structures of the bg network  <cit> .
the mechanisms underlying the therapeutic effects of stn-hfs are not fully elucidated.
stn neuron inhibition by hfs, with loss of the drive of the internal part of the globus pallidus and disinhibition of the thalamus , would be consistent with the classical bg model  <cit> .
however, far more complex effects and circuitry are probably involved.
for example, the direct activation of nearby thalamostriatal and pallidonigral fibres  <cit>  or direct or antidromic cortex activation  <cit> .
these mechanisms might lead to corticostriatal fiber activation and the observed increase in vglut <dig> levels.
similarly, the increase in vglut <dig> levels induced here by stn-hfs may reflect a release of the classical thalamic inhibition induced by dopamine lesions.
as vglut <dig> is found mostly in the thalamostriatal neurons specifically affected by da depletion, these data strongly suggest a major role for this pathway in the therapeutic effects of stn-hfs in pd  <cit> .
however, the normalization of vglut <dig> levels we observed in the somatosensory and premotor cortices may also play a non negligeable role in these effects of stn-hfs.
interestingly, vglut <dig> was also overexpressed in the snr following stn-hfs in 6-ohda-lesioned rats.
as vglut <dig> is the only vglut expressed by stn glutamatergic projections to the snr  <cit> , this confirms our hypothesis that information transmission via the transsubthalamic pathway is not completely blocked during stn-hfs  <cit>  although the effect on vglut <dig> expression in the snr might also be mediated by the modulation of thalamic afferents during stn-hfs.
vglut <dig> is expressed in striatal cholinergic interneurons linked to the dopaminergic, gabaergic and glutamatergic systems in the cortico-bg-cortical loop  <cit> .
it is therefore unsurprising that stn-hfs, which affects the whole bg network, affects vglut <dig> expression in the striatum and related structures, such as the cortex, hippocampus, nucleus accumbens, thalamus  and amygdala.
indeed, stn-hfs has been reported to affect not only dopamine, glutamate and gaba  <cit>  but also serotonin and cholinergic transmissions  <cit> .
consequently, it is difficult to explain the role of vglut <dig> in stn-hfs mechanisms.
however, vglut <dig> in cholinergic interneurons has been shown to increase acetylcholine tone and to release both glutamate and acetylcholine  <cit> .
this mechanism might explain the efficacy of stn-hfs, via complex modulation of the bg network  <cit> .
CONCLUSIONS
in conclusion, this is the first study of the expression of the three vglut subtypes in various brain structures in control and 6-ohda-lesioned rats subjected to stn-hfs.
we know that all the three types of vgluts are expected to be found in axon terminals and that immunoradioautography cannot really disclose between neuronal vs. axonal vs. glial expression of vgluts.
in situ hybridization looking for different vglut transcripts will probably provide some added morphological value to our present study.
these experiments are planned in our lab.
however, our present data can suggest that stn-hfs may achieve its therapeutic effect, at least in part, through normalization of the thalamostriatal and the thalamocortical pathways.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
conceived and designed the experiments: mf, sb, sem, ms. performed the experiments: mf, gd, cc.
analyzed the data: mf, cc, ms. contributed reagents/materials/analysis tools: sem, ms. wrote the paper: mf, sb, sem, ms. all authors read and approved the final manuscript.
acknowledgments
this work was supported by the institut national de la santé et de la recherche médicale, université joseph fourier and région rhône-alpes .
we thank, dr emilie lacombe for technical assistance and dr sebastien carnicella for critical reading of this manuscript.

