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).

A. Massie et al. / Neurochemistry International 57 (2010) 111–118 115

44.8 � 11.7%; contra: 35.0 � 8.7%; Fig. 3E) whereas VGLUT2 expres-sion was still unaffected at this post-lesion time (control: 89.5 � 7.4%;ipsi: 78.0 � 4.8%; contra: 87.5 � 7.8%; Fig. 3F). Sham lesioninginduced a small, though not significant, decrease in VGLUT2expression in the ipsilateral striatum and did not have any effecton striatal expression level of VGLUT1 (Fig. 3E and F).

2.4. In situ hybridization

VGLUT1 mRNA expression levels were measured in neocortexbetween Bregma +2.7 and Bregma �2.6 (Fig. 4A–C), whereas theVGLUT2 mRNA expression levels were evaluated in the Pf (lateraland medial) of the thalamus (Fig. 4D–I) three, five and twelveweeks post 6-OHDA injection into the MFB as well as in control andsham operated rats. For none of the vesicular glutamatetransporters and none of the conditions, we could detect asignificant change in mRNA expression level (Fig. 5). Yet, forVGLUT2 mRNA there was a tendency towards a decreased

Fig. 5. Bar diagram showing the relative optical density (ROD) of the hybridization signal

three, five and twelve weeks after 6-OHDA injection into the medial forebrain bundle.

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 probablythe result of the fact that the injection needle passes this nucleus inits way towards the MFB.

3. Discussion

In this study we describe time-dependent and bilateral changesin the striatal protein expression level of VGLUT1 as well asVGLUT2 upon dopamine depletion by unilateral injection of 6-OHDA into the MFB. Although no change in distribution patterncould be observed using immunohistochemistry, we detected abilateral upregulation of VGLUT1 and a downregulation of VGLUT2at a post-lesion time of three weeks. Twelve weeks after dopaminedepletion, VGLUT1 was bilaterally downregulated. We wereunable to correlate these changes in striatal protein expressionto changes in mRNA levels of the respective VGLUTs in the

for VGLUT1 (A) and VGLUT2 (B) in respectively neocortex and parafascicular nucleus

Results are expressed as mean � SEM of n = 6 animals per group.

A. Massie et al. / Neurochemistry International 57 (2010) 111–118116

corresponding projection areas. Both VGLUT1 and VGLUT2transcripts in respectively neocortex and Pf remained stableduring the whole time-window included in this study.

Although a consensus exists that changes in striatal glutama-tergic neurotransmission are linked to the pathogenesis ofParkinson’s disease, there is controversy as to the effect ofnigrostriatal damage on striatal extracellular glutamate concen-trations. Whereas some groups reported increased extracellularglutamate concentrations (Lindefors and Ungerstedt, 1990;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 thesecontradictory data are the result of different lesioning protocols aswell as different post-lesion times. Since Meshul et al. (1999)described biphasic changes in striatal glutamate after 6-OHDAinjection into the MFB, with an increase at three weeks and adecrease at twelve weeks, we investigated VGLUT transcript andprotein expression levels at these time intervals. Moreover, weincluded a post-lesion time of five weeks, since it was reported byRobelet et al. (2004) that neither changes in VGLUT expression norextracellular glutamate could be observed at this post-lesion time.

3.1. Bilateral effect on striatal VGLUT expression after unilateral 6-

OHDA injection

Similar as for the high-affinity glutamate reuptake transporters(Massie et al., submitted for publication) as well as for theextracellular glutamate concentrations (Lindefors and Ungerstedt,1990), we observed bilateral changes in the striatal proteinexpression levels of both VGLUTs after unilateral injection of 6-OHDA. Crosstalk between the two hemispheres can involve severalroutes. Although basal ganglia connectivity is largely sustained byipsilateral projections, several decussations have been described(Kerkerian-Le Goff et al., 2006). Not only a small part of thenigrostriatal pathway is crossing over to the contralateral striatum(Fass and Butcher, 1981) but also crossed thalamic efferents are asource of descending bilateral projections (Marini and Tredici,1995; Marini et al., 1999; Castle et al., 2005). Moreover, at the levelof the cortex, interhemispheric communication is commonthrough 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 weekspost-lesion is in line with the increase in VGLUT1 protein describedby Raju et al. (2008) in striatum of the MPTP-treated monkey aswell as by Kashani et al. (2007) in putamen of parkinsonianhumans. Although it was postulated by Baker et al. (2002) that inthe nucleus accumbens extracellular glutamate concentrations aremainly determined by glutamate release via the cystine/glutamateantiporter, an increase in glutamate filling of the synaptic vesiclesdue to increased expression of VGLUT1 may result in an increase ofquantal size (Wojcik et al., 2004) and could possibly also result instrengthening of corticostriatal transmission. Interestingly, allstudies in which increased extracellular glutamate has beenreported in the dopamine depleted striatum, have been conductedin rats at three to four weeks post 6-OHDA injection into thenigrostriatal pathway (Lindefors and Ungerstedt, 1990; Meshulet al., 1999; Jonkers et al., 2002). Most likely, the increasedexpression of VGLUT1, together with the increase in the cystine/glutamate antiporter expression that was observed before (Massieet al., 2008b), will contribute to such an increase in extracellularglutamate. At five weeks post-lesion, we observed a similarbilateral increase. This is in contrast to the findings of Robelet et al.

