Atualmente observa-se que a proporção de pessoas com 60 anos ou mais está crescendo mais rápido do que a de outras faixas etárias. Um dos resultados desta transição epidemiológica é o aumento das doenças cujo fator de risco é o envelhecimento, entre elas, a doença de Parkinson. Embora muitas regiões exibam os sinais neuropatológicos da doença de Parkinson, a degeneração dos neurônios, contendo neuromelanina, da substância negra é considerada como sendo uma característica importante, representando o critério cardinal para o diagnóstico. No entanto, ainda não está claro por que certas regiões do cérebro, como a substância negra, são vulneráveis em algumas doenças neurodegenerativas e alguns neurônios vizinhos, às vezes morfologicamente indistinguíveis, permanecem preservados. Análises moleculares de populações de neurônios podem conduzir a uma melhor compreensão sobre a fisiologia dos mesmos, bem como os mecanismos envolvidos nos processos de doença. Na era pós genômica, realizar análises proteômicas são de grande interesse científico, pois permitem avanços no conhecimento dos processos biológicos. A técnica de microdissecção e captura a laser tem sido uma ferramenta importante e cada vez mais utilizada para aquisição de populações puras de células a partir de secções histológicas, evitando que áreas não pertencentes ao tecido alvo sejam dissecadas. A união destes métodos pode contribuir de maneira relevante para o entendimento fisiopatológico dos neurônios contendo neuromelanina da substância negra. No entanto, para que a microdissecção e captura a laser e as análises proteômicas sejam eficazes, é imprescindível a aplicação de um protocolo bem estruturado. Dentro desse contexto, o presente trabalho tem como objetivo criar um protocolo de microdissecção a laser de neurônios contendo neuromelanina em indivíduos cognitivamente normais, para subsequente análise proteômica. Os casos utilizados neste estudo são provenientes do Banco de Encéfalos Humanos do Grupo de Estudos em Envelhecimento Cerebral. Para o desenvolvimento da nossa proposta, contamos com a colaboração do Centro de Proteômica Médica da Universidade de Bochum, Alemanha. O protocolo foi desenvolvido baseado em outros previamente descritos na literatura e otimizado de acordo com objetivos pretendidos. Analisamos o plano anatômico de amostragem do tecido, o método de congelamento, a espessura do corte para a microdissecção, a solução utilizada para a coleta do tecido durante a microdissecção e o método de digestão proteolítica para posteriores análises proteômicas. Através de ensaios comparativos, alcançamos os resultados desejados e os mesmos foram validados através de análises por espectrometria de massas. Consequentemente, também fomos capazes de reconhecer fatores técnicos que possivelmente impossibilitariam um efetivo estudo do proteoma / Currently the worldwide proportion of people aged 60 years and over is growing faster than any other age group. This strikingly epidemiological transition results in an increase of aging related diseases, including Parkinson\'s disease (PD). Although many brain areas exhibit the neuropathological signs of Parkinson\'s disease, the degeneration of neuromelanin containing cells in the substantia nigra is considered a hallmark feature, representing cardinal diagnostic criteria for PD. However, why certain brain regions -- such as the substantia nigra -- are vulnerable in some neurodegenerative diseases, while some neighboring morphologically indistinguishable neurons remain preserved, is still unclear. Molecular analysis of specific neuronal populations can lead us to a better understanding about the physiological role played by these neurons and mechanisms involved in disease\'s processes. In a post-genomic era, proteomic analyses are of great scientific interest since they allow a better understanding of the biological processes. The laser capture microdissection technique has also became an important tool in biological research, being increasingly used for acquisition of pure populations of cells from histological sections, preventing the dissection of areas outside the target tissue. The combination of these methods can significantly contribute to understand the pathophysiological role of the containing neuromelanin neurons of the substantia nigra. However, for an effective application of both techniques, laser capture microdissection and proteomic analysis, it is essential the application of an efficient protocol. In this context, this study aims to establish a protocol