 it is long recognized that the degeneration of dopaminergic neurons induces an abnormal activation of glutamate systems in the basal ganglia that is central to the pathophysiology of parkinson’s disease. glutamate mediated mechanisms are also thought to play a role in the development of dyskinesias with long-term administration of l- -dihydroxyphenylalanine, the most efficient treatment for pd. many experimental studies also evidence that dopamine denervation induces an increase in corticostriatal glutamate and that l-dopa-induced dyskinesia are linked to bg network glutamate transmission abnormalities. microdialysis studies have suggested that dopamine lesion may also increase glutamate transmission in the bg output structures, substantia nigra pars reticulata and entopeduncular nucleus, presumably as a result of the abnormal activation of the subthalamic nucleus. three subtypes of vesicular glutamate transporters have been identified: vglut and. these transporters mediate glutamate uptake inside presynaptic vesicles and are anatomical and functional markers of glutamatergic excitatory transmission. vglut1- are very similar in structure and function, but are used by different neuronal populations. vglut and vglut are expressed by the cortical and subcortical neurons respectively. vglut is expressed by nonglutamatergic neurons, such as cholinergic striatal interneurons, a gabaergic interneuron subpopulation from the cortex and hippocampus and serotoninergic neurons from the dorsal and medial raphe nuclei. since the 1990s, high frequency stimulation of the stn has become an effective surgical treatment of late-stage parkinson’s disease, improving all motor symptoms in pd patients, particularly in those who experience motor fluctuations. however, the mechanisms underlying the improvement in symptoms remain unclear. beyond its local effect on stn activity, we know that, by activating axons, stn-hfs may generate widespread and heterogeneous distal effects throughout the bg network. indeed, we have already reported in previous studies that in intact or 6-ohda -lesioned rats, stn-hfs increases extracellular glutamate in the striatum, the globus pallidus and the snr. the present study analyzed the effects of da depletion and for the first time those of stn-hfs on vglut1- expression in several bg nuclei, by using immunoradioautography with affinity-purified rabbit vglut vglut or vglut antiserum. we found that da-depletion did not affect vglut and vglut expression in almost all bg structures studied while that of vglut significantly decreased. interestingly, stn-hfs did not affect vglut1- expression in normal rats, but systematically increased their expression in most of the bg nuclei studied in da-depleted animals. according to the changes of vglut1- expression observed and to their known anatomical localization, we suggest that stn-hfs may achieve its therapeutic effect, at least in part, through normalization of the thalamostriatal and thalamocortical pathways. methods animals adult male sprague–dawley rats, weighing to 270 g, were housed in an animal room on a 12-hour light/dark cycle, with food and water supplied ad libitum. this study was carried out in strict accordance with the recommendations of the european community council directive of november concerning the care of laboratory animals, french ministry of agriculture regulations and french guidelines for the use of live animals in scientific investigations. the protocol was approved by the committee on the ethics of animal experiments of the “grenoble institute of neuroscience ethical committee” agreement number all surgery was performed under a mixture of xylazine and ketamaine and all efforts were made to minimize the number of animal used and their suffering. all operated rats were intraperitoneally treated with rimadyl to prevent post-surgery suffering. lesion procedure forty rats were anesthetized with a mixture of xylazine and ketamine and secured in a kopf stereotaxic apparatus. all animals received desipramine pretreatment, to protect noradrenergic neurons. lesioned animals received a unilateral injection of 9 μg of 6-hydroxydopamine dissolved in 3 μl of % sterile nacl supplemented with % ascorbic acid, administered at a flow rate of  μl · min- to the left snc. an identical procedure was used for controls but with the injection of nacl %. the stereotaxic coordinates for the injection site relative to the bregma were as follows: anteroposterior, -  mm; lateral, +  mm; dorsoventral, -  mm, with the incisor bar at  mm below the interaural plane, according to the stereotaxic atlas of paxinos and watson. after injections, animals were kept warm and allowed to recover from the anesthetic before being returned to the animal house for three weeks until the stimulation experiments. this time interval was left to allow the da system degeneration induced by the neurotoxin to stabilize. implantation of the stimulation electrode