Les ganglions de la base, un ensemble de noyaux sous-corticaux interconnectés, sont impliqués dans l'élaboration, le contrôle et la mémorisation de comportements cognitivo-moteurs. L'une des principales structures de sortie de ce réseau, la substance noire réticulée (SNr), intègre les différentes informations neuronales puis les transmet au cortex via un relais thalamique. Cependant, cette transmission nécessite une régulation fine de l'activité neuronale de la SNr car celle-ci exerce une inhibition constante de ces structures cibles en raison de son activité GABAergique spontanée. Parmi les acteurs de cette régulation, le glutamate et le GABA sont à l'origine d'un équilibre fin entre excitation et inhibition des neurones nigraux. De nombreuses études se sont intéressées aux mécanismes de régulation de l'activité neuronale de la SNr mais, paradoxalement, aucune ne s'est intéressée au rôle des astrocytes. L'objet de ce travail de thèse a donc été d'étudier les relations entre neurones et astrocytes au sein de la SNr, afin de définir une potentielle implication des astrocytes dans la régulation de l'activité neuronale de cette structure. Nous avons étudié les excitabilités calciques des astrocytes et électriques des neurones grâce aux techniques d'imagerie calcique et de patch-clamp, dans un modèle de tranche parasagittale de cerveau de rat préservant les connexions subthalamo-nigrales et pallido-nigrales. Nous avons ainsi montré que les astrocytes nigraux possèdent une activité calcique spontanée, à la fois autonome et dépendante des libérations toniques de glutamate et de GABA. D'autre part, nous avons mis en évidence que l'activité de ces cellules est modulée par la stimulation à haute fréquence du noyau sous-thalamique. Nous avons montré qu'en retour, ces activités calciques spontanées astrocytaires sont impliquées dans la régulation de la fréquence de décharge des neurones de la SNr. Enfin, nous avons mis en évidence que la recapture astrocytaire du glutamate, et probablement du GABA, intervient également dans la régulation de l'activité de décharge neuronale nigrale. En conclusion, ce travail met en évidence une communication bidirectionnelle entre les neurones et les astrocytes de la SNr. Cette communication semble jouer un rôle important dans la régulation de l'activité de cette structure. / Basal ganglia, a set of interconnected nuclei, are implicated in the elaboration, control and memorization of cognitive-motor behaviors. One of the main output structure of this network, the substantia nigra pars reticulata (SNr), integrates and conveys neuronal information to cortical areas via a thalamic relay. However, this transmission requires an accurate regulation of the SNr neuronal activity since this structure inhibits its targets due to its spontaneous GABAergic activity. Among the different actors of this regulation, glutamate and GABA provide a tight balance between excitation and inhibition of the SNr neuronal activity. Several studies have explored the different mechanisms involved in this regulation but paradoxically, none concerned the astrocyte functions. In this work, our aim was to study astrocyte-neuron relations in order to define a potential astrocyte implication in the regulation of the neuronal activity in the SNr. We studied calcium and electrical activities of astrocytes and neurons using calcium imaging and patch-clamp techniques in parasagittal rat brain slices, conserving subthalamo- and pallido-nigral projections. We showed that astrocytes in the SNr displayed spontaneous calcium activities, both dependent and independent of glutamatergic and GABAergic tonic neuronal transmissions. Moreover, we showed that astrocytes calcium activities were regulated by the subthalamic nucleus high frequency stimulation. Our results revealed that, in turn, astrocytes calcium activities were involved in the regulation of the neuronal firing rate. Finally, we showed that astrocyte glutamatergic, and maybe GABAergic, reuptake was involved in the regulation of the neuronal firing rate. To conclude, this study revealed a bidirectional communication between astrocytes and neurons in the SNr. This communication seems to be important in the regulation of the activity in this structure.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A hipóxia isquemia (HI) pré-natal é uma das principais causas de mortalidade e doenças neurológicas crônicas em neonatos, que podem apresentar déficits remanentes como: retardamento, paralisia cerebral, dificuldade de aprendizado ou epilepsia. Estes prejuízos, provavelmente, estão relacionados com o atraso no desenvolvimento neural, astrogliose e com a perda de neurônios e oligodendrócitos. Déficits funcionais e cognitivos estão associados à degeneração de vias dopaminérgicas e de estruturas hipocampais. A enzima tirosina hidroxilase (TH) é a enzima limitante na síntese de dopamina e seus níveis são alterados em eventos de HI. O óxido nítrico (NO) é um gás difusível que atua modulando diferentes sistemas, participando de eventos como plasticidade sináptica e neuromodulação no sistema nervoso central e é produzido