Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fluphenazine-induced orofacial dyskinesia (OD) is a putative animal model of tardive dyskinesia (TD) whose pathophysiology has been related to an increase in dopamine hypersensitivity and oxidative stress. Data from literature have shown that patients with TD present a decrease in dopamine transporter (DAT) expression. In a previously study, we have demonstrated that experimental animals presenting high intensity of vacuous chewing movements (VCM) induced by chronic treatment with haloperidol also presented a reduced dopamine uptake into striatum. Considering that one way to regulate DAT is through redox modulation, the first objective of the present study to determine if the chronic treatment with fluphenazine could induce an increase in oxidative stress index in brain regions (striatum and substantia nigra) and an alteration in levels of dopamine uptake in the striatum of rats treated acute and chronically with fluphenazine (Article 1). The fluphenazine treatment produced VCMs in the majority of the treated rats (87% after 24 weeks). Concomitant treatment with diphenyl diselenide decreased the prevalence of VCMs to 50%. Additionally, we separated the rats that developed (+VCM) or did not develop (-VCM) VCMs. We did not find any statistical differences among the groups when oxidative stress parameters were evaluated. Chronic fluphenazine treatment significantly decreased dopamine uptake. Concomitant treatment with diphenyl diselenide was not able to prevent this decrease in those rats that developed VCMs. Another objective of this work was to evaluate the role of dopamine (DA) and other monoamines and their metabolites on acute and chronic of OD induced by fluphenazine in rats (manuscript in preparation 1). The vacuous chewing movements (VCMs) or the levels of monoamines and its metabolites were quantified after 3 (acute) or 24 (chronic) weeks after beginning of treatment. The fluphenazine treatment produced VCMs in part of treated rats (50% after 3 weeks and about 85% after 24 weeks). There were not significant differences between the groups in monoamines levels neither in their metabolites in the striatum under acute fluphenazine treatment in +VCMs rats. However, we observed a trend to increase the levels of the DA metabolites, HVA (p=0.05) and DOPAC (p=0.06), after chronic treatment with fluphenazine. Our data suggest that an increase in DA metabolism could contribute to the maintenance of VCMs in rats. Moreover, development of VCMs seems not to be dependent of DA metabolism. Moreover, the use of diphenyl disselenide seems to be a promissory pharmacological therapy in the reduction of OD prevalence. / A discinesia orofacial (DO) induzida por flufenazina consiste num modelo de discinesia tardia (DT) cuja patofisiologia tem sido relacionada à hipersensibilidade dopaminérgica e ao estresse oxidativo. Dados da literatura demonstraram que pacientes com DT apresentam reduzida expressão do transportador de dopamina (TDA). Em um estudo prévio, nós demonstramos que animais experimentais que apresentam alta intensidade de movimentos de mascar no vazio (MMV) induzidos por tratamento crônico com haloperidol também apresentaram uma redução na captação de dopamina (DA) no estriado. Tendo em vista que uma das maneiras de reduzir a atividade dos TDA é via modulação redox, um primeiro objetivo deste estudo foi determinar se o tratamento crônico com flufenazina poderia induzir um aumento nos índices de estresse oxidativo em regiões cerebrais (estriado e substantia nigra) e quais os efeitos deste tratamento nos níveis de captação de DA no estriado de ratos tratados aguda e cronicamente com flufenazina (Artigo 1). O tratamento com flufenazina produziu MMV na maioria dos ratos tratados (87% após 24 semanas). O tratamento concomitante com disseleneto de difenila diminuiu a prevalência dos MMV para 50%. Além disso, separamos os animais que desenvolveram (+MMV) ou não desenvolveram (-MMV) MMV. Não encontramos nenhuma diferença estatística entre os grupos quando comparados parâmetros de estresse