rats from the two experimental groups were first anesthetized by the inhalation of a mixture of 3% isoflurane in air and mounted in a stereotaxic frame. the dorsal skull was exposed and holes were drilled for the implantation of the stimulation electrode into the left stn. during the implantation and stimulation procedure, anesthesia was maintained with an inhaled mixture of 1% isoflurane in air and body temperature was maintained at 37°c with a feedback-controlled heating pad. stereotaxic coordinates were chosen according to the atlas of paxinos and watson and were as follows relative to the bregma: ap, -  mm; l, +  mm; and dv, -  mm as previously described. electrical stimulation for electrical stimulation, we used a concentric stimulating bipolar electrode, with an outer diameter of 250 μm and a distance between the poles of 1 mm. stimuli were delivered under anesthesia during 4 hours with a world precision instrument acupulser and stimulus isolation units giving a rectangular pulse. this duration of stimulation was chosen to be sure that the proteic expression of vgluts can be detected and stabilized and almost corresponds to that used in previous studies analyzing mrna levels of different target proteins of basal ganglia circuits. as previously reported, the stimulation parameters matched those routinely used in parkinsonian patients. at the end of each experiment, an electrical lesion was created in the stn so that the position of the electrode could be checked post-mortem. in control rats the stimulation was never switched “on”. histology at the end of the electrical stimulation, all animals were perfused transcardially with % saline, under chloral hydrate anesthesia. brains were rapidly removed and frozen in cooled isopentane, then stored at −20°c. serial frontal sections were cut with a cryostat, collected on microscopic slides and stored at −20°c. tissue sections from different bg nuclei and related structures, nucleus accumbens, motor and somatosensory cortices, thalamus, subthalamic nucleus, globus pallidus and substantia nigra pars reticulata ) were selected to analyze changes in vglut expression. the correct location of the stimulation electrode was checked by collecting several subthalamic tissue sections and counterstaining with cresyl violet. the tip of the electrode was systematically implanted directly in the stn at the top of its dorsal part. these histological controls were systematically carried out for all the animals in each experimental group. all animals with incorrectly positioned stimulation electrodes were excluded. th-immunohistochemistry we assessed the extent of the dopaminergic denervation induced by nigral 6-ohda injection by th immunostaining on striatal and nigral sections from the fixed brains of lesioned animals. th immunostaining was carried out as previously described. briefly, striatal and nigral tissue sections from 6-ohda-lesioned rats were mounted on silane-coated microscope slides. tissue sections were postfixed in 4% paraformaldehyde, thoroughly washed with tris buffered-saline and incubated for 1 hour in % triton x- in tbs and 3% normal goat serum. they were then incubated with primary antisera diluted in tbst supplemented with 1% normal goat serum for 24 h, at 4°c. the antiserum was diluted 1: for th staining. antibody binding was detected with avidin-biotin-peroxidase conjugate, with 3’-diaminobenzidine as the chromagen. the detection reaction was allowed to proceed for one to three minutes, as previously described. sections were dehydrated in a series of graded ethanol solutions, cleared in xylene, mounted in dpx and covered with a coverslip for microscopy. vglut 1– immunoradioautography tissue sections were air-dried, post-fixed by immersion in fixative, and then washed in pbs. nonspecific binding sites were saturated by incubation with 3% bovine serum albumin in pbs, 1% ngs and 2 mm nai. sections were incubated overnight at 4°c in buffer a supplemented with affinity-purified rabbit vglut vglut or vglut antiserum, and then for 1 hour with an affinity-purified goat anti-rabbit igg in buffer a supplemented with % sodium azide. the sections were rinsed in water, dried and placed against x-ray films for to 11 days. the specificity of all antisera used in this study have been previously validated by our group. for each labeled section, a background value was estimated by measuring optical density in the corpus callosum, since this structure is devoided of specific staining for vglut 1– antibodies. this background value was then systematically subtracted from the optical density values obtained for each corresponding section. quantification and statistical analysis for the evaluation of the extent of da-denervation, striatal and nigral th immunostained sections were directly processed by using the calopix software of the computerized image analysis system. six th-immunostained sections from each structure and for each rat were used for