em grandes quantidades em eventos de injúria e inflamação, como é o caso da HI. O presente estudo teve por objetivos avaliar, utilizando o modelo criado por Robinson e colaboradores em 2005, os efeitos da HI sobre o comportamento motor e avaliar o desenvolvimento de estruturas encefálicas relacionadas a este comportamento como a substância negra (SN) e o complexo hipocampal. A HI foi induzida a partir do clampeamento das artérias uterinas da rata grávida, por 45 minutos no décimo oitavo dia de gestação (grupo HI). Em um grupo de fêmeas a cirurgia foi realizada, mas não houve clampeamento das artérias (grupo SHAM). A avaliação do comportamento motor foi realizada com os testes ROTAROD e de campo aberto em animais de 45 dias. Os encéfalos foram processados histologicamente nas idades de P9, P16, P23 e P90, sendo então realizada imunohistoquímica para TH e histoquímica para NADPH diaforase (NADPH-d), para avaliação do NO. Nossos resultados demonstraram redução da imunorreatividade para a TH em corpos celulares na SN aos 16 dias no grupo HI e aumento na imunorreatividade das fibras na parte reticulada aos 23 dias, com a presença de corpos celulares imunorreativos nesta região no grupo HI. Demonstramos também aumento do número de células marcadas para NADPH-d no giro dentado nos animais HI, nas idades analisadas, assim como aumento na intensidade de reação no corno de Ammon (CA1 e CA3) aos 9 dias no grupo HI, e posterior redução nesta marcação aos 23 e 90dias neste mesmo grupo. Nos testes comportamentais, observamos diminuição da atividade motora no grupo HI com uma melhora do desempenho ao longo dos testes no ROTAROD, sem entretanto atingir o mesmo nível do grupo SHAM. Os animais HI não apresentaram maior nível de ansiedade em relação ao grupo SHAM, descartando a hipótese das alterações observadas nos testes de motricidade estarem relacionadas a fatores ansiogênicos. O modelo de clampeamento das artérias uterinas da fêmea se mostrou uma ferramenta importante no estudo das alterações decorrentes do evento de HI pré-natal, por produzir diversos resultados que são similares aos ocorridos em neonatos que passam por este evento. / Perinatal hypoxia-ischemia (HI) is one of the major causes of mortality and chronic neurological diseases in newborns that can show permanent effects such as mental retardation, cerebral palsy, learning difficulty and epilepsy. It is probable that these impairs may be related to a delay in the neural development, astrogliosis and to the death of neurons and oligodendrocytes. Cognitive and functional deficits are related to degeneration of dopaminergic pathways and hippocampus. The enzyme tyrosine hydroxylase (TH) is a limiting step in the dopamine synthesis and its levels are impaired in HI insults. Nitric oxide (NO) is a diffusible gas that acts by modulating different systems and participates in several phenomena such as synaptic plasticity and neuromodulation in the central nervous system and is produced in higher levels in events of injury and inflamation as in the case of HI. This study aimed to evaluate the effects of HI on the motor behavior and to evaluate the development of brain structures related to this behavior as the substantia nigra (SN) and the hippocampal complex, using the model developed by Robinson and colleagues in 2005. HI was induced by clamping the uterine arteries of pregnant rats, for 45 minutes, on the eighteenth day of gestation (group HI). In a group of females, the surgery was performed, but no clamping of the arteries (group SHAM) was made. Assessment of motor behavior was performed with the ROTAROD test and open field test in animals of 45 days (P45) of age. The brains were processed histologically at ages P9, P16, P23 and P90, and then submitted to immunohistochemistry for TH and NADPH diaphorase (NADPH-d) histochemistry for evaluation of NOS. Our results demonstrated an apparent decrease in TH immunoreactivity in cell bodies in the SN at P16 in the HI group and an increase in immunoreactivity of the fibers in the SN pars reticulata at P23 with the presence of TH immunoreactive cell bodies at this same region in the HI group. We also showed an increase in the number of NADPH-d stained cells in the dentate gyrus in the HI group, at all ages, as also an increase in the intensity of staining in the Ammons horn (CA1 and CA3) at P9 in the HI group and, after that, a decrease in this staining at P23 and P90 in this same group. In the behavioral tests we observed a decrease in the motor activity in the HI group with a partial recovery all over the several sessions in the ROTAROD test, however this group did not reach the same performance as the SHAM group. HI animals did not show a higher level of anxiety when compared to SHAM animals, ruling out the hypothesis that anxiogenic factors may be impairing the results in the motor behavior tests. Our results showed that the model of uterine arteries clamping could be an important tool in the study of the effects of perinatal HI, by producing several consequences that are very similar to the effects observed in newborn children who suffered an HI event.