for laser microdissection of containing neuromelanin neurons in cognitively normal individuals for subsequent proteomic analyses. We selected cases from the Brain Bank of the Brazilian Aging Brain Study. A collaboration with the Medical Proteome Center, University of Bochum, Germany took part during the development of our proposal. Our protocol was developed based on previous published protocols and optimized according the intended aims. We analyzed anatomical planes for neuronal collection, freezing methods, thickness of tissue for microdissection sections, solution for tissue collection during laser microdissection and the proteolytic digestion methods. Through our comparative tests, we have achieved the desired results and validated them by mass spectrometry analyses. Consequently, we were also able to exclude technical factors that could possibly preclude one effective proteome analysis

Midbrain dopamine (DA) neurons play a key role in a wide range of behaviours, from motor control, motivation, reward and reinforcement learning. Disorders of midbrain dopaminergic signaling is involved in a variety of nervous system disorders including Parkinson’s disease, schizophrenia and drug addiction. Understanding the basis of how dopaminergic neuronal activity in the substantia nigra pars compacta (SNc) governs movements, requires a deep appreciation of how afferent inputs of various neurotransmitter systems create a neuronal circuit that precisely modulates DA neuronal excitability. Two brainstem cholinergic neuclei, the laterodorsal tegmental nucleus (LDT) and the pedunculopontine tegmental nucleus (PPT), have major cholinergic projections to the SNc, despite the fact that the precise mechanisms of cholinergic modulation of DA neuronal activity mediated by nAChRs remain unclear. To dissect out the modulatory roles of the cholinergic system in regulating DAergic neuronal activity in the SNc and locomotion, we employed optogenetics along with electrophysiological and behavioural approaches. My results from whole-cell recordings from lateral and medial SNc DA neurons revealed that lateral DA neurons received predominantly excitatory nAChR mediated cholinergic neurotransmission (monosynaptic nicotinic or disynaptic glutamatergic responses) resulting in greater excitability of DA neurons both at 5 and 15 Hz blue LED light stimulation of cholinergic terminals. However, medial SNc DA neurons received predominantly biphasic current responses that were both inhibitory GABAergic and excitatory nAChR mediated cholinergic neurotransmission. This led to a net inhibition of action potential firing of DA neurons at 5 Hz blue LED light stimulation of cholinergic terminals, while at 15 Hz stimulation there was an initial inhibition followed by a significant increase of the baseline action potential firing frequency. Furthermore, in vivo optogenetic experiments showed that activation of the cholinergic system in the medial SNc resulted in decreased locomotion, while for the lateral SNc led to increased locomotion. Together our findings provide new insights into the role of the cholinergic system in modulating DA neurons in the SNc. The cholinergic inputs to different subregions of the SNc may regulate the excitability of the DA neurons differentially within a tight range from excitation to inhibition which may translate into different kinds of locomotor behaviour. / Graduate

Zusammenfassung der Arbeit Dissertation zur Erlangung des akademischen Grades Dr. med. Titel: Quantifizierung und zelluläre Lokalisation von Eisen in der Substantia nigra bei Patienten mit Parkinsonscher Erkrankung und gesunden Kontrollen eingereicht von Isabel Weigelt angefertigt an der Medizinischen Fakultät der Universität Leipzig Paul Flechsig Institut für Hirnforschung, Abteilung Molekulare und Zelluläre Mechanismen der Neurodegeneration betreut von PD Dr. Dr. Markus Morawski Prof. Dr. Thomas Arendt November 2019 Das Spurenelement Eisen ist aufgrund seiner Schlüsselrolle in der Neurotransmittersynthese oder der Myelinisierung essentiell für Neuronen. Sein ubiquitäres Vorkommen erfordert eine Regulierung der Eisenhomöostase in engen Grenzen. Hohe Konzentrationen an freiem Eisen sind jedoch in der Lage, über den Weg der Fenton-Reaktion das oxidative Stresslevel der Zelle drastisch zu erhöhen und die Bildung vermehrter reaktiver Sauerstoffspezies zu triggern; betroffene Zellen degenerieren. Eine Erhöhung der Eisenkonzentration wird bei zahlreichen neurodegenerativen Erkrankungen beschrieben, so auch bei