quantification. the loss of th immunostaining in the snc or in the striatum was evaluated by comparing the total surface of both structures, as revealed by the th immunolabelling, in normal and lesioned animals. for quantification of vglut1- contents, four ap levels ) were choosen. for each rat, three stained sections of the same ap level were used for quantification and the triplicate od values obtained for each structure analyzed were averaged. immunoradioautograms obtained from x-ray films were analyzed with autoradio v software. values of optical densities measured from each structure analyzed are expressed as a mean ± standard error in table  histograms presented in figures show the mean ± standard error of the mean of optical densities expressed as a percentage of control values. data were analyzed for each brain structure by kruskal-wallis tests with sigmastat software. post-hoc analyses were carried out with the dunn’s method. a, modifications of vglut1- expression induced by unilateral 6-ohda-lesion of snc. *p<, vs control group. b, modifications of vglut1- expression induced by unilateral stn-hfs in 6-ohda lesioned rats. *p<, vs non stimulated 6-ohda-lesioned group. values of optical densities measurements are expressed as a mean ± standard error. data were analyzed for each brain structure by kruskal-wallis tests with sigmastat software. post-hoc analyses were carried out with the dunn’s method. bold numbers correspond to significant differences. ipsilateral side: related to the lesion and/or stimulation side; controlateral side: related to the lesion and/or stimulation side; acb, accumbens nucleus; cpu, caudate putamen; gp, globus pallidus; pm cx, premotor cortex; snr, substantia nigra pars reticulata; ss cx, somatosensory cortex; stn, subthalamic nucleus; thalamus.  histological controls of the extent of the dopamine lesion and of electrode location three weeks after the unilateral injection of 6-ohda, all lesioned animals presented a substantial loss of th immunostaining in the ipsilateral snc and the striatum, as shown by comparison with the contralateral side or with control animals. an analysis of densitometric measurements of th immunostaining showed an absence of statistical difference between the two lesioned groups. in da-depleted animals, the loss of snc th + neurons was evaluated by comparing the total snc surface on the intact side with the homologous area on the lesioned side. a loss of 92 ± 5% of th immunolabeled surface was measured. in the striatum of the same rats, the loss of da nerve terminals, as revealed by th immunostaining mainly affected the dorsal part of the striatum. this loss affected around 83 ± 4% of the striatal surface as compared to the total striatal surface of the control side. in this denervated striatal area, th immunolabeling, as evaluated by a mean of densitometric values, was decreased by 85 ± 5% when compared to the controlateral intact side. the correct implantation of the stimulation electrode in the stn is illustrated in figure 1c-e. figure 1e shows, at a higher magnification, the small electrical lesion created at the end of the experiment, indicating the point stimulated. regional distribution of vglut1- in control rats vglut1- expression was qualitatively analyzed in control rats that had been neither lesioned nor stimulated, to ensure the validity and specificity of the immunoradioautographical staining. immunoradioautograms from the different sections showed a distribution of vglut1- similar to that previously reported, confirming the validity of our vglut1- staining procedure and the lack of cross-reactivity between the antibodies used. vglut immunostaining was dense in almost all the structures studied, including, especially, the striatum, nucleus accumbens, cortex, the motor part of the thalamus and hippocampus. by contrast, no vglut labeling was found in the globus pallidus, the substantia nigra and in most of the brainstem. vglut proteins were detected in almost the same set of structures as vglut although the density of vglut immunostaining was slightly lower than that for vglut in striatal, cortical and thalamic areas, whereas the opposite was observed in many sub-cortical structures. these data are consistent with the well-described complementary pattern of expression of vglut and vglut in the rat brain. vglut staining, unlike that for vglut was detectable in the substantia nigra pars reticulata, hypothalamic nuclei and midbrain, which displayed widespread staining. different, complementary patterns of immunostaining for vglut and vglut were observed in the hippocampus. the density of vglut proteins was highest in layers iv and vi of the cortex and in the hypothalamus, the central gray matter and the superior colliculus. vglut staining was weaker than that for vglut and vglut but was also observed in many different areas. vglut levels were moderate in the striatum, but high in the hippocampus, with a complementary