O Colículo Superior (SC) é conhecido por apresentar diversas funções que modulam a caça predatória. Neste estudo, investigamos as funções do SC em ratos expostos a caça de insetos. Primeiramente, verificamos que o comportamento predatório induz uma distinta ativação da porção lateral do SC (SCl). Para entender as potenciais funções dessa região colicular, foi analisado o comportamento predatório antes e após lesões bilaterais iontoforéticas por NMDA do SCl. Animais com SCl lesados ficaram menos motivados a perseguirem as baratas, falharam para se orientarem na direção do movimento das presas e quando tentaram capturar as presas, eles apresentaram sérios déficits para capturá-las e segurá-las eficientemente. Por outro lado, animais com lesões da porção medial do SC (SCm) apresentaram apenas um aumento da latência para iniciar a caça, enquanto os outros parâmetros não diferiram significantemente dos animais intactos. Posteriormente, examinamos as conexões eferentes do SCl e do SCm usando como traçador anterógrado a leucoaglutinina do Phaseolus vulgaris. Notamos projeções densas do SCl para a região rostral da coluna lateral da matéria cinzenta periaquedutal (PAGl), um setor criticamente envolvido no controle dos aspectos motivacionais relacionados aos comportamentos de caça predatória e forrageamento. Além disso, o SCl se projeta densamente para o tálamo dorsal, especificamente para os núcleos ventral lateral, central medial e paracentral do tálamo, os quais sabemos que se projetam para setores estriatais ou para áreas motoras corticais, que provavelmente estão envolvidas no ajuste da ação motora durante a captura das presas. O SCm, por sua vez, aferenta densamente a coluna dorsolateral da PAG, núcleo cuneiforme, e núcleos reticulares mesencefálico e pontino, que são setores envolvidos na elaboração de respostas defensivas, além disso, o SCm se projeta esparsamente para os núcleos posterior lateral e suprageniculado do complexo geniculado medial / The superior colliculus is classically known to present a number of functions that fit hunting behavior. In the present study, we investigate the potential roles of the superior colliculus in rats displaying insect hunting. First, we have found that predatory hunting induces a distinct activation of the lateral region of the intermediate layer of the superior colliculus (SCl). To understand the potential roles of this collicular region, we analyzed the hunting performance before and after iontophoretic NMDA lesions bilaterally placed into the SCl. Animals with SCl lesions were clearly less motivated to pursue the roaches, failed to orient themselves toward the moving prey, and whenever the SCl-lesioned rats tried to catch the roaches, they presented serious deficits to capture and hold them efficiently. Next, we examined the SCl efferents connections using Phaseolus vulgaris leucoagglutinin as an anterograde tracer. Of particular relevance, we noted that the SCl projects to the rostral lateral periaqueductal gray, a site critically involved in controlling motivational drive to chase prey and forage. In addition, the SCl also present particularly strong projections to the dorsal thalamus, aimed at the ventral lateral, ventral medial, central medial and paracentral nuclei of thalamus, all of which known to project either to striatal sites or to cortical motor areas, likely to be involved in adjusting the motor action during prey capture. Therefore, the SCl, which seems to present cells responding to prey displacement in the temporal field, presents important arms to the periaqueductal gray and dorsal thalamic sites, influencing, respectively, the motivational drive and the motor skills to hunt

