Modification d'expression de NR2B lors de dyskinésies de la patte avant chez le rat induites par traitement chronique à la L-DOPA ou par stimulation à haute fréquence du Noyau Subthalamique

La stimulation à haute fréquence (SHF) du noyau subthalamique (NST) joue un rôle essentiel chez les patients Parkinsoniens dans l'amélioration des troubles moteurs pour lesquels la dopa-thérapie n'est plus satisfaisante. Tout comme l'administration à long terme de L-DOPA, la SHF du NST, peut aussi, selon l'intensité de stimulation, évoquer des mouvements dyskinétiques. Ces dyskinésies sont considérées comme un phénomène d'apprentissage moteur pathologique, secondaire à une altération de la transmission glutamatergique et sont sous-tendues par des modifications durables d'expression génique, notamment dans le striatum. L'objectif de ce travail de thèse est d'étudier et de comparer les mécanismes moléculaires des dyskinésies induites par la L-DOPA à celles induites par la SHF, en se focalisant plus particulièrement sur la sous unité NR2B des récepteurs NMDA. Dans un premier temps, nous avons montré par immunohistochimie que la sous unité NR2B est hyperphosphorylée dans le NST et l'EP suite à l'induction de dyskinésie par la SHF du NST chez l'animal sain. Ces résultats ont été confirmés par la suite dans un modèle animal de la maladie de Parkinson, le rat 6-OHDA. La comparaison de ces modifications avec celles observées chez le rat 6-OHDA rendus dyskinétique par un traitement chronique à la L-DOPA nous permet de suggérer que l'induction des dyskinésies est associée à une hyperphosphorylation de NR2B au sein d'une voie subthalamo-entopédonculaire alors qu'une activation de NR2B dans le striatum semble être impliquée dans l'expression des dyskinésies. Enfin, nos résultats mettent également en évidence une implication différentielle des deux structures de sorties des ganglions de la base dans les processus akinétiques et dyskinésiogènes.High frequency stimulation of the subthalamic nucleus (STN-HFS) alleviates parkinsonian motor symptoms and indirectly improves dyskinesia by decreasing L-DOPA requirement. However, inappropriate stimulation can also trigger dyskinetic movements Dyskinesia are thought to be a pathological learning process due to an overactive glutamate transmission within the basal ganglia. Moreover, several molecular changes seem to be involved in this process. The aim of the present study is to compare the molecular mechanisms of dyskinesia induced by L-DOPA and by STN-HFS, by focusing more particularly on the NR2B-containing NMDA receptor. We show by immunohistochemistry that NR2B subunit is hyperphosphorylated within the STN and the EP during a dyskinesiogenic STN-HFS in normal rats. Similar results are obtained from 6-OHDA rats, a model of Parkinson disease. Comparison of these results with those observed in 6-OHDA dyskinetic rats chronically treated with L-DOPA suggest that dyskinesia induction is associated with an hyperphosphorylation of NR2B within a subthalamo-entopeduncular network while activation of NR2B within the striatum seem to be involved in the expression of dyskinesia. A different implication of the two output of the basal ganglia in akinetic and dyskinesiogenic process is also demonstrated. STAR Date de soutenance : 8 juillet 2011 Thèse sur travaux: nonSAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Author manuscript, published in "Neurobiology of Disease 2012;48(3):379-90"

DOI: 10.1016/j.nbd.2012.06.009 Neurobiology of disease- Regular article Effects of L-DOPA and STN-HFS dyskinesiogenic treatments on NR2

The Homer-1 protein Ania-3 interacts with the plasma membrane calcium pump.

International audienceThe Homer family of scaffold proteins couples NMDA receptors to metabotropic glutamate receptors and links extracellular signals to calcium release from intracellular stores. Ania-3 is a member of the Homer family and is rapidly inducible in brain in response to diverse stimuli. Here, we report the identification of the plasma membrane Ca2+ ATPase (PMCA) as a novel Ania-3/Homer-associated protein. Ania-3/Homer interacts with the b-splice forms of all PMCAs (PMCA1b, 2b, 3b, and 4b) via their PDZ domain-binding COOH-terminal tail. Ectopically expressed Ania-3 colocalized with the PMCA at the plasma membrane of polarized MDCK epithelial cells, and endogenous Ania-3/Homer and PMCA2 are co-expressed in the soma and dendrites of primary rat hippocampal neurons. The interaction between Ania-3/Homer and PMCAs may represent a novel mechanism by which local calcium signaling and hence synaptic function can be modulated in neurons