oxidativo. O tratamento crônico, mas não agudo, com flufenazina diminuiu significativamente a captação de DA nos animais que apresentaram MMV. O tratamento concomitante com disseleneto de difenila não foi capaz de prevenir esta redução naqueles ratos que desenvolveram MMV. Um outro objetivo deste trabalho foi avaliar a participação da DA, de outras monoaminas e de seus metabólitos no modelo agudo e crônico de DO induzida por flufenazina em ratos (manuscrito em preparação 1). O tratamento com flufenazina produziu MMV na maioria dos animais tratados (50% após 3 semanas e cerca de 85% após 24 semanas. Não houve diferença estatisticamente significativa entre os grupos controle e tratado com flufenazina agudamente com relação aos níveis de monoaminas e seus metabólitos no estriado de ratos apresentando MMV+. Observamos uma tendência a um aumento nos níveis dos metabólitos da DA, HVA (p=0.05) e DOPAC (p=0.06), após tratamento crônico com flufenazina. Em conjunto, estes resultados indicam que a redução no transporte de DA pode ser um possível mecanismo relacionado à manutenção da DO crônica em ratos. O metabolismo da DA parece ter participação na manutenção da DO, mas não no desenvolvimento. Além disso, o uso do disseleneto de difenila parece ser terapia farmacológica promissora para a redução da prevalência da DO.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Schizophrenia is a psychiatric disorder that affects 1% of world population and its pharmacologic treatment consists in the use of antipsychotics. It is known that chronic treatment with classical antipsychotics, such as haloperidol, can cause motors disturbers, among which stands out the tardive dyskinesia (TD). The TD pathophysiology has been associated with oxidative stress increase in areas of the brain related to the movements control. So, studies have proposed the use of natural compounds with antioxidants properties to decrease the production of reactive species on TD or on animals models of orofacial dyskinesia (OD). Bauhinia forficata (B. forficata), a plant used on folk medicine such as hypoglycemic, and presents antioxidant properties, could be used to reduce the oxidative stress present on OD. Thus, the first aim of this study was evaluate the effect of B. forficata on in vitro lipid peroxidation induced by pro-oxidants and also, the possible protector effect on OD, through of the quantifications of vacuous chewing movements (VCM), and on locomotor and exploratory activities decrease induced by haloperidol in rats (manuscript 1). B. forficata prevented the lipid peroxidation by pro-oxidant agents nitroprusside sodium, Fe2+/EDTA and Fe2+. Furthermore, adult male rats received haloperidol (38 mg/kg) each 28 days for 16 weeks and B. forficata decoction (2.5 g/L) on the place of drinking water or water to drink everyday for 16 weeks. The haloperidol treatment increased the VCM and reduced the locomotor and exploratory activities relative to control group on open field test. B. forficata co-treatment was not able to prevent the motor alterations induced by haloperidol, as well as only partially prevented VCM in rats. On the other hand, B. forficata caused an increase on locomotor activity. These results showed that B. forficata has antioxidant potential, but this effect is associated partially to protection against VCM induced by haloperidol in rats (manuscript 1). Some studies have showed that chronic treatment with classic antipsychotics can cause metabolic side effects. B. forficata which is known on folk medicine by glycemia decrease, could prevent the metabolic side effects caused by use of antipsychotics. So, the other propound of this study was to investigate the weight gain, glycemic levels and other metabolic parameters in rats chronically treated with haloperidol and the possible effect of B. forficata on these metabolic side effects (manuscript 2). After 16 weeks of treatment, the animals treated with haloperidol showed high glycemic prevalence and high glucose levels. B. forficata co-treatment elevated glucose and triglycerides levels. No difference