Parkinsonscher Erkrankung (PD). Die aktuelle Datenlage zu quantitativen Eisenkonzentrationen in den verschiedenen Zelltypen innerhalb der menschlichen SN ist jedoch rar. Elementanalysen, vor allem von Eisen, bieten eine ideale Möglichkeit, um die Rolle des Eisens sowie der Eisenspeicherformen im Rahmen der Pathogenesemechanismen weiter zu entschlüsseln und so besser beurteilen zu können. Ziel der Arbeit war es, neben der Beschreibung typischer Veränderungen der Zellen bei PD die Verteilung der Haupteisenspeicherformen Ferritin und Neuromelanin darzustellen sowie eine eventuelle Kolokalisation mit Gliazellarten aufzudecken. Quantitativ sollte eine Bestimmung des Gesamteiseins und der differentiellen ortsaufgelösten zellulären Eisenkonzentration erfolgen, um direkte Veränderungen in den Eisenkonzentrationen zwischen Kontrollen und PD identifizieren zu können. Hierfür erfolgte zu qualitativen Analysezwecken zunächst eine klassische sowie Fluoreszenz-immunhistochemische Darstellung zellspezifischer Marker (anti-HuC/D, -IBA-1, -GFAP, -CNPase, -OLig2, -MBP, -Ferritin, -HLA) nach Standardprotokoll mit einer Ni-DAB Verstärkung. Histochemische Färbungen nach Perls und Turnbull wurden zum semiquantitativen Eisennachweis durchgeführt. Als Methode zur ortsaufgelösten quantitativen Eisenbestimmung kam die Ionenstrahlmikroskopie (Partikel-induzierte Röntgen-Emission, PIXE) zur Anwendung. Die wichtigsten Ergebnisse sind nachfolgend dargestellt: Immunhistochemie und Histochemie • Typische zelluläre Veränderungen bei PD konnten in Übereinstimmung mit bestehender Literatur immunhistochemisch bestätigt werden. So findet sich der höchste Neuronenverlust im Nigrosom 1, die Gliazellen zeigen deutliche Aktivierungszeichen hinsichtlich ihrer Verteilung und Morphologie. • Eine Kolokalisation von Ferritin mit Oligodendrozyten konnte sowohl über klassische Immunhistochemie als auch über eine Fluoreszenz-Doppelmarkierung gezeigt werden. • Bei PD nimmt die Dichte von Ferritin-positiven Oligodendrozyten zu. • Als Zeichen für eine Aktivierung der Oligodendrozyten bei PD sind die erhöhte Oigodendrozytendichte und degenerative Veränderungen der Myelinfasern in Kombination mit einer erhöhten MBP-Immunreaktivität denkbar. • Bei PD sind semiquantitativ höhere Bestände nicht-chelatierten Fe3+ nachweisbar, das flächenmäßig dargestellte eisenreiche Gebiet der SN zeigt in > 50% der Fälle mit PD eine größere Ausdehnung (Färbung nach Perls). Das Nigrosom 1 ist bei PD nicht mehr anhand einer schwächeren Farbreaktion im Vergleich zur gesamten SN auszumachen. Ionenstrahlmikroskopie • Mit dem Ionenstrahlmikroskop wurden erstmals selektiv gefärbte Zellarten in der humanen SN bei PD und Kontrollen hinsichtlich ihrer zellulären Eisenkonzentration vergleichend quantifiziert. • Das Gesamteisen im Nigrosom 1 beträgt bei den Kontrollen ca. 2,49 mmol/l, bei PD ca. 3,00 mmol/l. • Neuronen zeigen bei PD eine um ca. 107 % erhöhte Eisenkonzentration (KO: 1,98 mmol/l). • Mikrogliazellen vergrößern im Rahmen ihrer Aktivierung bei PD ihre zellulären Eisenbestände um ca. 26 % (KO: 3,19 mmol/l). • Die zelluläre Eisenkonzentration der Astrozyten ist bei PD nicht signifikant verändert (KO: 4,31 mmol/l; PD: 4,11 mmol/l). • Oligodendroglia weisen in Abhängigkeit des verwendeten Zellmarkers unterschiedliche zelluläre Eisenkonzentrationen auf. Olig2-positive Zellen akkumulieren bei PD im Durchschnitt 250% mehr Eisen (KO: 2,76 mmol/l), CNPase-positive Oligodendrozyten ca. 64 % mehr Eisen (KO: 1,81). • Neuromelanin ist bei PD infolge der Neurodegeneration zunehmend extrazellulär zu finden und deutlich stärker mit Eisen beladen (KO: 8,73 mmol/l; PD: 14,21 mmol/l). • Ferritin-positive Oligodendrozyten besitzen unter den Gliazellen den höchsten zellulären Eisengehalt (6,01 mmol/l). Bei PD sinkt die zelluläre Eisenkonzentration jedoch auf ca. 51 %, was eine Entleerung der Ferritinspeicher mutmaßen lässt. Eine Erhöhung der Eisenkonzentration ist in nahezu allen untersuchten Zellarten zu beobachten. Eine vermutete Entleerung der Ferritinspeicher impliziert in Kombination mit der steigenden zellulären Eisenkombination letztlich eine Vergrößerung des LIP der Zellen in der SN bei PD. Diese Hypothese wird durch die bei PD entschieden intensiver ausfallende Perls-Färbung zum Nachweis nicht-chelatierten Fe3+ gestützt. Eisen kommt unumstritten eine nicht zu vernachlässigende Rolle bei neurodegenerativen Prozessen zu. Die Quantifizierung von Eisen in verschiedenen Zelltypen gibt Aufschluss über die Veränderung der Eisenkonzentrationen bei PD. Dennoch