Christophe Melon

Chronic l-DOPA treatment increases extracellular glutamate levels and GLT1 expression in the basal ganglia in a rat model of Parkinson's disease

There is growing experimental evidence for the implication of glutamate‐mediated mechanisms both in the pathophysiology of Parkinsons disease and in the development of dyskinesias with long‐term administration of l‐3,4‐dihydroxyphenylalanine (l‐DOPA). However, the impact of this treatment on glutamate transmission in the basal ganglia has been poorly investigated. In this study, we examined the effects of 6‐hydroxydopamine‐induced lesion of nigral dopamine neurons with or without subsequent chronic l‐DOPA treatment on several parameters of glutamate system function in the rat striatum and substantia nigra pars reticulata. All the lesioned animals treated with l‐DOPA developed severe dyskinesias. Extracellular glutamate levels, measured by microdialysis in freely moving conditions, and gene expression of the glial glutamate transporter GLT1, assessed by in situ hybridization, were unaffected by dopamine lesion or l‐DOPA treatment alone, but were both markedly increased on the lesion side of rats with subsequent l‐DOPA treatment. No change in the expression of the vesicular glutamate transporters vGluT1 and vGluT2 was measured in striatum. These data show that chronic l‐DOPA treatment leading to dyskinesias increases basal levels of glutamate function in basal ganglia. The l‐DOPA‐induced overexpression of GLT1 may represent a compensatory mechanism involving astrocytes to limit glutamate overactivity and subsequent toxic processes.

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

Parkinsons 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.

Effects of prolonged iron overload and low frequency electromagnetic exposure on spatial learning and memory in the young rat

Low-frequency electromagnetic fields (EMF) have been suggested to affect the brain via alterations of blood-brain barrier permeability to iron. Because of an immature blood-brain barrier, the young brain may be particularly vulnerable to EMF exposure. It is therefore possible that behavioral and neurotoxic effects resulting from EMF-induced iron excess in the brain would be greater in young adults. The objective of the present study was to investigate the interaction between low-frequency EMF and iron overload in young rats. In Experiment 1, we tested the effects of iron overload on spatial learning and memory. Iron treatment did not affect performance in a reference (Morris water maze) and a working memory task (8-arm radial maze). In contrast, detection of a spatial change in an object exploration task was impaired. These effects correlated with modifications of the serotoninergic metabolism. In Experiment 2, the combination of EMF exposure and iron overload was tested. As in Experiment 1, rats were not impaired in reference and working memory tasks but were mildly impaired in the detection of the spatial change. Overall, the results showed an effect of iron overload on spontaneous spatial memory processes. However, low-frequency EMF exposure did not potentiate the effects of iron overload in young rats.

Spatial learning, monoamines and oxidative stress in rats exposed to 900 MHz electromagnetic field in combination with iron overload.

The increasing use of mobile phone technology over the last decade raises concerns about the impact of high frequency electromagnetic fields (EMF) on health. More recently, a link between EMF, iron overload in the brain and neurodegenerative disorders including Parkinsons and Alzheimers diseases has been suggested. Co-exposure to EMF and brain iron overload may have a greater impact on brain tissues and cognitive processes than each treatment by itself. To examine this hypothesis, Long-Evans rats submitted to 900 MHz exposure or combined 900 MHz EMF and iron overload treatments were tested in various spatial learning tasks (navigation task in the Morris water maze, working memory task in the radial-arm maze, and object exploration task involving spatial and non spatial processing). Biogenic monoamines and metabolites (dopamine, serotonin) and oxidative stress were measured. Rats exposed to EMF were impaired in the object exploration task but not in the navigation and working memory tasks. They also showed alterations of monoamine content in several brain areas but mainly in the hippocampus. Rats that received combined treatment did not show greater behavioral and neurochemical deficits than EMF-exposed rats. None of the two treatments produced global oxidative stress. These results show that there is an impact of EMF on the brain and cognitive processes but this impact is revealed only in a task exploiting spontaneous exploratory activity. In contrast, there are no synergistic effects between EMF and a high content of iron in the brain.