Preclinical evaluation of [18F]2FNQ1P as the first fluorinated serotonin 5-HT6 radioligand for PET imaging

International audiencePurposeBrain serotonin 6 receptor (5-HT6) is one of the most recently identified serotonin receptors. It is a potent therapeutic target for psychiatric and neurological diseases, e.g. schizophrenia and Alzheimer’s disease. Since no specific fluorinated radioligand has yet been successfully used to study this receptor by positron emission tomography (PET) neuroimaging, the objective of the present study was to study the first 5-HT6 18F-labelled radiotracer.Methods2FNQ1P, inspired by the quinolone core of a previous radiotracer candidate, GSK215083, was selected according its 5-HT6 affinity and selectivity and was radiolabelled by 18F nucleophilic substitution. The cerebral distribution of [18F]2FNQ1P was studied in vivo in rats, cats and macaque monkeys.ResultsThe chemical and radiochemical purities of [18F]2FNQ1P were >98 %. In rats, in vitro competition with the 5-HT6 antagonist, SB258585, revealed that the radioligand was displaced dose dependently. Rat microPET studies showed low brain uptake of [18F]2FNQ1P, reversed by the P-glycoprotein inhibitor, cyclosporin. On the contrary, PET scans in cats showed good brain penetration and specific striatal binding blocked after pretreatment with unlabelled 2FNQ1P. PET scans in macaque monkeys confirmed high specific binding in both cortical and subcortical regions, specifically decreased by pretreatment with the 5-HT6 receptor antagonist, SB258585.Conclusion2FNQ1P was initially selected because of its suitable characteristics for 5-HT6 receptor probing in vitro in terms of affinity and specificity. Although in vivo imaging in rats cannot be considered as predictive of the clinical characteristics of the radiotracer, [18F]2FNQ1P appeared to be a suitable 5-HT6 PET tracer in feline and primate models. These preclinical results encourage us to pursue the clinical development of this first fluorinated 5-HT6 PET radiotracer

Coordinated and spatial upregulation of arc in striatonigral neurons correlates with L-dopa-induced behavioral sensitization in dyskinetic rats.

International audienceAlthough oral administration of L-Dopa remains the best therapy for Parkinson disease, its long-term administration causes the appearance of abnormal involuntary movements such as dyskinesia. Although persistent striatal induction of some genes has already been associated with such pathologic profiles in hemiparkinsonian rats, molecular and cellular mechanisms underlying such long-term adaptations remain to be elucidated. In this study, using a rat model of L-Dopa-induced dyskinesia, we report that activity regulated cytoskeletal (Arc)-associated protein is strongly upregulated in the lesioned striatum and that the extent of its induction further varies according to the occurrence or absence of locomotor sensitization. Moreover, Arc is preferentially induced, along with FosB, nur77, and homer-1a, in striatonigral neurons, which express mRNA encoding the precursor of dynorphin. Given the likely importance of Arc in the regulation of cytoskeleton during synaptic plasticity, its upregulation supports the hypothesis that a relationship exists between cytoskeletal modifications and the longlasting action of chronically administrated L-Dopa

Ventral pallidum encodes contextual information and controls aversive behaviors

Successful avoidance of aversive outcomes is crucial for the survival of animals. Although accumulating evidence indicates that an indirect pathway in the basal ganglia is involved in aversive behavior, the ventral pallidum (VP), which is an important component of this pathway, has so far been implicated primarily in appetitive behavior. In this study, we used single-cell recordings and bicuculline (GABAA antagonist) injections to elucidate the role of VP both in the encoding of aversive context and in active avoidance. We found 2 populations of neurons that were preferentially activated by appetitive and aversive conditioned stimuli (CSs). In addition, VP showed appetitive and aversive outcome anticipatory activities. These activity patterns indicate that VP is involved in encoding and maintaining CS-induced aversive contextual information. Furthermore, the disturbance of VP activity by bicuculline injection increased the number of error trials in aversive trials. In particular, the subjects released the response bar prematurely, showed no response at all, or failed to avoid the aversive outcome. Overall, these results suggest that VP plays a central role in controlling CS-induced negative motivation to produce avoidance behavior

Forelimb dyskinesia mediated by high-frequency stimulation of the subthalamic nucleus is linked to rapid activation of the NR2B subunit of N-methyl-D-aspartate receptors.