bleiben genaue Mechanismen, die zu einem Anstieg des Eisens führen, Gegenstand weiterer Forschungen.:1 Einleitung 1 1.1 Eisen 1 1.1.1 Überblick über den systemischen Eisenstoffwechsel 1 1.1.2 Eisenverteilung und Eisenstoffwechsel im Gehirn 2 1.1.3 Der labile Eisenpool 4 1.2 Parkinsonsche Erkrankung 5 1.2.1 Symptomatik und Pathogenese 5 1.2.2 Äthiopathogenese 6 1.2.3 Substantia nigra 7 1.2.3.1 Topografie und Gliederung des Kerngebietes 7 1.2.3.2 Zellverlust innerhalb der Substantia nigra bei Parkinsonscher Erkrankung 9 1.2.3.3 Einbindung der Substantia nigra in die Basalganglienschleife 11 1.2.4 Veränderungen der Gliazellen bei Parkinsonscher Erkrankung 11 1.2.4.1 Veränderungen der Mikroglia 11 1.2.4.2 Veränderungen der Astroglia 13 1.2.4.3 Veränderungen der Oligodendroglia und der Myelinfasern 14 1.2.5 Eisen – Spurenelement mit Schlüsselrolle in der Pathogenese des M. Parkinson? 15 1.3 Methodische Einleitung 18 1.3.1 Quantifizierungsmöglichkeiten von Eisen 18 2 Fragestellung und Zielsetzung 20 3 Material und Methoden 22 3.1 Material 22 3.1.1 Humanes Probenmaterial 22 3.1.2 Chemikalien 24 3.1.3 Reagenzien 25 3.1.4 Geräte und Verbrauchsmaterialien 27 3.2 Methoden 28 3.2.1 Gewebefixierung 28 3.2.2 Paraffineinbettung 28 3.2.3 Herstellung der Paraffinschnitte 29 3.2.4 Färbungen und Immunhistochemie 29 3.2.5 „Avidin-Biotin-Complex“-Methode für die Immunhistochemie 30 3.2.6 Darstellung verschiedener Gehirnzelltypen mit der ABC-Methode 33 3.2.6.1 Darstellung von Neuronen mittels anti-Humanem Neuronalem Protein (anti-HuC/D) 33 3.2.6.2 Darstellung von Astroglia mittels anti-Glial Fibrillary Acidic Protein (anti-GFAP) 34 3.2.6.3 Darstellung von Mikroglia mittels anti-HLA-DP,-DQ,-DR und anti-IBA-1 34 3.2.6.4 Darstellung von Oligodendroglia mittels anti-CNPase und anti-Olig2 34 3.2.6.5 Darstellung von Myelin mittels anti-Myelin Basic Protein (anti-MBP) 35 3.2.6.6 Darstellung von Ferritin mittels anti-Ferritin Heavy Chain (Y-16) 35 3.2.7 Fluoreszenz-Immunhistochemie 35 3.2.8 Histochemische Färbungen zum semiquantitativen Eisennachweis 37 3.2.8.1 Färbung nach Perls zum Nachweis von Fe3+ 37 3.2.8.2 Färbung nach Turnbull zum Nachweis von Fe2+ 38 3.2.9 Identifikation anatomischer Regionen der Substantia nigra und Anfertigung der Abbildungen 39 3.2.10 Ionenstrahlmikroskopie 39 3.2.10.1 Technische Aspekte der Ionenstrahlmikroskopie 40 3.2.10.2 Analysemethoden am LIPSION 43 3.2.10.3 Grundlagen der protoneninduzierten Röntgenemission (PIXE) 44 3.2.10.4 Grundlagen der Rutherford-Rückstreuung (RBS) 45 3.2.10.5 Probenpräparation für die Nanosonde 45 3.2.10.6 Kombination von Immunhistochemie und Ionenstrahlmikroskopie 46 3.2.10.7 Der Messvorgang an der Nanosonde 47 3.2.10.8 Auswertung der experimentell ermittelten Daten 49 3.2.11 Quantitative MR-Bildgebung 53 3.2.12 Statistische Auswertung 53 4 Ergebnisse 54 4.1 Immunhistochemie 54 4.1.1 Darstellung von Neuronen mittels anti-Humanem Neuronalem Protein (anti HuC/D) 54 4.1.2 Darstellung von Astroglia mittels anti-Glial Fibrillary Acidic Protein (anti GFAP) 56 4.1.3 Darstellung von Myelin mittels anti-Myelin Basic Protein (anti MBP) 58 4.1.4 Darstellung von Mikroglia mittels anti-ionized calcium binding adaptor molecule 1 (anti IBA 1) und anti HLA DP, DQ, DR 60 4.1.5 Darstellung von Oligodendroglia mittels anti-CNPase und anti-Olig2 64 4.1.6 Darstellung von Ferritin-reaktiven Zellen mittels anti-Ferritin Heavy Chain (Y-16) 66 4.1.7 Fluoreszenz-Doppelmarkierung zur Darstellung der zellulären Lokalisation immunhistochemisch detektierter Ferritinbestände (Ferritin - IBA-1, Ferritin - GFAP, Ferritin - Olig2) 68 4.2 Semiquantitativer Eisennachweis 70 4.2.1 Nachweis von Fe3+ nach Perls (DAB) 70 4.2.2 Nachweis von Fe2+ nach Turnbull 73 4.3 Ionenstrahlmikroskopie 76 4.3.1 Ortsaufgelöste Elementdarstellung und Bestimmung der Eisenkonzentration in ausgewählten Regionen von Interesse 76 4.3.2 Eisenkonzentration verschiedener Gehirnzelltypen im Nigrosom 1 79 4.3.3 Eisenkonzentration ausgewählter Eisenspeicherformen im Nigrosom 1 81 4.3.4 Gesamteisenkonzentration im Nigrosom 1 sowie Anteil der untersuchten Zellarten und Eisenspeicherformen am Gesamteisen 82 4.3.5 Elementprofile ausgewählter Zellarten mit deren Eisenspeicherformen 83 4.4 Kurzzusammenfassung der Ergebnisse 86 4.4.1 Klassische Immunhistochemie, Histochemie und Fluoreszenz-IHC 86 4.4.2 Ionenstrahlmikroskopie 86 5 Diskussion 88 5.1 Methodische und vergleichende Betrachtungen 88 5.1.1 Hirngewebe 88 5.1.2 Ionenstrahlmikroskopie 88 5.1.3 Immunhistochemie 92 5.2 Diskussion der Ergebnisse 93 5.2.1 Quantifizierung von Eisen im Nigrosom 1 der SNpc 93 5.2.2 Degeneration dopaminerger Neuronen innerhalb der Substantia nigra 100 5.2.3 Veränderungen der Gliazellen bei Morbus Parkinson 100 5.2.4 Interpretationsmöglichkeiten der Perls-Färbung 104 5.3 Ausblick 106 6 Zusammenfassung 108 7 Literaturverzeichnis 111 8 Eigenständigkeitserklärung 132 9 Lebenslauf 133 10 Danksagung 135