was observed on cholesterol, urea, creatinine, alanine aminotransferase (AST), aspartate aminotransferase (ALT) and lactate dehydrogenase (LDH). Haloperidol treatment caused weight gain and promoted a significant decrease on brain/body weight rate. In conclusion, B. forficata co-treatment and haloperidol can increase the number of hyperglycemic animals. Thus, is necessary to emphasize the importance of more toxicology studies on chronic treatment with B. forficata to avoid health implications of population. / A esquizofrenia é uma desordem psiquiátrica que atinge cerca de 1% da população mundial e seu tratamento farmacológico consiste na utilização de antipsicóticos. Sabe-se que o tratamento crônico com antipsicóticos clássicos, como o haloperidol, pode causar distúrbios motores, dentre os quais se destaca a discinesia tardia (DT). A fisiopatologia da DT tem sido associada ao aumento do estresse oxidativo em áreas do cérebro relacionadas ao controle dos movimentos. Desta forma, estudos têm proposto o uso de compostos naturais com propriedades antioxidantes para diminuir a produção de espécies reativas na DT ou em modelos animais de discinesia orofacial (DO). A Bauhinia forficata (B. forficata), uma planta utilizada na medicina popular como hipoglicemiante, e que apresenta propriedades antioxidantes, poderia ser utilizada para reduzir o estresse oxidativo presente na DO. Logo, o primeiro objetivo deste estudo foi avaliar o efeito da B. forficata na peroxidação lipídica in vitro induzida por diferentes pró-oxidantes, e também o possível efeito protetor da B. forficata na DO, através da quantificação dos movimentos de mascar no vazio (MMV), e na diminuição da atividade locomotora e exploratória induzidos por haloperidol em ratos (manuscrito 1). A B. forficata preveniu a formação de peroxidação lipídica induzida pelos agentes pró-oxidantes nitroprussiato de sódio, Fe2+/EDTA e Fe2+. Além disso, ratos adultos machos receberam haloperidol (38 mg/kg) a cada 28 dias por 16 semanas, e o decocto de B. forficata (2,5 g/L) no lugar da água de beber ou água para beber todos os dias por 16 semanas. O tratamento com o haloperidol aumentou o número de MMV, e reduziu as atividades locomotora e exploratória dos animais em relação ao grupo controle no teste do campo aberto. O co-tratamento com B. forficata não foi capaz de prevenir as alterações motoras induzidas por haloperidol, bem como preveniu apenas parcialmente os MMV em ratos. Por outro lado, a B. forficata sozinha causou um aumento na atividade locomotora. Os resultados desse estudo mostraram que a B. forficata tem potencial antioxidante, mas esse efeito está associado apenas parcialmente à proteção contra os MMV induzidos pelo haloperidol em ratos (manuscrito 1). Alguns estudos têm mostrado que o tratamento crônico com antipsicóticos clássicos pode causar efeitos colaterais metabólicos. A B. forficata, conhecida na medicina popular por diminuir a glicemia, poderia prevenir os efeitos colaterais metabólicos causados pelo uso de antipsicóticos. Assim, outra proposta deste estudo foi investigar o ganho de peso, os níveis glicêmicos e outros parâmetros metabólicos em ratos tratados cronicamente com haloperidol e o possível efeito da B. forficata nesses efeitos colaterais metabólicos (manuscrito 2). Após 16 semanas de tratamento, os animais tratados com haloperidol apresentaram alta prevalência glicêmica e altos níveis séricos de glicose. O co-tratamento com B. forficata elevou os níveis de glicose e de triglicerídeos. Nenhuma diferença foi observada nos níveis de colesterol, uréia, creatinina, alanina aminotransferase, aspartato aminotransferase (AST) e lactato desidrogenase (LDH). O tratamento com haloperidol causou aumento no ganho de peso corporal e promoveu significante diminuição na taxa peso do cérebro/corpo. Em conclusão, o co-tratamento com B. forficata e haloperidol pode aumentar o número de animais hiperglicêmicos. Assim, é necessário enfatizar a importância de mais estudos toxicológicos sobre o tratamento crônico com B. forficata para evitar implicações na saúde da população.