distribution for vglut and vglut. effects of 6-ohda-lesion and/or stn-hfs on vglut1- expression da lesion and stn stimulation were unilaterally performed in this study. however, we found similar changes of vgluts expression on both sides, as revealed by the optical density measurements of immunoreactive signals for vglut1- in all structures examined. in order to simplify the presentation of our data, we decided to only show the results obtained from the ipsilateral side on figures  and as precised in materials and methods, changes in vgluts expression induced by 6-ohda lesion and stn stimulation were first analyzed by kruskal-wallis tests with sigmastat software. results of these tests for each brain structure are presented in legends of figures  and post-hoc analyses were then carried out with the dunn’s method. effect of the 6-ohda-lesion on vglut1- expression da depletion did not affect vglut and vglut expression whatever the brain structure analyzed. slight changes were observed in the striatum, nucleus accumbens, somatosensory cortex and thalamus, but they were not statistically significant. by contrast, 6-ohda lesioning induced a significant decrease in vglut expression of nearly 50% with respect to that in sham-operated rats. this decrease was particularly strong in the stn, thalamus, and cortical areas which displayed decreases of 55%, 48% and 42% with respect to control rats, respectively. effect of stn-hfs on vglut1- expression in sham-operated control rats and in 6-ohda-lesioned animals no significant change in vglut1- expression was detected after four hours of stn-hfs in sham-operated control rats rats. levels of vglut1- expression were similar between the two experimental groups in all different structures studied, as show in figures  and on the contrary, stn-hfs induced a marked increase in vglut1- expression in 6-ohda-lesioned rats. this increase affected almost all the brain structures studied. vglut levels were always higher for all brain areas studied in stimulated 6-ohda rats when compared to non-stimulated 6-ohda-rats except for the nucleus accumbens and stn. the largest differences were found in the somatosensory cortex and the motor part of the thalamus. for many brain structures analyzed, vglut levels measured in stimulated 6-ohda-rats were comparable to those detected in control rats. interestingly, for the striatum and the thalamus, vglut levels remained moderately overexpressed versus controls. stn-hfs induced a significant increase in vglut expression in all structures studied in 6-ohda-lesioned rats when compared to non-stimulated 6-ohda-rats. however this increase did not affect the globus pallidus.. thus, stn-hfs more or less completely reversed the decrease in vglut expression induced by the da-depletion in all structures analyzed. stn-hfs induced a strong upregulation of vglut expression in 6-ohda-lesioned rats. interestingly, this effect was particularly marked in the thalamus and the somatosensorial cortex. discussion the key findings of this study were: 1) da depletion decreased vglut in all brain structures studied; 2) stn-hfs did not affect vglut1- expression in control rats whatever the brain structure analyzed; 3) stn-hfs increased vglut1- expression in 6-ohda-lesioned rats in almost all structures analyzed. thereby stn-hfs: i) normalized vglut levels after the decrease induced by da depletion, and ii) significantly increased vglut levels above those detected in control animals. this was also true for vglut levels but only for the striatum. this remodeling suggests that the mode of action of stn-hfs results from a global effect on basal ganglia network and related structures and that its therapeutic efficacy may to be linked, at least in part, to the normalization of thalamostriatal and thalamocortical neurotransmissions. bilateral effects of unilateral da lesioning and stn-hfs on vglut expression as stated above, unilateral da lesioning and stn-hfs caused similar changes in vglut expression on both sides of the brain. bilateral effects of unilateral da lesioning have already been reported in the striatum for tissue concentrations of glutamate, extracellular glutamate content assessed by microdialysis or voltammetry, glutamate receptor mrna and the glial glutamate transporter glt-. similarly, unilateral stn-hfs has been reported to induce bilateral increases in striatal and nigral glutamate content. these bilateral effects may result from crossed glutamatergic projections from the cortex or the thalamus innervating the bg on the contralateral side, consistent with cross-talk in cortico-bg-cortical loops. effect of 6-ohda-snc-lesioning on vglut expression we found here that 6-ohda-lesions had no effect on vglut or vglut levels in any of the structures studied. by contrast, vglut levels decreased significantly three weeks after lesioning. at first glance, our observations contrast with previous reported data showing that dopamine depletion