INTRODUÇÃO: O diagnóstico da doença de Parkinson (DP) pode ser um desafio, principalmente nas fases precoces da doença. O diagnóstico acurado desta condição requer mais que a avaliação clínica isolada. A Tomografia computadorizada do crânio de fóton único (SPECT) e a ultrassonografia transcraniana (USTC) podem ser úteis na diferenciação entre a DP e as síndromes parkinsonianas atípicas ou entre a DP e o tremor essencial. O presente estudo objetivou investigar a acurácia da USTC combinada com o teste de olfação Sniffin\' Sticks (SST-16) para diferenciar pacientes com DP de controles saudáveis e comparar com a acurácia do SPECT com 99mTc- TRODAT-1 (TRODAT). MÉTODOS: Trata-se de um estudo transversal que incluiu pacientes com DP segundo critérios do United Kingdom Parkinson\'s disease Society e um grupo controle de indivíduos saudáveis pareados para idade e gênero. Os pacientes foram examinados por um especialista em distúrbios do movimento e submetidos a SPECT encefálico com TRODAT, USTC e SST-16. Curvas Receiver Operating Characteristic (ROC) foram obtidas para definir os pontos de corte dos métodos avaliados para detecção de DP. RESULTADOS: Vinte indivíduos com DP (13 homens e 7 mulheres) e 9 participantes saudáveis foram admitidos no estudo. A idade mediana de início dos sintomas foi de 56,5 anos e a mediana do tempo de duração da doença foi de 5 anos. Maior área de ecogênica da substância negra (SN) foi observada no grupo com DP (p=0,013). Área ecogênica da SN de 0,22 cm2 foi definida pela curva ROC para detecção de DP, com acurácia de 79%. O ponto de corte do potencial de ligação do TRODAT no striatum foi 0,90, com acurácia de 99% para o diagnóstico de DP. Escore do SST-16 maior ou igual a 10 pontos foi o ponto de corte para detecção de DP, com acurácia de 85,8%. A combinação da USTC com teste da olfação levou à acurácia de 95% para detecção de DP. CONCLUSÃO: A combinação da USTC com SST-16 eleva a capacidade de ! detecção da DP. A acurácia da USTC combinada ao SST-16 para identificar pacientes com DP idiopática aproximou-se da acurácia do SPECT com TRODAT / INTRODUCTION: Diagnosing Parkinson\'s disease (PD) can be challenging, especially in the early stages of the disease. An accurate diagnosis requires more than clinical findings alone. Brain single-photon emission computed tomography (SPECT) and transcranial sonography (TCS) are helpful for diagnosing PD and differentiating it from atypical parkinsonian syndromes as well as essential tremor. This study aimed to investigate the accuracy of TCS combined with the Sniffin\' sticks olfactory test (SST-16) for differentiation between idiopathic PD patients and healthy controls compared to that of 99mTc-TRODAT-1 SPECT (TRODAT). METHODS: A cross-sectional study included PD patients diagnosed in accordance with United Kingdom PD Society Brain Bank criteria and a control group of age and sex-matched healthy subjects. All patients were examined by a movement disorder specialist and underwent brain SPECT using TRODAT, TCS examination and SST-16 test. Receiver Operating Characteristic (ROC) curves were used to calculate cut-off points for TCS, Striatal TRODAT binding potentials and SST-16. The area under the ROC curve determined the accuracy of the method. RESULTS: Twenty patients with PD (13 males and 7 females) and nine healthy subjects were included. Median age of PD onset was 56.5 years with median disease duration of 5 years. A larger substantia nigra (SN) echogenic area was observed in the PD group (p=0.013). SN echogenic area cut-off point of 0.22 cm2 was obtained from a ROC curve for PD diagnosis. Considering this cut-off point, TCS accuracy was estimated at 79.2% for PD diagnosis. The cut-off value of 0.90 for striatal TRODAT binding was associated with 99% accuracy for the diagnosis of PD. SST-16 values equal or greater than 10 points showed a 85.8% accuracy for PD diagnosis. Combination of both SST-16 and TCS improved the accuracy to 95% for PD diagnosis. CONCLUSION: Combined assessment of SST-16 and TCS are reliable and highly accurate for distinguishing PD patients from healthy controls. The accuracy of TCS combined with SST-16 for differentiation between idiopathic PD patients and healthy controls is similar to that of SPECT TRODAT