Parkinson’s Disease is a disorder of the midbrain dopaminergic system with characteristic neurodegenerative patterns, recognized for its motor symptoms. The neurodegeneration is most prevalent in the substantia nigra pars compacta, while dopaminergic neurons in neighboring structures are comparatively spared. There are many possible explanations for this disparity, including differences in tolerance to oxidative stress, and vulnerability to α-synuclein aggregates. The substantia nigra is part of the basal ganglia, a network of nuclei in the midbrain and base of the forebrain which are responsible for coordinating voluntary movement. Dopamine has an inhibitory effect in the basal ganglia. It dampens signals to remove noise, so the basal ganglia circuitry is not hyperactive. In the absence of dopamine, the flow of information through the basal ganglia is disrupted. This results in tremor, bradykinesia, and rigidity, known as the classic triad. No cure currently exists and therapies are unable to slow disease progression, so treatments are aimed at symptom management. Degenerative processes in Parkinson’s Disease occur rapidly, early in the disease progression, with about 60% neuronal death in the substantia nigra prior to diagnosis. There is a need for biomarkers or other signs which can be used to clinically to diagnose the disease at an earlier stage. In conclusion this paper provides suggestions for future lines of research.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24786号 / 医博第4978号 / 新制||医||1066(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 花川 隆, 教授 村井 俊哉, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No description available.

Les processus compensatoires qui accompagnent les atteintes du système dopaminergique (DA-ergic) nigrostrié illustrent les capacités adaptatives du cerveau adulte. Cette neuroplasticité permet le maintien de la transmission dopaminergique pendant un certain temps de sorte que les symptômes moteurs cardinaux de la Maladie de Parkinson (MP), qui se caractérise par une dégénérescence progressive des neurones DA-ergiques de la substantia nigra (SN), ne se manifestent qu'après une perte neuronale très importante. De ce fait, le diagnostic présymptomatique est une question cruciale pour le développement de traitements neuroprotecteurs. Un autre exemple de neuroplasticité est illustré par la production de nouveaux neurones dans le cerveau adulte (neurogenèse adulte). Cette neurogenèse s’observe principalement dans deux zones: le continuum zone sous-ventriculaire (SVZ)-bulbe olfactif (OB) et le gyrus denté (DG) de l'hippocampe, et se trouve altérée chez les patients parkinsoniens. Ces dernières années, le traitement chirurgical par la stimulation à haute fréquence (SHF) du noyau sous-thalamique (NST) s'est avéré être une option thérapeutique très efficace pour ces patients. Dans ce contexte, mon travail de thèse a été axé sur l’étude de la neuroplasticité dans différents modèles de la maladie de Parkinson et de son traitement avec les objectifs principaux: 1) Développer un modèle de MP présymptomatique; 2) étudier les mécanismes compensatoires impliquant le système nigrostrié; 3) Déterminer les effets de la SHF-NST sur la neurogenèse adulte dans la SVZ-OB et le DG.Dans la première étude, nous avons développé des modèles expérimentaux de la MP à différents stades, basés sur l’administration de MPTP chez la souris. Nous avons montré que le passage du stade avancé présymptomatique au stade symptomatique précoce correspondant au seuil d’atteinte des systèmes DA-ergiques associé à l’apparition des déficits moteurs, se caractérise par : (a) une diminution de DA dans les terminaisons striatales épargnées par la lésion; (b) une augmentation de DA et d’expression de la tyrosine hydroxylase dans les cellules de la SN; (c) une augmentation du renouvellement de la DA dans le striatum et une augmentation moindre dans la SN.La deuxième étude est basée sur un modèle de lésion DA-ergique extensive par injection intranigrale de 6-hydroxydopamine chez le rat, imitant les stades tardifs de la MP. Nous avons étudié séparément les étapes de prolifération et de survie des