is associated with an increase in synaptic glutamate release and with high striatal extracellular glutamate levels and glutamatergic activity and greater thalamostriatal activity, two to four weeks after lesioning of nigral dopaminergic neurons. however, other studies have reported an absence of change in glutamate levels. these differences may be accounted for by differences in the extent of the dopamine lesion, lesion sites, methodologies and time courses. we cannot exclude the possibility that different cellular mechanisms underlie presynaptic glutamate processes and extracellular glutamate release after lesioning. indeed, striatal extracellular glutamate levels have been reported to depend on a complex balance between vesicular release and non vesicular release via glutamate transporters on both neurons and glia and the cysteine-glutamine antiporter. dopamine depletion leads to complex, biphasic changes in striatal glutamatergic transmission over the first few weeks, possibly stabilizing over three months. contradictory data have been reported, for the cysteine-glutamate antiporter and glial transporters for example. furthermore, the changes in vgluts expression induced by 6-ohda lesions are also complex. vglut levels increase in the three weeks following the injection but then decrease, whereas vglut levels decrease and then normalize. in monkeys, mptp treatment increases vglut expression but does not affect vglut levels. in postmortem samples of parkinsonian patients vglut and vglut levels are increased in the putamen while vglut levels is lowered in the prefrontal and temporal cortex. however, the decrease in vglut levels observed here in all the brain structures of 6-ohda rats closely parallels the thalamic hypoactivity induced by the strengthening of gabaergic inputs from the snr and ep/gpi by the stn overactivity observed in da-depleted bg networks. furthermore, neuronal degeneration has been observed postmortem in the thalamic nuclei of pd patients and in the parafascicular nucleus in 6-ohda-lesioned rats. vglut is massively expressed by thalamic nuclei and have be postulated as selective marker of thalamo-striatal activity. this observation support the notion of a potential decreased glutamatergic afferences from the thalamus. effect of stn-hfs on vglut1- expression in control and 6-ohda-lesioned rats stn-hfs had no effect on vglut1- expression in any of the brain structures studied in control rats. this suggests that in the absence of dopamine depletion, stn-hfs did not affect vglut1- expression. these data are rather surprising since we reported in previous microdialysis study that in intact rats stn-hfs increases extracellular glutamate in the striatum, the globus pallidus and the snr. however, the duration of stimulation used here was longer than that used in our previous studies. thus, we can speculate that increase of extracellular glutamate levels induced by stn-hfs in physiological conditions mainly involves non vesicular release. by contrast, after 6-ohda lesions, stn-hfs induced an increase in vglut1- expression in almost all the structures analyzed. in these da depleted conditions, we cannot exclude the possibility that the balance between non vesicular and vesicular release of glutamate is disturbed, involving more glutamate transporters on both neurons and glia. indeed, it is well documented that following da nigrostriatal lesion, there is an increase in the number of glial cells, including astrocytes and microglia. therefore, the non vesicular release may be due to an increase in membrane transporters, such as glial glutamate transporters and the neuronal glutamate transporter eeac. thus, our previous findings concerning increased extracellular glutamate levels in the striatum, globus pallidus and snr of 6-ohda-lesioned rats are consistent and confirm that stn-hfs affects not only its direct targets, but also more distant structures of the bg network. the mechanisms underlying the therapeutic effects of stn-hfs are not fully elucidated. stn neuron inhibition by hfs, with loss of the drive of the internal part of the globus pallidus and disinhibition of the thalamus, would be consistent with the classical bg model. however, far more complex effects and circuitry are probably involved. for example, the direct activation of nearby thalamostriatal and pallidonigral fibres or direct or antidromic cortex activation. these mechanisms might lead to corticostriatal fiber activation and the observed increase in vglut levels. similarly, the increase in vglut levels induced here by stn-hfs may reflect a release of the classical thalamic inhibition induced by dopamine lesions. as vglut is found mostly in the thalamostriatal neurons specifically affected by da depletion, these data strongly suggest a major role for this pathway in the therapeutic effects of stn-hfs in pd. however, the normalization of vglut levels we observed in the somatosensory and premotor cortices