Progressive Parkinsonism by acute dysfunction of excitatory amino acid transporters in the rat substantia nigra.

Parkinson's disease (PD) is characterized by the progressive degeneration of substantia nigra (SN) dopamine neurons, involving a multifactorial cascade of pathogenic events. Here we explored the hypothesis that dysfunction of excitatory amino acid transporters (EAATs) might be involved. Acutely-induced dysfunction of EAATs in the rat SN, by single unilateral injection of their substrate inhibitor l-trans-pyrrolidine-2,4-dicarboxylate (PDC), triggers a neurodegenerative process mimicking several PD features. Dopamine neurons are selectively affected, consistent with their sustained excitation by PDC measured by slice electrophysiology. The anti-oxidant N-acetylcysteine and the NMDA receptor antagonists ifenprodil and memantine provide neuroprotection. Besides oxidative stress and NMDA receptor-mediated excitotoxicity, glutathione depletion and neuroinflammation characterize the primary insult. Most interestingly, the degeneration progresses overtime with unilateral to bilateral and caudo-rostral evolution. Transient adaptive changes in dopamine function markers in SN and striatum accompany cell loss and axonal dystrophy, respectively. Motor deficits appear when neuron loss exceeds 50% in the most affected SN and striatal dopamine tone is dramatically reduced. These findings outline a functional link between EAAT dysfunction and several PD pathogenic mechanisms/pathological hallmarks, and provide a novel acutely-triggered model of progressive Parkinsonism.

Changes to interneuron-driven striatal microcircuits in a rat model of Parkinson's disease.

Striatal interneurons play key roles in basal ganglia function and related disorders by modulating the activity of striatal projection neurons. Here we have injected rabies virus (RV) into either the rat substantia nigra pars reticulata or the globus pallidus and took advantage of the trans-synaptic spread of RV to unequivocally identify the interneurons connected to striatonigral- or striatopallidal-projecting neurons, respectively. Large numbers of RV-infected parvalbumin (PV+/RV+) and cholinergic (ChAT+/RV+) interneurons were detected in control conditions, and they showed marked changes following intranigral 6-hydroxydopamine injection. The number of ChAT+/RV+ interneurons innervating striatopallidal neurons increased concomitant with a reduction in the number of PV+/RV+ interneurons, while the two interneuron populations connected to striatonigral neurons were clearly reduced. These data provide the first evidence of synaptic reorganization between striatal interneurons and projection neurons, notably a switch of cholinergic innervation onto striatopallidal neurons, which could contribute to imbalanced striatal outflow in parkinsonian state.

High-frequency stimulation of the subthalamic nucleus potentiates L-DOPA-induced neurochemical changes in the striatum in a rat model of Parkinson's disease.

This study examined the cellular changes produced in the striatum by chronic L-DOPA treatment and prolonged subthalamic nucleus high-frequency stimulation (STN-HFS) applied separately, successively, or in association, in the 6-hydroxydopamine-lesioned rat model of Parkinson's disease (PD). Only animals showing severe L-DOPA-induced dyskinesias (LIDs) were included, and STN-HFS was applied for 5 d at an intensity efficient for alleviating akinesia without inducing dyskinesias. L-DOPA treatment alone induced FosB/deltaFosB immunoreactivity, exacerbated the postlesional increase in preproenkephalin, reversed the decrease in preprotachykinin, and markedly increased mRNA levels of preprodynorphin and of the glial glutamate transporter GLT1, which were respectively decreased and unaffected by the dopamine lesion. STN-HFS did not affect per se the postlesion changes in any of these markers. However, when applied in association with L-DOPA treatment, it potentiated the positive modulation exerted by L-DOPA on all of the markers examined and tended to exacerbate LIDs. After 5 d of L-DOPA withdrawal, the only persisting drug-induced responses were an elevation in preprodynorphin mRNA levels and in the number of FosB/deltaFosB-immunoreactive neurons. Selective additional increases in these two markers were measured when STN-HFS was applied subsequently to L-DOPA treatment. These data provide the first evidence that STN-HFS exacerbates the responsiveness of striatal cells to L-DOPA medication and suggest that STN-HFS acts specifically through an L-DOPA-modulated signal transduction pathway associated with LIDs in the striatum. They point to striatal cells as a primary site for the complex interactions between these two therapeutic approaches in PD and argue against a direct anti-dyskinetic action of STN-HFS.