(2004) who described no effect of striatal dopamine deprivation onVGLUT1 expression. Possibly, this discrepancy is related totechnical details. Whereas we inject 6-OHDA into the MFB, inthe former study the injection was placed in the substantia nigra.The decreased expression of VGLUT1 that we observed twelveweeks after 6-OHDA lesioning, correlates to the decreasedextracellular glutamate concentrations as described by Meshulet al. (1999).

3.3. No effect of 6-OHDA lesioning on cortical VGLUT1 transcript levels

We were unable to link these striatal protein changes tochanges in cortical VGLUT1 transcript levels. Most probably, thechanges in VGLUT1 mRNA expression in cortical neurons project-ing to the striatum are camouflaged by VGLUT1 expressingintracortical projecting neurons. Indeed, only a subpopulation ofthe cortical neurons projects to the striatum. Moreover, asdescribed by Barroso-Chinea et al. (2008), VGLUT1 cannot beconsidered as a definitive marker for glutamatergic striatalafferents from cortical sources since thalamostriatal afferentsarising from the ventral thalamic nuclei also express VGLUT1transcript. However, VGLUT1 hybridization signal was faint in theventral thalamic nuclei compared to the neocortex, makingquantification rather difficult and inaccurate. Yet, at first sight,no strong increase in signal could be seen in 6-OHDA lesioned ratswith a short post-lesion time (personal observation). Furthermore,the striatal VGLUT1 protein changes might also depend on post-transcriptional mechanisms.

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 longbeen known to contribute to the basal ganglia circuitry, from afunctional point of view it remains poorly characterized (Barroso-Chinea et al., 2008). In the current study, we detected a bilateraldecrease in striatal VGLUT2 protein expression at three weekswhereas at five and twelve weeks we did not detect any significantchange, in line with the observations of Robelet et al. (2004). Adecrease in VGLUT2 expression, a marker for thalamostriatalafferents, might reflect the neuronal loss that has been found in theCM/Pf of patients with Parkinson’s disease (Henderson et al.,2000a,b). Though, we do not detect any significant loss of VGLUT2transcript in Pf at any post-lesion time. However, neuronal lossmight be masked by an increase in VGLUT2 transcript per cell asthe loss of Pf neurons innervating the dopamine depleted striatumcan be accompanied by a marked increase in the metabolic activityof the remaining thalamostriatal projecting neurons (Aymerichet al., 2006; Kashani et al., 2007). Possibly, this increase inmetabolic activity is already apparent at the mRNA level at threeweeks after lesioning and will result in a restoration of striatalVGLUT2 protein from five weeks on. Another possible explanationfor the discrepancy between striatal protein expression and Pftranscript levels could be linked to the fact that, although the mainsources of thalamostriatal projections are the intralaminarthalamic nuclei, they also arise from midline and specific relaythalamic nuclei (Smith et al., 2004).

3.5. No effect of 6-OHDA lesioning on VGLUT2 transcript levels in the

parafascicular nucleus

In rodents, some controversy exists as to the effect of striataldopamine depletion on Pf neurodegeneration and VGLUT2 mRNAexpression. The lack of effect of striatal dopamine depletion onVGLUT2 transcript in the Pf is in contradiction to the observationsof Sedaghat et al. (2009) who described an overall reduction of

A. Massie et al. / Neurochemistry International 57 (2010) 111–118 117

VGLUT2 hybridization density, linked to a reduced density ofneurons, in Pf ipsilateral to intranigral 6-OHDA injection at a post-lesion time of one and five months. Also, one month after 6-OHDAinjection into the MFB more than 50% loss of Pf neurons projectingto the striatum was observed (Aymerich et al., 2006). However, asdescribed above, in the latter study degeneration of thalamicneurons was accompanied by hyperactivity and increased VGLUT2mRNA expression in surviving neurons. Systemic MPTP adminis-tration in mice (Freyaldenhoven et al., 1997) as well as striatalinjection of MPP+ in rats (Ghorayeb et al., 2002) induced significantloss of neurons in midline and intralaminar thalamic nuclei,whereas in the striatum of the dopamine depleted monkey nosignificant change in relative prevalence of VGLUT2 thalamos-triatal terminals was observed (Raju et al., 2008). Here again, it isobvious that the discrepancy that can be observed in the availablereports is most probably the result of the use of different animalmodels.

4. Conclusion

The data presented in this paper, together with our previousfindings for the glial high-affinity Na+/K+-dependent glutamatetransporters and xCT, the specific subunit of the cystine/glutamateantiporter, 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) andsupport the idea of a biphasic change in striatal glutamatergicneurotransmission in the hemi-Parkinson rat model (Meshul et al.,1999).

In conclusion, changes in VGLUT expression will certainlycontribute to the aberrant glutamatergic neurotransmission in thehemi-Parkinson rat model. Moreover, in post-mortem tissue ofParkinson patients identical results were obtained for VGLUT1 asin this study at three to five weeks post-lesion (Kashani et al.,2007). This strongly supports the idea that VGLUT1 might be aninteresting target in the search for new therapeutic strategies forthe treatment of Parkinson’s disease and should encourage thesearch for specific in vivo ligands of the VGLUTs.

Acknowledgements

This work was supported by grants of the Brussels Capital-Region (Prospective Research for Brussels), Fonds voorWetenschappelijk Onderzoek-Flanders and the Vrije UniversiteitBrussel. AS is a research assistant of the FWO-Flanders and KV is aresearch assistant of the IWT-Flanders. The authors wish toacknowledge Mr. G. De Smet, Ms. R. Vanlaer and Ms. R. Berckmansfor excellent technical assistance.

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