may also play a non negligeable role in these effects of stn-hfs. interestingly, vglut was also overexpressed in the snr following stn-hfs in 6-ohda-lesioned rats. as vglut is the only vglut expressed by stn glutamatergic projections to the snr, this confirms our hypothesis that information transmission via the transsubthalamic pathway is not completely blocked during stn-hfs although the effect on vglut expression in the snr might also be mediated by the modulation of thalamic afferents during stn-hfs. vglut is expressed in striatal cholinergic interneurons linked to the dopaminergic, gabaergic and glutamatergic systems in the cortico-bg-cortical loop. it is therefore unsurprising that stn-hfs, which affects the whole bg network, affects vglut expression in the striatum and related structures, such as the cortex, hippocampus, nucleus accumbens, thalamus and amygdala. indeed, stn-hfs has been reported to affect not only dopamine, glutamate and gaba but also serotonin and cholinergic transmissions. consequently, it is difficult to explain the role of vglut in stn-hfs mechanisms. however, vglut in cholinergic interneurons has been shown to increase acetylcholine tone and to release both glutamate and acetylcholine. this mechanism might explain the efficacy of stn-hfs, via complex modulation of the bg network.  in conclusion, this is the first study of the expression of the three vglut subtypes in various brain structures in control and 6-ohda-lesioned rats subjected to stn-hfs. we know that all the three types of vgluts are expected to be found in axon terminals and that immunoradioautography cannot really disclose between neuronal vs. axonal vs. glial expression of vgluts. in situ hybridization looking for different vglut transcripts will probably provide some added morphological value to our present study. these experiments are planned in our lab. however, our present data can suggest that stn-hfs may achieve its therapeutic effect, at least in part, through normalization of the thalamostriatal and the thalamocortical pathways. competing interests the authors declare that they have no competing interests. authors’ contributions conceived and designed the experiments: mf, sb, sem, ms. performed the experiments: mf, gd, cc. analyzed the data: mf, cc, ms. contributed reagents/materials/analysis tools: sem, ms. wrote the paper: mf, sb, sem, ms. all authors read and approved the final manuscript. acknowledgments this work was supported by the institut national de la santé et de la recherche médicale, université joseph fourier and région rhône-alpes. we thank, dr emilie lacombe for technical assistance and dr sebastien carnicella for critical reading of this manuscript.
here we report that: 1) dopamine-depletion did not affect vglut and vglut expression but significantly decreased that of vglut in almost all bg structures studied; 2) stn-hfs did not change vglut1- 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 vglut 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. chronic high-frequency stimulation of the subthalamic nucleus is the neurosurgical therapy of choice for the management of motor deficits in patients with advanced pd. high frequency stimulationsubthalamic nucleusparkinson’s diseasebasal ganglia6-ohda-lesionratglutamatevesicular glutamate transporters they provide evidence for the involvement of vglut 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. vesicular glutamate transporters import glutamate into synaptic vesicles and are key anatomical and functional markers of glutamatergic excitatory transmission.
 it has been suggested that glutamatergic system hyperactivity may be related to the pathogenesis of parkinson’s disease. vesicular glutamate transporters import glutamate into synaptic vesicles and are key anatomical and functional markers of glutamatergic excitatory transmission. both vglut and vglut have been identified as definitive markers of glutamatergic neurons, but vglut is also expressed by non glutamatergic neurons. vglut and vglut are thought to be expressed in a complementary manner in the cortex and the thalamus, in glutamatergic neurons involved in different physiological functions. chronic high-frequency stimulation of the subthalamic nucleus 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- expression in different brain nuclei involved in motor circuits, namely the basal ganglia network, in normal and 6-hydroxydopamine lesioned rats.  here we report that: 1) dopamine-depletion did not affect vglut and vglut expression but significantly decreased that of vglut in almost all bg structures studied; 2) stn-hfs did not change vglut1- 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 vglut 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 vglut 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. high frequency stimulationsubthalamic nucleusparkinson’s diseasebasal ganglia6-ohda-lesionratglutamatevesicular glutamate transporters 