Tardive dyskinesia (TD), first appearing in humans after introduction of the phenothiazine class of antipsychotics in the 1950s, is now recognized as an abnormality resulting predominately by long-term block of dopamine (DA) D2 receptors (R). TD is thus reproduced in primates and rodents by chronic administration of D2-R antagonists. Through a series of studies predominately since the 1980s, it has been shown in rodent modeling of TD that when haloperidol or other D2-R antagonist is added to drinking water, rats develop spontaneous oral dyskinesias, vacuous chewing movements (VCMs), after ~3 months, and this TD is associated with an increase in the number of striatal D2-R. This TD persists for the duration of haloperidol administration and another ~2 months after haloperidol withdrawal. By neonatally lesioning dopaminergic nerves in brain in neonatal rats with 6-hydroxydopamine (6-OHDA), it has been found that TD develops sooner, at ~2 months, and also is accompanied by a much higher number of VCMs in these haloperidol-treated lesioned rats, and the TD persists lifelong after haloperidol withdrawal, but is not associated with an increased D2-R number in the haloperidol-withdrawn phase. TD apparently is related in part to supersensitization of both D1-R and serotoninergic 5-HT2-R, which is also a typical outcome of neonatal 6-OHDA (n6-OHDA) lesioning. Testing during the haloperidol-withdrawn phase in n6-OHDA rats displaying TD reveals that receptor agonists and antagonists of a host of neuronal phenotypic classes have virtually no effect on spontaneous VCM number, except for 5-HT2-R antagonists which acutely abate the incidence of VCMs in part. Extrapolating to human TD, it appears that (1)5-HT2-R supersensitization is the crucial alteration accounting for persistence of TD, (2) dopaminergic-perhaps age-related partial denervation-is a risk factor for the development of TD, and (3) 5-HT2-R antagonists have the therapeutic potential to alleviate TD, particularly if/when an antipsychotic D2-R blocker is withdrawn.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum do mundo, e seu tratamento atual se baseia principalmente no uso de medicações que facilitam a transmissão dopaminérgica nos núcleos da base. A L-DOPA é a principal medicação usada no tratamento da DP, contudo seu uso crônico está associado ao surgimento de complicações motoras, como as discinesias induzidas por L-DOPA (DIL). As DIL ocorrem em cerca de 50% dos pacientes com DP que usaram LDOPA por cerca de 4 a 6 anos, e podem causar uma série de impactos negativos aos pacientes. Pela incapacidade adicional que esta complicação traz aos pacientes com DP, a definição de fatores que possam predizer o surgimento das DIL é de importância direta para o clínico que prescreve L-DOPA rotineiramente. O objetivo deste estudo foi determinar os principais fatores de risco clínicos e genéticos para o desenvolvimento de DIL em uma casuística de pacientes brasileiros com DP. Um estudo transversal foi realizado em pacientes brasileiros de dois centros (Ribeirão Preto e São Paulo) como parte do projeto \"Latin American Research Consortium on the Genetics of PD\" (LARGE-PD), incluindo apenas pacientes com DP e em uso de L-DOPA. A avaliação foi baseada em um exame neurológico completo e em uma entrevista semi-estruturada feita por um médico neurologista especialista em Distúrbios de Movimentos. A presença de DIL foi considerada se a pontuação fosse >= 1 no item 32 da Parte IV na MDS-UPDRS. Baseados em estudos prévios, nós escolhemos oito polimorfismos tipo single nucleotide polymorphism (SNP) nestes genes: COMT, MAOB, ANKK1, DRD3, DAT1, BDNF, ADORA2A and NOS1. A genotipagem foi realizada através de ensaios TaqMan SNP. Foram realizados modelos de regressão logística e análises de sobrevivência para identificarmos os fatores de risco clínicos e genéticos associados ao surgimento de DIL. Ao todo, foram analisados 199 pacientes (sexo masculino - 59%; idade média 61.8 anos), sendo 96 indivíduos (48.2%) com DIL. Através de um modelo de regressão logística multivariado com 7 variáveis independentes, o fenótipo clínico motor do tipo postural instability with gait disorder (PIGD) (OR 0.17, IC 95% 0.07-0.39; p < 0.001), longos períodos de tratamento com L-DOPA (OR 1.31, IC 95% 1.17-1.47; p < 0.001), altas doses diárias equivalentes de L-DOPA (OR 1.00, IC 95% 1.000-1.002; p = 0.04) e a início precoce dos sintomas da 11 DP (OR 1.04, IC 95% 1.01-1.07; p = 0.009) foram os fatores de risco clínicos mais associados ao surgimento de DIL. Dentre os fatores de risco genéticos, apenas o alelo T do SNP rs1799836 no gene da MAOB esteve associado ao aumento na chance de surgimento de DIL (OR 1.51, IC 95% 1.00-2.28; p = 0.05). Nossos resultados mostraram que a predição de surgimento de DIL em pacientes com DP em uso de L-DOPA pode ser feita através de alguns fatores de risco clínicos (fenótipo clínico motor, duração do tratamento com L-DOPA, dose diária equivalente de L-DOPA e idade de início de sintomas) e genéticos, como o SNPrs1799836 no gene da MAOB. Novos estudos com amostras maiores e desenhos prospectivos longitudinais são necessários para se explorar a associação entre estes preditores e o surgimento de DIL. / Parkinson\'s disease (PD) is the second most common neurodegenerative disease in the world, and its treatment is mainly based on drugs involved on dopaminergic neurotransmission in basal ganglia. L-DOPA is the major medication used on the management of PD, but its chronic use is associated with the onset of motor complications, as L-DOPA-induced dyskinesias (LID). LID occur in approximately 50% of patients using L-DOPA over 4 and 6 years, and they cause negative impacts on the quality of life of patients PD. To predict the onset of LID based on clinical and genetic risk may be an useful tool for clinicians which prescribe L-DOPA. The aim of this study was to determinate the main clinical and genetic risk factors for the onset of LID in Brazilian PD patients. A cross-sectional study was conducted with Brazilian PD patients from two centers (Ribeirao Preto and Sao Paulo) as part of the Latin American Research consortium on the Genetics of PD (LARGE-PD), which enrolled only PD patients using L-DOPA. PD patients were submitted to neurological examination and semi-structured interviews performed by movement disorders specialists. Presence of LID was confirmed if UPDRS Part IV had a score >= 1 on item 32. Based on previous studies, we chose eight Single Nucleotide Polymorphisms (SNP) in the following genes: COMT, MAOB, ANKK1, DRD3, DAT1, BDNF, ADORA2A and NOS1. Genotyping was performed using TaqMan SNP genotyping assays. We performed logistic regression and survival analysis to identify clinical and genetic risk factors associated with LID onset We enrolled 199 PD patients (males - 59%; mean age 61.8 years), and 96 patients (48.2%) had LID. At a multivariate model with 7 independent variables, postural instability with gait disorder (PIGD) clinical phenotype (OR 0.17, CI 95% 0.07-0.39; p < 0.001), longer duration of L-DOPA therapy (OR 1.31, Cl 95% 1.17-1.47; p < 0.001), higher L-DOPA equivalent daily doses (OR 1.00, CI 95% 1.000-1.002; p = 0.04), as also as early onset of PD (OR 1.04, CI 95% 1.01-1.07; p = 0.009) were the clinical risk factor more associated with onset of LID. Regarding genetic risk factors, only MAOB SNP rs1799836 was associated with LID, with the T allele increasing the risk of developing LIDs (OR 1.51, CI 95% 1.00-2.28; p = 0.05). Our results showed onset of LID can be predicted based on some clinical (motor clinical phenotype, duration of L-DOPA therapy, L-DOPA equivalent daily dose and age at PD onset) and genetic risk factors, as MAOB SNP rs1799836. Further studies in larger samples, using longitudinal and prospective designs, are needed to explore the association between these predictors and onset of LID.