The Basal Ganglia consist of a number of different nuclei that form a diverse circuitry of GABAergic, dopaminergic and glutamatergic neurons. This complex network is further organized in subcircuits that govern limbic and motor functions in humans and other vertebrates. Due to the interconnection of the individual structures, dysfunction in one area or cell population can affect the entire network, leading to synaptic and molecular alterations in the circuitry as a whole. The studies in this doctoral thesis aimed at characterizing restricted subpopulations of neurons in the Basal Ganglia circuitry and their importance in the wider function of the network. To this end, we identified subpopulations of neurons in the subthalamic nucleus (STN), substantia nigra (SN) and ventral tegmental area (VTA), characterized their molecular profile and investigated their physiological role in the circuitry. Within the mouse STN, reduction of glutamatergic neurotransmission in a subpopulation expressing Paired-like homeodomain transcription factor 2 (Pitx2) led to structural alterations in the nucleus as well as biochemical alterations of the dopaminergic system in the Nucleus accumbens (NAc) and changes in reward-related behavior. In the ventral midbrain, we identified and characterized novel marker genes selective to the VTA or SN. Of these, transient receptor potential cation channel subfamily V member 1 (TrpV1) marks a population of mainly glutamatergic neurons in the VTA which project to the NAc, while gastrin releasing peptide (Grp) is expressed in a population of dopaminergic neurons neuroprotected in Parkinson's disease. Furthermore, we discovered that disruption of glutamatergic co-release of dopaminergic neurons expressing dopamine transporter (DAT), diminishes fast EPSCs and glutamate release but does not affect the acquisition of reward-related behavioral tasks. To selectively quantify glutamate release from specific subpopulations, we devised a technique combining glutamate-amperometry and optogenetics. This was used to measure glutamate released from Pitx2-expressing synaptic terminals in the Globus pallidus as well as DAT- or TrpV1-expressing terminals in the NAc. In summary, this doctoral thesis has furthered understanding of the function and importance of specific subpopulations within the Basal Ganglia circuitry and provides a novel means to investigate glutamate in the intact rodent brain within clearly defined, restricted cell populations.

The role of metal accumulation in the pathophysiology of neurodegenerative diseases has been a hot topic in recent years due to the possibility of its treatment by chelating agents. Although the mechanisms of neurodegeneration are well known, the role of metal accumulation is still unclear. The main limitation are unsatisfactory methods for in vivo metal imaging; the most widely used technique is magnetic resonance imaging (MRI). Our aim was to assess the possibility of using transcranial sonography (TCS) in differential diagnosis of neurodegenerative diseases and to further explore the underlying factors of echogenicity. In the first study, using TCS fusion with MRI, we focused on location verification of the commonly assessed structures (substantia nigra and nucleus lentiformis) and exclusion of possible focal structural changes affecting the echogenicity in WD and PD patients. Moreover, obtained MRI were used for semi-quantitative comparison with TCS images. Although TCS has been confirmed to be highly beneficial in differential diagnosis of Wilson's disease and it should be recommended as a screening method for extrapyramidal patients with atypical course of the disease, the direct relationship between TCS and metal deposits could not be proven. The obtained results from the ultrasound fusion...