nouvelles cellules sur des animaux non lésés et des animaux lésés avec ou sans SHF subchronique (8 jours) du NST. Nous avons pu montrer une régulation spécifique des étapes de prolifération et de survie suite à la lésion dopaminergique, et des effets stimulateurs de la SHF du NST sur la survie des cellules néoformées, suggérant un effet neuroprotecteur de ce traitement. / The compensatory processes that accompany a lesion of the nigrostriatal dopaminergic (DA-ergic) system serve to maintain its function and illustrate adult brain neuroplasticity. The typical motor symptoms of Parkinson’s diseases (PD), characterized by progressive degeneration of DA-ergic neurons of substantia nigra (SN), appear only after substantial neuronal loss. Therefore presymptomatic diagnosis is a crucial issue for future neuroprotective therapies. Another good manifestation of neuroplasticity is adult neurogenesis, known to persist in two areas: the subventricular zone (SVZ) – the olfactory bulb (OB) continuum, and the dentate gyrus (DG) of the hippocampus, and to be altered in PD. In recent years, the surgical treatment by high frequency stimulation (HFS) of the subthalamic nucleus (STN) has proven to be an efficient therapeutic option for PD patients. In this context, my PhD work was focused on neuroplasticity under the functional deficiency of the nigrostriatal DA-ergic system (parkinsonism) and its treatment with the following main objectives: 1 - Develop a model of presymptomatic parkinsonism; 2 - study compensatory mechanisms in nigrostriatal system; 3 - Characterize the effects of subchronic STN HFS on adult neurogenesis. In the first part, we have developed models of presymptomatic parkinsonism based on MPTP administration in mice, as defined by sub-threshold DA depletion and degeneration of DA-ergic axons in the striatum followed by a loss of DA-ergic cell bodies in the SN (advanced presymptomatic stage). In the early symptomatic stage, these parameters reach a threshold that is associated with the appearance of motor deficiency. We have shown that the transition from the advanced presymptomatic stage to the early symptomatic stage is characterized by: (a) a decrease of DA content in surviving DA-ergic axons in the striatum; (b) an increase of DA content and TH-expression in surviving neuronal cell bodies in the SN; (c) an increase of DA turnover in the striatum and much less increase in the SN. The last part of my work is based on extensive DA lesion in rats, using intranigral 6-hydroxydopamine injection mimicking late PD stages, to determine a possible effect of STN-HFS on adult neurogenesis. We have completed series of animals with DA lesion either sham implanted or subsequently treated for 8 days by STN-HFS to be compared with unlesioned rats, and studied selective phases of neurogenesis: proliferation and survival. This study demonstrates selective regulation of cell proliferation and survival following DA depletion and provides the first evidence that prolonged STN-HFS might have a neuroprotective action as shown by the selective increase in survival of newly formed cells following this treatment.

A doença de Parkinson (DP) é uma desordem neurodegenerativa caracterizada clinicamente por tremor, rigidez, acinesia (ou bradicinesia) e instabilidade postural. Patofisiologicamente, a DP é classificada como uma sinucleinopatia associada à perda de neurônios dopaminérgicos na substância negra (SN), mas outros neurônios do tronco encefálico podem estar degenerados na DP, contribuindo não só para as alterações motoras, mas também não motoras observadas. Dentre as alterações não motoras, as alterações respiratórias estão presentes tais como obstrução das vias aéreas superiores, pneumonia e ainda a apnéia obstrutiva do sono uma das principais causas de morte na DP. Os mecanismos que levam à degeneração de neurônios envolvidos no controle respiratório ainda não estão bem esclarecidos, mas dados recentes do nosso laboratório mostraram que no modelo de DP induzido pela injeção no caudado-putâmen (CPu) de 6-hidroxidopamina (6-OHDA), um agente neurotóxico seletivo para células catecolaminérgicas, observou-se intensa redução na frequência respiratória e ventilação basais e induzidas pela ativação do quimiorreflexo central por hipercapnia. Além disso, observou-se também intensa redução do número de neurônios bulbares envolvidos no controle neural da respiração, como os neurônios Phox2b+ da região do núcleo retrotrapezóide (RTN), que estão envolvidos com a inspiração e o quimiorreflexo