Preferential vulnerability of mesencephalic dopamine neurons to glutamate transporter dysfunction.

Nigral depletion of the main brain antioxidant GSH is the earliest biochemical event involved in Parkinson's disease pathogenesis. Its causes are completely unknown but increasing number of evidence suggests that glutamate transporters [excitatory amino acid transporters (EAATs)] are the main route by which GSH precursors may enter the cell. In this study, we report that dopamine (DA) neurons, which express the excitatory amino acid carrier 1, are preferentially affected by EAAT dysfunction when compared with non-DA neurons. In rat embryonic mesencephalic cultures, l-trans-pyrrolidine-2,4-dicarboxylate, a substrate inhibitor of EAATs, is directly and preferentially toxic for DA neurons by decreasing the availability of GSH precursors and lowering their resistance threshold to glutamate excitotoxicity through NMDA-receptors. In adult rat, acute intranigral injection of l-trans-pyrrolidine-2,4-dicarboxylate induces a large regionally selective and dose-dependent loss of DA neurons and alpha-synuclein aggregate formation. These data highlight for the first time the importance of excitatory amino acid carrier 1 function for the maintenance of antioxidant defense in DA neurons and suggest its dysfunction as a candidate mechanism for the selective death of DA neurons such as occurring in Parkinson's disease.

High-resolution neuroanatomical tract-tracing for the analysis of striatal microcircuits.

Although currently available retrograde tracers are useful tools for identifying striatal projection neurons, transported tracers often remained restricted within the neuronal somata and the thickest, main dendrites. Indeed, thin dendrites located far away from the cell soma as well as post-synaptic elements such as dendritic spines cannot be labeled unless performing intracellular injections. In this regard, the subsequent use of anterograde tracers for the labeling of striatal afferents often failed to unequivocally elucidate whether a given afferent makes true contacts with striatal projections neurons. Here we show that such a technical constraint can now be circumvented by retrograde tracing using rabies virus (RV). Immunofluorescence detection with a monoclonal antibody directed against the viral phosphoprotein resulted in a consistent Golgi-like labeling of striatal projection neurons, allowing clear visualization of small-size elements such as thin dendrites as well as dendritic spines. The combination of this retrograde tracing together with dual anterograde tracing of cortical and thalamic afferents has proven to be a useful tool for ascertaining striatal microcircuits. Indeed, by taking advantage of the trans-synaptic spread of RV, different subpopulations of local-circuit neurons modulating striatal efferent neurons can also be identified. At the striatal level, structures displaying labeling were visualized under the confocal laser-scanning microscope at high resolution. Once acquired, confocal stacks of images were firstly deconvoluted and then processed through 3D-volume rendering in order to unequivocally identify true contacts between pre-synaptic elements (axon terminals from cortical or thalamic sources) and post-synaptic elements (projection neurons and/or interneurons labeled with RV).

Differential organization of cortical inputs to striatal projection neurons of the matrix compartment in rats.

In prior studies, we described the differential organization of corticostriatal and thalamostriatal inputs to the spines of direct pathway (dSPNs) and indirect pathway striatal projection neurons (iSPNs) of the matrix compartment. In the present electron microscopic (EM) analysis, we have refined understanding of the relative amounts of cortical axospinous vs. axodendritic input to the two types of SPNs. Of note, we found that individual dSPNs receive about twice as many axospinous synaptic terminals from IT-type (intratelencephalically projecting) cortical neurons as they do from PT-type (pyramidal tract projecting) cortical neurons. We also found that PT-type axospinous synaptic terminals were about 1.5 times as common on individual iSPNs as IT-type axospinous synaptic terminals. Overall, a higher percentage of IT-type terminals contacted dSPN than iSPN spines, while a higher percentage of PT-type terminals contacted iSPN than dSPN spines. Notably, IT-type axospinous synaptic terminals were significantly larger on iSPN spines than on dSPN spines. By contrast to axospinous input, the axodendritic PT-type input to dSPNs was more substantial than that to iSPNs, and the axodendritic IT-type input appeared to be meager and comparable for both SPN types. The prominent axodendritic PT-type input to dSPNs may accentuate their PT-type responsiveness, and the large size of axospinous IT-type terminals on iSPNs may accentuate their IT-type responsiveness. Using transneuronal labeling with rabies virus to selectively label the cortical neurons with direct input to the dSPNs projecting to the substantia nigra pars reticulata, we found that the input predominantly arose from neurons in the upper layers of motor cortices, in which IT-type perikarya predominate. The differential cortical input to SPNs is likely to play key roles in motor control and motor learning.