Distinct effects of mGlu4 receptor positive allosteric modulators at corticostriatal vs. striatopallidal synapses may differentially contribute to their antiparkinsonian action

Metabotropic glutamate 4 (mGlu4) receptor is a promising target for the treatment of motor deficits in Parkinsons disease (PD). This is due in part to its localization at key basal ganglia (BG) synapses that become hyperactive in this pathology, particularly striatopallidal synapses. In this context, mGlu4 receptor activation using either orthosteric agonists or positive allosteric modulators (PAMs) improves motor symptoms in rodent PD models in certain conditions. However, literature data show that mGlu4 receptor PAMs have no effect at striatopallidal GABAergic synapses (unless combined with an orthosteric agonist) and on the firing activity of pallidal neurons, and fail to provide significant motor improvement in relevant PD models. This questions the mechanistic hypothesis that mGlu4 receptor PAMs should act at striatopallidal synapses to alleviate PD motor symptoms. To shed light on this issue, we performed brain slice electrophysiology experiments. We show that Lu AF21934, an mGlu4 PAM small-molecule probe-compound, was ineffective at striatopallidal synapses at all concentrations tested, while it significantly inhibited corticostriatal synaptic transmission. Similarly, Lu AF21934 did not affect electrophysiology readouts at striatopallidal synapses in the presence of haloperidol or in 6-hydroxydopamine-lesioned rats. Interestingly, co-application of Lu AF21934 with a glutamate transporter inhibitor revealed a significant inhibitory action at striatopallidal synapses. Possibly, this effect could rely on increased level/permanence of glutamate in the synaptic cleft. Such differential efficacy of mGlu4 receptor PAMs at corticostriatal vs. striatopallidal synapses raises several issues regarding the synaptic target(s) of these drugs in the BG, and challenges the mechanisms by which they alleviate motor deficits in experimental PD models.

Progressive brain metabolic changes under deep brain stimulation of subthalamic nucleus in parkinsonian rats.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an efficient neurosurgical treatment for advanced Parkinsons disease. Non‐invasive metabolic neuroimaging during the course of DBS in animal models may contribute to our understanding of its action mechanisms. Here, DBS was adapted to in vivo proton magnetic resonance spectroscopy at 11.7 T in the rat to follow metabolic changes in main basal ganglia structures, the striatum, and the substantia nigra pars reticulata (SNr). Measurements were repeated OFF and ON acute and subchronic (7 days) STN‐DBS in control and parkinsonian (6‐hydroxydopamine lesion) conditions. Acute DBS reversed the increases in glutamate, glutamine, and GABA levels induced by the dopamine lesion in the striatum but not in the SNr. Subchronic DBS normalized GABA in both the striatum and SNr, and glutamate in the striatum. Taurine levels were markedly decreased under subchronic DBS in the striatum and SNr in both lesioned and unlesioned rats. Microdialysis in the striatum further showed that extracellular taurine was increased. These data reveal that STN‐DBS has duration‐dependent metabolic effects in the basal ganglia, consistent with development of adaptive mechanisms. In addition to counteracting defects induced by the dopamine lesion, prolonged DBS has proper effects independent of the pathological condition.

Metabolic, synaptic and behavioral impact of 5‐week chronic deep brain stimulation in hemiparkinsonian rats

The long‐term effects and action mechanisms of subthalamic nucleus (STN) high‐frequency stimulation (HFS) for Parkinsons disease still remain poorly characterized, mainly due to the lack of experimental models relevant to clinical application. To address this issue, we performed a multilevel study in freely moving hemiparkinsonian rats undergoing 5‐week chronic STN HFS, using a portable constant‐current microstimulator. In vivo metabolic neuroimaging by 1H‐magnetic resonance spectroscopy (11.7 T) showed that STN HFS normalized the tissue levels of the neurotransmission‐related metabolites glutamate, glutamine and GABA in both the striatum and substantia nigra reticulata (SNr), which were significantly increased in hemiparkinsonian rats, but further decreased nigral GABA levels below control values; taurine levels, which were not affected in hemiparkinsonian rats, were significantly reduced. Slice electrophysiological recordings revealed that STN HFS was, uniquely among antiparkinsonian treatments, able to restore both forms of corticostriatal synaptic plasticity, i.e. long‐term depression and potentiation, which were impaired in hemiparkinsonian rats. Behavior analysis (staircase test) showed a progressive recovery of motor skill during the stimulation period. Altogether, these data show that chronic STN HFS efficiently counteracts metabolic and synaptic defects due to dopaminergic lesion in both the striatum and SNr. Comparison of chronic STN HFS with acute and subchronic treatment further suggests that the long‐term benefits of this treatment rely both on the maintenance of acute effects and on delayed actions on the basal ganglia network.