International audienceDyskinesia is a major side-effect of chronic l-DOPA administration, the reference treatment for Parkinson's disease. High-frequency stimulation of the subthalamic nucleus (STN-HFS) alleviates parkinsonian motor symptoms and indirectly improves dyskinesia by decreasing the L-DOPA requirement. However, inappropriate stimulation can also trigger dyskinetic movements, in both human and rodents. We investigated whether STN-HFS-evoked forelimb dyskinesia involved changes in glutamatergic neurotransmission as previously reported for L-DOPA-induced dyskinesias, focusing on the role of NR2B-containing N-methyl-D-aspartate receptors (NR2B/NMDARs). We applied STN-HFS in normal rats at intensities above and below the threshold for triggering forelimb dyskinesia. Dyskinesiogenic STN-HFS induced the activation of NR2B (as assessed by immunodetection of the phosphorylated residue Tyr(1472)) in neurons of the subthalamic nucleus, entopeduncular nucleus, motor thalamus and forelimb motor cortex. The severity of STN-HFS-induced forelimb dyskinesia was decreased in a dose-dependent manner by systemic injections of CP-101,606, a selective blocker of NR2B/NMDARs, but was either unaffected or increased by the non-selective N-methyl-D-aspartate receptor antagonist, MK-801

Subthalamic stimulation-induced forelimb dyskinesias are linked to an increase in glutamate levels in the substantia nigra pars reticulata.

International audienceThe neurobiological mechanisms by which high-frequency stimulation of the subthalamic nucleus (STN-HFS) alleviates the motor symptoms of Parkinson's disease (PD) remain unclear. In this study, we analyzed the effects of STN-HFS on motor behavior in intact or hemiparkinsonian rats (6-hydroxydopamine lesion of the substantia nigra pars compacta) and investigated the correlation between these effects and extracellular glutamate (Glu) and GABA levels, assessed by intracerebral microdialysis in the substantia nigra pars reticulata (SNr). STN-HFS at an intensity corresponding to the threshold inducing contralateral forelimb dyskinesia, increased Glu levels in the SNr of both intact and hemiparkinsonian rats. In contrast, STN-HFS at half this intensity did not affect Glu levels in the SNr in intact or hemiparkinsonian rats but increased GABA levels in hemiparkinsonian rats only. STN-HFS-induced forelimb dyskinesia was blocked by microinjection of the Glu receptor antagonist kynurenate into the SNr and facilitated by microinjection of a mixture of the Glu receptor agonists AMPA and NMDA into the SNr. These new neurochemical data suggest that STN-HFS-induced forelimb dyskinesia is mediated by glutamate, probably via the direct activation of STN axons, shedding light on the mechanisms of STN-HFS in PD

Characterization and Reliability of [18F]2FNQ1P in Cynomolgus Monkeys as a PET Radiotracer for Serotonin 5-HT6 Receptors

Brain serotonin-6 receptor (5-HT6R) is the one of the most recently identified serotonin receptors. Accumulating evidence suggests that it is a potent therapeutic target for psychiatric and neurological diseases. Since [18F]2FNQ1P was recently proposed as the first fluorinated positron emission tomography (PET) radioligand for this receptor, the objective of the present study was to demonstrate its suitability for 5-HT6R neuroimaging in primates. [18F]2FNQ1P was characterized by in vitro autoradiography and in vivo PET imaging in cynomolgus monkeys. Following in vivo PET imaging, tracer binding indices were computed using the simplified reference tissue model and Logan graphical model, with cerebellum as reference region. The tracer binding reproducibility was assessed by test–retest in five animals. Finally, specificity was assessed by pre-injection of a 5-HT6R antagonist, SB258585. In vitro, results showed wide cerebral distribution of the tracer with specificity toward 5-HT6Rs as binding was effectively displaced by SB258585. In vivo brain penetration was good with reproducible distribution at cortical and subcortical levels. The automated method gave the best spatial normalization. The Logan graphical model showed the best tracer binding indices, giving the highest magnitude, lowest standard deviation and best reproducibility and robustness. Finally, 5-HT6R antagonist pre-injection significantly decreased [18F]2FNQ1P binding mainly in the striatum and sensorimotor cortex. Taken together, these preclinical results show that [18F]2FNQ1P is a good candidate to address 5-HT6 receptors in clinical studies