Adenylyl cyclases are key points for the concurrent integration of diverse signaling pathways. Controlling production of the second messenger cAMP, adenylyl cyclases provide an important mechanism for the regulation of physiological functions by amplifying signaling events to stimulate downstream effectors. While different isoforms of adenylyl cyclase exhibit distinct patterns of expression and regulation, of particular interest are two groups of Ca2+ regulated isoforms that are highly expressed in the central nervous system. Adenylyl cyclase type 5 (AC5) is a Ca2+ inhibited isoform that is highly expressed in the striatum, and whose activity is involved in the regulation of movement, pain, and metabolism. Adenylyl cyclase type 8 (AC8) is stimulated by Ca2+ in a calmodulin dependent manner, and appears to be involved with long-term memory, anxiety, and reward pathways. Studying the signaling characteristics of these adenylyl cyclase isoforms is necessary for improving our scientific understanding of biological pathways, as well identifying therapeutic targets that can be exploited for treatment of disease. In this work, we investigated changes in the protein interaction network of AC5 following prolonged Gi/o-mediated inhibition that results in heterologous sensitization. The diversity of signaling pathways and multitude of protein interactions that have been implicated in the development of the heterologous sensitization response prompted the development of a novel screening strategy to capture and identify AC5-protein interactions which occur following prolonged Gi/o-mediated inhibition. We utilized bimolecular fluorescence complementation (BiFC) in conjunction with fluorescence activated cell sorting (FACS) and Next Generation sequencing to capture, identify, and characterize novel AC5 interacting partners. We further studied the effects of increased AC5 activity by functionally characterizing a series of gain-of-function mutations that have been identified in patients diagnosed with Familial Dyskinesia and Facial Myokymia (FDFM). Our results demonstrate that the AC5 mutants exhibit enhanced activity to Gs-mediated stimulation and reduced inhibition by Gi/o-coupled receptors. We further suggest that this dysregulation of AC5 in striatal medium spiny neurons likely results in an imbalance in the direct and indirect striatal signaling pathways that coordinate the initiation and maintenance of movement. Genetic models of AC8 regulation have implicated its activity in signaling pathways that may regulate comorbid long-term anxiety and ethanol consumption. Therefore, we developed and conducted a high-throughput screen and validation paradigm of small molecules for the discovery of AC8 selective inhibitors. The screening effort identified two lead compounds that demonstrate enhanced efficacy and selectivity over AC1 compared to currently available adenylyl cyclase inhibitors.

Le traitement substitutif par la L-DOPA, indiqué dans la maladie de Parkinson, induit à terme des complications motrices appelées les dyskinésies induites par la L-DOPA. L'apparition des dyskinésies est due, au moins en partie, à la mise en place d'une plasticité aberrante dans le striatum, qui fait suite à des modifications transcriptionnelles induites par la L-DOPA. Une analyse du transcriptome nous a permis d'identifier le gène Nptx2, codant pour la neuropentraxine Narp, comme étant un candidat potentiellement impliqué dans l'apparition des dyskinésies. L'objectif de ce travail était d'étudier la régulation et le rôle de Narp dans l'apparition des dyskinésies, dans un modèle de souris lésée à la 6-hydroxydopamine. Nous avons montré que les dyskinésies induites par la L-DOPA sont diminuées chez des souris invalidées pour Nptx2 (Narp-KO). Par ailleurs, l'injection dans le striatum dorsal d'un adénovirus exprimant une forme dominante négative de Narp, induit une réduction importante des scores de dyskinésies. Dans le striatum, Narp est exprimé par les neurones épineux de taille moyenne et par les interneurones à parvalbumine. Après une stimulation dopaminergique, l'augmentation de l'expression de Nptx2 s'accompagne d'un enrichissement de Narp au niveau synaptique. Nos travaux montrent donc que Narp joue un rôle important dans le développement des dyskinésies et suggèrent qu'il pourrait être impliqué dans la plasticité synaptique des neurones du striatum, comme cela a été montré dans l'hippocampe. Ces résultats permettent d'ouvrir de nouvelles perspectives thérapeutiques pour retarder l'apparition de ces complications motrices chez les patients parkinsoniens. / Dopaminergic replacement therapy in Parkinson’s disease is hampered by the occurrence of L-DOPA-induced dyskinesia (LID). One major hypothesis is that LID result from L-DOPA-induced aberrant plasticity in the striatum due to modifications of the transcriptional program. Using a microarray-based approach, we identified Narp as a putative candidate implicated in LID induction. Thus, we investigated Narp involvement in LID by examining abnormal involuntary movements (AIM) development in Narp genetically-ablated mice or upon intrastriatal injection of a dominant negative form of Narp. Interestingly, the total AIM score was greatly reduced in these two models of impaired Narp expression. Hence, my results highlight Narp as an important actor in LID development. Then, I further examined Narp regulatory mechanisms in the striatum and I demonstrated that dopamine stimulation leads to increased Narp expression both at the transcriptional level and at the protein level through its accumulation within the synaptic compartment. These findings advance knowledge about mechanisms underlying dyskinesia with the hope of delaying their appearance in patients.