It was generally accepted that dopamine receptors D1 (D1R)- and D2 (D2R)-expressing neurons are homogeneously and randomly distributed throughout the striatum. However, in reporter transgenic mice, the specific subregions of the caudal lateral striatum have been reported: the D1R-poor zone, in which D2R-expressing neurons are predominant, and the D2R-poor zone, in which D1R-expressing neurons are predominant. The present study demonstrated the presence of these distinct subregions not only in rodents but also in marmosets using endogenous dopamine receptors. We also showed that direct pathway medium spiny neurons in these distinct subregions preferentially project to parvalbumin-positive GABAergic neurons in the dorsal part of the substantia nigra pars lateralis. / 博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

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Parkinson’s disease is an age-related neurodegenerative disorder. The major symptoms of Parkinson’s disease are closely linked to the pathology of the disease. The main pathology of Parkinson’s disease consists of the degeneration of neurons of the substantia nigra pars compacta (SNpc), which leads to reduced amounts of dopamine in the brain. One of the treatment strategies in Parkinson’s disease is to conserve dopamine by inhibiting the enzymes responsible for its catabolism. The monoamine oxidase (MAO) B isoform catalyses the oxidation of dopamine in the central nervous system and is therefore an important target for Parkinson’s disease treatment. Inhibition of MAO-B provides symptomatic relief for Parkinson’s disease patients by increasing endogenous dopamine levels as well as enhancing the levels of dopamine after administration of levodopa (L-dopa), the metabolic precursor of dopamine. Recent studies have shown that phthalide can be used as a scaffold for the design of reversible MAO inhibitors. Although phthalide is a weak MAO-B inhibitor, substitution on the C5 position of phthalide yields highly potent reversible MAO-B inhibitors. In the present study, sesamol and benzodioxane were used as scaffolds for the design of MAO inhibitors. The structures of sesamol and benzodioxane closely resemble that of phthalide, which suggests that these moieties may be useful for the design of MAO inhibitors. This study may be viewed as an exploratory study to discover new scaffolds for MAO inhibition. Since substitution at C5 of phthalide with a benzyloxy side chain yielded particularly potent MAO inhibitors, the sesamol and benzodioxane derivatives possessed the benzyloxy substituent in the analogous positions to C5 of phthalide. These were the C5 and C6 positions of sesamol and benzodioxane, respectively. The sesamol and benzodioxane derivatives were synthesised by reacting sesamol and 6- hydroxy-1,4-benzodioxane, respectively, with an appropriate alkyl bromide in the presence of potassium carbonate (K2CO3) in N,N-dimethylformamide (DMF). 6-Hydroxy-1,4- benzodioxane, in turn, was synthesised from 1,4-benzodioxan-6-carboxaldehyde. The structures of the compounds were verified with nuclear magnetic resonance (NMR) and mass spectrometry (MS) analyses, while the purities were estimated by high-pressure liquid chromatography (HPLC). Sixteen sesamol and benzodioxane derivatives were synthesised. To determine the inhibition potencies of the synthesised compounds the recombinant human MAO-A and MAO-B enzymes were used. The inhibition potencies were expressed as the corresponding IC50 values. The results showed that the sesamol and benzodioxane derivatives are highly potent and selective inhibitors of MAO-B and to a lesser extent MAOA. The most potent MAO-B inhibitor was 6-(3-bromobenzyloxy)-1,4-benzodioxane with an IC50 value of 0.045 μM. All compounds examined displayed selectivity for the MAO-B isoform over MAO-A. Generally the benzodioxane derivatives were found to be more potent inhibitors of human MAO-A and MAO-B than the sesamol derivatives. The reversibility and mode of MAO-B inhibition of a representative derivative, 6-(3- bromobenzyloxy)-1,4-benzodioxane, was examined by measuring the degree to which the enzyme activity recovers after dialysis of enzyme-inhibitor complexes, while Lineweaver- Burk plots were constructed to determine whether the mode of inhibition is competitive. Since MAO-B activity is completely recovered after dialysis of enzyme-inhibitor mixtures, it was concluded that 6-(3-bromobenzyloxy)-1,4-benzodioxane binds reversibly to the MAO-B enzyme. The Lineweaver-Burk plots constructed were linear and intersected on the y-axis. Therefore it may be concluded that 6-(3-bromobenzyloxy)-1,4-benzodioxane is a competitive MAO-B inhibitor. To conclude, the C6-substituted benzodioxane derivatives are potent, selective, reversible and competitive inhibitors of human MAO-B. These compounds are therefore promising leads for the future development of therapy for Parkinson’s disease. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2015