Intralaminar thalamic nuclei lesions: widespread impact on dopamine denervation-mediated cellular defects in the rat basal ganglia.

Intralaminar thalamic nuclei represent a major site of non-dopaminergic degeneration in Parkinson disease, but the impact of this degeneration on the pathophysiological functioning of basal ganglia remains unknown. To address this issue, we compared the effects of 6-hydroxydopamine-induced lesions of nigral dopamine neurons alone or combined with ibotenate-induced lesions of intralaminar thalamic neurons on markers of neuronal metabolic activity in the rat basal ganglia using in situ hybridization histochemistry. Thalamic lesions prevented most of the dopamine denervation-induced changes (i.e. the increases in mRNA levels of enkephalin and GAD67 in the striatum, of GAD67 in the globus pallidus and entopeduncular nucleus, and of cytochrome oxidase subunit-I in the subthalamic nucleus), but did not affect the downregulation of striatal substance P and upregulation of GAD67 in the substantia nigra pars reticulata. We also provide immunohistochemical evidence that thalamic lesions markedly decreased striatal expression of the vesicular glutamate transporter vGluT2, confirming the association of this transporter with the thalamic projections to the basal ganglia. Altogether, these data reveal a major antagonistic influence of thalamic and dopaminergic afferents onto the basal ganglia and suggest that degeneration of thalamic neurons in Parkinson disease may represent an important factor counteracting expression of the defects associated with the dopamine denervation.

The added value of rabies virus as a retrograde tracer when combined with dual anterograde tract-tracing.

Rabies virus (RV) has widely been used as a trans-synaptic retrograde tracer to analyze chains of connected neurons. The use of antibodies directed against the viral nucleoprotein enables viral nucleocapsids to be visualized within the cell soma, as well as within the thickest main dendrites. However, through this approach it is often difficult to accurately define post-synaptic elements (thin dendrites and/or dendritic spines). This limitation can now easily been circumvented by taking advantage of antibodies directed against a soluble viral phosphoprotein that spreads throughout the cytoplasm of the infected neuron, thereby producing Golgi-like immunofluorescent labeling of first-order projection neurons that are infected with RV. Furthermore, when combined with anterograde tracers such as Phaseolus vulgaris-leucoagglutinin (PHA-L) and biotinylated dextran amine (BDA), this procedure to detect RV facilitates the accurate visualization of both the pre- and post-synaptic elements. Finally, this method of viral detection is sufficiently sensitive to detect weakly labeled second-order neurons, which can then be further characterized neurochemically. Several examples are provided to illustrate why retrograde trans-synaptic tracing using RV can be regarded as an important breakthrough in the analysis of brain circuits, providing an unprecedented level of resolution.

Enhanced delivery of gamma-secretase inhibitor DAPT into the brain via an ascorbic acid mediated strategy.

Inhibition of gamma-secretase, one of the enzymes responsible for the cleavage of the amyloid precursor protein (APP) to produce pathogenic Abeta peptides, is an attractive approach for the treatment of Alzheimer's disease. We designed a gamma-secretase inhibitor bearing an ascorbic acid moiety which allows a specific delivery of the drug to the brain. Through, on the one hand, Abeta peptide production measurements by specific in vitro assays (gamma-secretase cell free assay and cell based assay on HEK 293 APP transfected cells) and on the other hand through pharmacokinetic studies on animal models, the new inhibitor shows a good pharmacokinetic profile as well as a potent gamma-secretase inhibitory activity in vitro. From the obtained results, it is expected that drug will be mainly delivered to the CNS with a low diffusion in the peripheral tissues. Consequently the side effects of this gamma-secretase inhibitor on the immune cells could be reduced.