La lévodopa est actuellement la thérapie de référence pour les patients atteints de la maladie de Parkinson et permet le rétablissement artificiel des niveaux de dopamine. Cependant, ce traitement s'accompagne de mouvements involontaires anormaux invalidants, ou dyskinésies. Au cours de ma thèse, j'ai étudié l'implication des cellules de Purkinje de la région cérébelleuses crus II, via les voies cérébello-thalamo-cortico-striatale et cérébello-thalamo-striatale, dans l'émergence et le traitement des mouvements involontaires anormaux oro-linguaux induits par la lévodopa dans un modèle murin de la maladie de Parkinson. Mes résultats suggèrent que la stimulation chronique et spécifique des cellules de Purkinje par une technique d'optogénétique semble à même de corriger et de prévenir le phénotype dyskinétique et ce en association avec une modulation de l'activité du noyau thalamique intralaminaire parafasciculaire, du cortex moteur primaire oral et du striatum dorsal. Les voies cérébello-thalamo-cortico-striatale et cérébello-thalamo-striatale semblent ainsi impliquées dans le traitement correctif et préventif des dyskinésies induites par la lévodopa dans la maladie de Parkinson. / Levodopa is currently the gold standard treatment for Parkinson’s disease patients and artificially restores dopamine levels. However, it induces debilitating abnormal involuntary movements, or dyskinesia. During my thesis, I assessed the involvement of the cerebellar crus II region Purkinje cells, via the cerebello-thalamo-cortico-striatal and cerebello-thalamo-striatal pathways, in the onset and the treatment of levodopa induced oro-lingual abnormal involuntary movements, in a mouse model of Parkinson’s disease. My results suggest that the chronic and specific stimulation of Purkinje cells, using optogenetics, seems able to correct and prevent the dyskinetic phenotype, together with modulations of the activity of the parafascicular intralaminar thalamic nucleus, primary oral motor cortex and dorsal striatum, thus arguing for the involvement of the cerebello-thalamo-cortico-striatal and the cerebello-thalamo-striatal pathways in the corrective and preventive treatment for levodopa induced dyskinesia in Parkinson’s disease.

Parkinson’s disease is a disorder mainly characterized by progressive degeneration of dopamine producing neurons in the substantia nigra of the midbrain. The most commonly used treatment strategy is to pharmacologically restore the lost function by the administration of the dopaminergic precursor L-DOPA. Another treatment strategy is to replace the degenerated neurons with immature fetal ventral mesencephalic tissue, or ultimately stem cell-derived tissue. Grafting trials have, however, revealed poor reinnervation capacity of the grafts, leaving much of the striata dopamine-denervated. An additional drawback is the upcoming of dyskinesia (involuntary movements), a phenomenon also observed during L-DOPA treatment of Parkinson’s disease patients. Attempts to characterize nerve fiber formation from dopamine neurons have demonstrated that the nerve fibers are formed in two morphologically diverse outgrowth patterns, one early outgrowth seen in the absence of astrocytes and one later appearing outgrowth seen in co-existence with astrocytes. The overall objective of this thesis has been to study the dopaminergic outgrowth including guidance of nerve fiber formation, and to look into the mechanisms of L-DOPA-induced dyskinesia. The first paper in this thesis characterizes the different outgrowth patterns described above and their relation to different glial cells. The study demonstrated the two different outgrowth patterns to be a general phenomenon, applying not only to dopamine neurons. Attempts of characterization revealed no difference of origin in terms of dopaminergic subpopulations, i.e. A9 or A10, between the outgrowth patterns. Furthermore, the “roller-drum” technique was found optimal for studying the dual outgrowth sequences. The second and the third paper also utilized the “roller-drum” technique in order to promote both patterns of neuronal fiber formation. The effects of glial cell line-derived neurotrophic factor (GDNF) on the formation of dopamine nerve fibers, was investigated. Cultures prepared from gdnf knockout mice revealed that dopaminergic neurons survive and form nerve fiber outgrowth in the absence of GDNF. The dopaminergic nerve fibers exhibited an outgrowth pattern consistent with that previous observed in rat. GDNF was found to exert effect on the glial-associated outgrowth whereas the non-glial-associated was