central. Dessa forma, o objetivo do presente trabalho foi investigar se a existência de uma via entre os neurônios da SN e do RTN poderia ser responsável por essa neurodegeneração. Realizamos injeções de traçadores anterógrados e retrógrados na SN e no RTN de ratos para verificar a existência de projeções diretas entre essas regiões, entretanto observamos que não há projeções diretas entre a SN e o RTN, mas há projeções indiretas entre essas duas regiões, utilizando a Substância Cinzenta Periaquedutal (PAG) como região intermediária. Além disso, observamos que no modelo de DP induzido pela injeção bilateral de 6-OHDA no CPu ocorre uma redução no número de varicosidades catecolaminérgicas na PAG e de neurônios que são ativados pelo quimiorreflexo central que se projetam da PAG para o RTN. Nossos experimentos eletrofisiológicos mostraram que a inibição bilateral da PAG pela injeção de muscimol não gera alterações respiratórias basais como ocorre no modelo da DP; entretanto, nesses animais, pudemos também observar, apesar de ser menor do que ocorre com animais submetidos ao modelo da DP, inibição de alterações respiratórias induzidas por hipercapnia. Nossos dados anatômicos mostraram que a comunicação entre os neurônios da SN e do RTN envolve neurônios da PAG e que essa via pode estar reduzida no modelo da DP, o que pode contribuir para a redução de neurônios do RTN; e que a redução neuronal desta via pode alterar as respostas respiratórias frente à ativação do quimiorreflexo central. / Parkinson\'s disease (PD) is a neurodegenerative disorder characterized clinically by tremor, rigidity, akinesia (or bradykinesia) and postural instability. Pathophysiologically, PD is classified as a synucleinopathy predominantly associated with loss of dopaminergic neurons in the substantia nigra (SN), but other brainstem neurons may also be degenerate in PD, contributing not only to the motor but also non-motor alterations observed in this pathology. Among the non-motor changes observed respiratory changes are present and can be characterized as upper airway obstruction, pneumonia and obstructive sleep apnea are one of the main causes of death in PD. The mechanisms that lead to the degeneration of neurons involved in respiratory control are still not well understood but data in the literature have demonstrated the loss of receptors in a region considered to be the respiratory rate generator in postmortem brains of humans. In the model animal DP of 6-hydroxydopamine (6-OHDA), a selective neurotoxic agent for catecholaminergic cells, there was an intense reduction in basal respiratory rate and ventilation, in addition to a intense reduction of neurons involved in neural control of breathing: Phox2b+ neurons in the retrotrapezoid nucleus (RTN) region. Thus, the aim of the present study was to investigate whether the existence of a pathway between SN and RTN neurons could be responsible for this bulbar neurodegeneration. We performed experiments using the injection of anterograde and retrograde tracers in the SN and the RTN to verify the existence of direct projections between these regions in rats. However, our results showed that there are no direct projections between the SN and the RTN, but there are indirect projections between these two regions, using Periaquedutal Gray Substance (PAG) as the intermediate region. In addition, we observed that in the PD model induced by the bilateral injection of 6-OHDA in CPu, a reduction of the projections PAG neurons for RTN and that are activated by the central chemoreflex. Our electrophysiological experiments have shown that in the 6-OHDA PD model there is a reduction of the cardiorespiratory responses induced by the activation of the central chemoreflex, since the bilateral inhibition of the PG of control animals does not alter these cardiorespiratory responses. Therefore, our anatomical results showed that the communication between SN and RTN neurons involves PAG neurons and that this pathway may be reduced in the PD model, which may contribute to the reduction of RTN neurons; and that the neuronal reduction of this pathway may alter respiratory responses to activation of the central chemoreflex.