Bee Venom Alleviates Motor Deficits and Modulates the Transfer of Cortical Information through the Basal Ganglia in Rat Models of Parkinson's Disease.

Recent evidence points to a neuroprotective action of bee venom on nigral dopamine neurons in animal models of Parkinson’s disease (PD). Here we examined whether bee venom also displays a symptomatic action by acting on the pathological functioning of the basal ganglia in rat PD models. Bee venom effects were assessed by combining motor behavior analyses and in vivo electrophysiological recordings in the substantia nigra pars reticulata (SNr, basal ganglia output structure) in pharmacological (neuroleptic treatment) and lesional (unilateral intranigral 6-hydroxydopamine injection) PD models. In the hemi-parkinsonian 6-hydroxydopamine lesion model, subchronic bee venom treatment significantly alleviates contralateral forelimb akinesia and apomorphine-induced rotations. Moreover, a single injection of bee venom reverses haloperidol-induced catalepsy, a pharmacological model reminiscent of parkinsonian akinetic deficit. This effect is mimicked by apamin, a blocker of small conductance Ca2+-activated K+ (SK) channels, and blocked by CyPPA, a positive modulator of these channels, suggesting the involvement of SK channels in the bee venom antiparkinsonian action. In vivo electrophysiological recordings in the substantia nigra pars reticulata (basal ganglia output structure) showed no significant effect of BV on the mean neuronal discharge frequency or pathological bursting activity. In contrast, analyses of the neuronal responses evoked by motor cortex stimulation show that bee venom reverses the 6-OHDA- and neuroleptic-induced biases in the influence exerted by the direct inhibitory and indirect excitatory striatonigral circuits. These data provide the first evidence for a beneficial action of bee venom on the pathological functioning of the cortico-basal ganglia circuits underlying motor PD symptoms with potential relevance to the symptomatic treatment of this disease.

Inhibition of the mitochondrial glutamate carrier SLC25A22 in astrocytes leads to intracellular glutamate accumulation

The solute carrier family 25 (SLC25) drives the import of a large diversity of metabolites into mitochondria, a key cellular structure involved in many metabolic functions. Mutations of the mitochondrial glutamate carrier SLC25A22 (also named GC1) have been identified in early epileptic encephalopathy (EEE) and migrating partial seizures in infancy (MPSI) but the pathophysiological mechanism of GC1 deficiency is still unknown, hampered by the absence of an in vivo model. This carrier is mainly expressed in astrocytes and is the principal gate for glutamate entry into mitochondria. A sufficient supply of energy is essential for the proper function of the brain and mitochondria have a pivotal role in maintaining energy homeostasis. In this work, we wanted to study the consequences of GC1 absence in an in vitro model in order to understand if glutamate catabolism and/or mitochondrial function could be affected. First, short hairpin RNA (shRNA) designed to specifically silence GC1 were validated in rat C6 glioma cells. Silencing GC1 in C6 resulted in a reduction of the GC1 mRNA combined with a decrease of the mitochondrial glutamate carrier activity. Then, primary astrocyte cultures were prepared and transfected with shRNA-GC1 or mismatch-RNA (mmRNA) constructs using the Neon® Transfection System in order to target a high number of primary astrocytes, more than 64%. Silencing GC1 in primary astrocytes resulted in a reduced nicotinamide adenine dinucleotide (Phosphate) (NAD(P)H) formation upon glutamate stimulation. We also observed that the mitochondrial respiratory chain (MRC) was functional after glucose stimulation but not activated by glutamate, resulting in a lower level of cellular adenosine triphosphate (ATP) in silenced astrocytes compared to control cells. Moreover, GC1 inactivation resulted in an intracellular glutamate accumulation. Our results show that mitochondrial glutamate transport via GC1 is important in sustaining glutamate homeostasis in astrocytes. Main Points: The mitochondrial respiratory chain is functional in absence of GC1 Lack of glutamate oxidation results in a lower global ATP level Lack of mitochondrial glutamate transport results in intracellular glutamate accumulation