not affected. Astrocytic proliferation was inhibited using cytosine β-D-arabinofuranoside, resulting in reduced glial-associated outgrowth. The non-glial-associated dopaminergic outgrowth was on the other hand promoted, and was retained over longer time in culture. Furthermore, the non-glial-associated nerve fibers were found to target the fetal frontal cortex. Different developmental stages were shown to promote and affect the outgrowths differently. Taken together, these data indicate and state the importance of astrocytes and growth factors for neuronal nerve fiber formation and guidance. It also stresses the importance of fetal donor age at the time for transplantation. The fourth and fifth studies focus on L-DOPA dynamics and utilize in vivo chronoamperometry. In study four, 6-OHDA dopamine-depleted rats were exposed to chronic L-DOPA treatment and then rated as dyskinetic or non-dyskinetic. The electrochemical recordings demonstrated reduced KCl-evoked release in the intact striatum after chronic L-DOPA treatment. Time for maximal dopamine concentration after L-DOPA administration was found to be shorter in dyskinetic animals than in non-dyskinetic animals. The serotonergic nerve fiber content in the striatum was evaluated and brains from dyskinetic animals were found to exhibit significantly higher nerve fiber density compared to non-dyskinetic animals. Furthermore, the mechanisms behind the conversion of L-DOPA to dopamine in 6-OHDA dopamine-depleted rats were studied. Local administration of L-DOPA in the striatum increased the KCl-evoked dopamine release in the intact striatum. Acute application of L-DOPA resulted sometimes in a rapid conversion to dopamine, probably without vesicle packaging. This type of direct conversion is presumably occurring in non-neuronal tissue. Furthermore, KCl-evoked dopamine releases were present upon local application of L-DOPA in the dopamine-depleted striatum, suggesting that the conversion to dopamine took place elsewhere, than in dopaminergic nerve fibers. In conclusion, these studies state the importance of astrocytes for neuronal nerve fiber formation and elucidate the complexity of L-DOPA conversion in the brain.

Deep brain stimulation (DBS) has emerged as a potential intervention for treatment-resistant tardive dyskinesia (TD). Despite promising case reports, no consensus exists regarding optimal stimulation parameters, neuroanatomical target for DBS in TD, or mechanisms underlying its anti-dyskinetic effects. We used vacuous chewing movements (VCMs) in rats treated chronically with haloperidol (HAL) as a TD model to address some of these issues. We show that acute DBS applied to the subthalamic nucleus (STN) or the entopeduncular nucleus (EPN) suppresses VCMs without affecting locomotor activity. Using immediate early gene mapping with zif268 as an index of neuronal activity, we found that STN-DBS induced decreases in activity throughout the basal ganglia, whereas EPN-DBS increased activity in projection regions. While chemical inactivation of the STN or EPN with the GABAA agonist muscimol also suppressed VCMs, muscimol infusion did not mimic the changes in neuronal activity induced by DBS, suggesting that DBS is not equivalent to functional inactivation. We next examined the contribution of serotonin (5-HT) and dopamine (DA) to the anti-dyskinetic effects of DBS. Decreasing 5-HT transmission pharmacologically or with serotonergic lesions decreased VCMs. Using microdialysis and zif268 mapping, we determined that STN- but not EPN-DBS decreased 5-HT release and activity of raphe neurons. However, when the decrease in 5-HT induced by STN-DBS was prevented by pre-treating rats with fluoxetine or fenfluramine, we found that decreasing 5-HT is not necessary for the anti-dyskinetic effects of DBS. STN-DBS transiently increased striatal DA release in intact rats only, whereas EPN-DBS had no effect on DA release. Moreover, pharmacologically elevating DA levels did not suppress VCMs. Together these findings lead us to conclude that increased DA release does not contribute to the anti-dyskinetic effects of DBS. Finally, we compared depressive- and anxiety-like behaviours induced by chronic DBS of the EPN and STN, since adverse psychiatric effects of DBS have become a significant clinical concern. STN-DBS but not EPN-DBS induced depressive-like behaviour in a learned helplessness task. We established that the chronic HAL VCM model preparation may be used to explore mechanisms underlying anti-dyskinetic and psychiatric effects of DBS, and provided the first investigations into these mechanisms.