by Cheng Wai Ming. / Thesis submitted in: October, 1998. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 54-68 (2nd gp.)). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iv / ABSTRACT --- p.v / ABSTRACT (Chinese) --- p.vii / Chapter CHAPTER 1 --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Ionotropic glutamate receptors --- p.1 / Chapter 1.1.1 --- AMP A receptor --- p.3 / Chapter 1.1.1.1 --- Structure of AMP A receptor --- p.3 / Chapter 1.1.1.2 --- Electrophysiological properties of AMPA receptor --- p.4 / Chapter 1.1.1.3 --- Pharmacology of AMPA receptors --- p.6 / Chapter 1.1.1.4 --- Kinetics of AMPA receptors --- p.8 / Chapter 1.1.2 --- NMDA receptor --- p.9 / Chapter 1.1.2.1 --- Structure of NMDA receptor --- p.9 / Chapter 1.1.2.2 --- Electrophysiological properties of NMDA receptor --- p.10 / Chapter 1.1.2.3 --- Pharmacology of NMDA receptor --- p.11 / Chapter 1.1.2.4 --- Kinetics of NMDA receptor --- p.12 / Chapter 1.2. --- The basal ganglia and the SNR --- p.12 / Chapter 1.3 --- Excitatory glutamatergic inputs on SNR --- p.16 / Chapter 1.4 --- Aim of study --- p.17 / Chapter CHAPTER 2 --- Electrophysiological properties of SNR neurones --- p.18 / Chapter 2.1 --- Introduction --- p.18 / Chapter 2.2 --- Methods --- p.19 / Chapter 2.2.1 --- In vitro slice preparation and maintenance --- p.19 / Chapter 2.2.2 --- Whole-cell patch-clamp recording --- p.20 / Chapter 2.2.3 --- Solutions and drugs --- p.21 / Chapter 2.2.4 --- Histological methods --- p.21 / Chapter 2.2.5 --- Data analysis --- p.22 / Chapter 2.3 --- Results --- p.22 / Chapter 2.3.1 --- Passive membrane properties of SNR neurones --- p.22 / Chapter 2.3.2 --- Firing rate and action potential characteristics --- p.23 / Chapter 2.3.3 --- Firing patterns --- p.23 / Chapter 2.3.4 --- Weak hyperpolarization activated inward rectification --- p.24 / Chapter 2.3.5 --- Slow aflerhyperpolarization --- p.25 / Chapter 2.3.6 --- Current-frequency relationship --- p.25 / Chapter 2.3.7 --- Morphology of labelled SNR neurones --- p.25 / Chapter 2.4 --- Discussion and conclusion --- p.26 / Chapter CHAPTER 3 --- AMPA and NMDA induced membrane responses --- p.30 / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Methods --- p.31 / Chapter 3.2.1 --- In vitro slice preparation and maintenance --- p.31 / Chapter 3.2.2 --- Whole-cell patch-clamp recording --- p.31 / Chapter 3.2.3 --- Solutions and drugs --- p.31 / Chapter 3.2.4 --- Drug application --- p.32 / Chapter 3.2.5 --- Immunocytochemistry --- p.32 / Chapter 3.2.6 --- Data analysis --- p.33 / Chapter 3.3 --- Results --- p.33 / Chapter 3.3.1 --- AMPA induced responses in SNR GABA neurones --- p.33 / Chapter 3.3.1.1 --- AMPA induced membrane depolarization --- p.33 / Chapter 3.3.1.2 --- AMPA induced membrane current --- p.34 / Chapter 3.3.1.3 --- Current-voltage relationship --- p.34 / Chapter 3.3.1.4 --- Effect of NBQX --- p.35 / Chapter 3.3.1.5 --- Effects of JSTX and spermine --- p.35 / Chapter 3.3.2 --- NMDA-induced response in SNR GABA neurones --- p.36 / Chapter 3.3.2.1 --- NMDA induced membrane depolarization --- p.36 / Chapter 3.3.2.2 --- NMDA induced membrane current --- p.36 / Chapter 3.3.2.3 --- APV blocked NMDA-induced current --- p.36 / Chapter 3.3.2.4 --- Effect of glycine on NMDA induced response --- p.37 / Chapter 3.3.2.5 --- Mg2+-sensitivity --- p.37 / Chapter 3.3.2.6 --- Current-voltage relationship --- p.38 / Chapter 3.3.3 --- GluR2 subunit immunostaining --- p.38 / Chapter 3.4 --- Discussion and conclusion --- p.39 / Chapter 3.4.1 --- AMPA receptors in SNR neurones --- p.39 / Chapter 3.4.2 --- NMDA receptors in SNR neurones --- p.41 / Chapter 3.4.3 --- Functional significance --- p.41 / Chapter CHAPTER 4 --- Glutamate-mediated synaptic currents in SNR --- p.43 / Chapter 4.1 --- Introduction --- p.43 / Chapter 4.2 --- Methods --- p.44 / Chapter 4.2.1 --- In vitro slice preparation and maintenance --- p.44 / Chapter 4.2.2 --- Electrophysiological recordings --- p.44 / Chapter 4.2.3 --- Electrical stimulation --- p.45 / Chapter 4.2.4 --- Solutions and drugs --- p.45 / Chapter 4.2.5 --- Data analysis --- p.46 / Chapter 4.3 --- Results --- p.46 / Chapter 4.3.1 --- Characteristics of spontaneous EPSCs --- p.46 / Chapter 4.3.1.1 --- General characteristics --- p.46 / Chapter 4.3.1.2 --- Kinetics --- p.47 / Chapter 4.3.1.3 --- Pharmacology --- p.47 / Chapter 4.3.2 --- Characteristics of evoked EPSCs --- p.48 / Chapter 4.3.2.1 --- General characteristics --- p.48 / Chapter 4.3.2.2 --- Pharmacological characterization --- p.49 / Chapter 4.3.2.3 --- Effects of bicuculline --- p.50 / Chapter 4.4 --- Discussion and conclusion --- p.50 / Chapter 4.4.1 --- Excitatory transmission onto SNR neurones --- p.50 / Chapter 4.4.2 --- Source of excitatory drive --- p.51 / Chapter 4.4.3 --- Interaction with GABA inputs --- p.52 / Chapter 4.4.4 --- Functional significance --- p.52 / REFERENCES --- p.54