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

by Priscilla, Ka-Yee Chan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 77-100). / Abstract also in Chinese. / ACKNOWLEDGEMENTS --- p.4 / ABSTRACT --- p.5 / ABSTRACT (Chinese) --- p.7 / PUBLICATION --- p.8 / ABBREVIATION --- p.9 / Chapter CHAPTER 1 --- INTRODUCTION --- p.10 / Chapter 1.1 --- Overview of the study --- p.10 / Chapter 1.2. --- Substantia nigra pars reticulata (SNR) --- p.12 / Chapter 1.2.1 --- SNR and the basal ganglia / Chapter 1.2.2 --- GABA neurotransmission in SNR / Chapter 1.2.3 --- SNR and epilepsy / Chapter 1.3 --- GABAb receptors --- p.18 / Chapter 1.3.1 --- GABA receptors / Chapter 1.3.2 --- GABAb receptors and their classification / Chapter 1.3.3 --- Agonists and antagonists of GABAb receptor / Chapter 1.3.4 --- Distribution of GAB AB receptor / Chapter 1.3.5 --- GABAb receptors in epilepsy and the involvement of SNR / Chapter 1.4 --- Cannabinoid receptors --- p.24 / Chapter 1.4.1 --- Cannabinoid receptors and their classification / Chapter 1.4.2 --- Agonists and antagonists of cannabinoid receptor / Chapter 1.4.3 --- Distribution of cannabinoid receptors / Chapter 1.4.4 --- Cannabinoid receptors in epilepsy and the involvement of SNR / Chapter CHAPTER 2 --- METHODS --- p.31 / Chapter 2.1 --- Brain slice preparation and maintenance --- p.31 / Chapter 2.2 --- Experimental set-up --- p.32 / Chapter 2.2.1 --- Visualization of neurones / Chapter 2.2.2 --- Electrophysiological recordings / Chapter 2.2.3 --- Evoked stimulation / Chapter 2.2.4 --- Drug preparation and administration / Chapter 2.3 --- Identification of GAB A and dopamine neurones --- p.36 / Chapter 2.4 --- Data analysis --- p.37 / Chapter 2.4.1 --- Construction of dose-response curve / Chapter 2.4.2 --- Analysis of synaptic currents / Chapter 2.4.3 --- Statistics / Chapter CHAPTER 3 --- RESULTS --- p.39 / Chapter 3.1 --- Basic characteristics of IPSCs in SNR --- p.39 / Chapter 3.1.1 --- Spontaneous and miniature IPSCs / Chapter 3.1.2 --- Evoked IPSCs / Chapter 3.2 --- GABAb receptors in SNR --- p.42 / Chapter 3.2.1 --- Postsynaptic GABAb receptors in SNR neurones / Chapter 3.2.1.1 --- Baclofen-activated postsynaptic response / Chapter 3.2.1.2 --- Effects of GABAb receptor antagonist on IPSCs / Chapter 3.2.2 --- Presynaptic GABAb receptors / Chapter 3.2.3 --- Effects of GAB A uptake blocker / Chapter 3.3 --- Cannabinoid receptors in SNR --- p.51 / Chapter 3.3.1 --- Postsynaptic cannabinoid receptors in SNR neurones / Chapter 3.3.2 --- Presynaptic action of cannabinoids / Chapter CHAPTER 4 --- DISCUSSION and CONCLUSION --- p.55 / Chapter 4.1 --- General properties of IPSCs --- p.55 / Chapter 4.2 --- GABAb receptors in SNR neurones --- p.58 / Chapter 4.2.1 --- Postsynaptic GABAB receptors in SNR neurones / Chapter 4.2.2 --- GABAb component in spontaneous and evoked IPSCs / Chapter 4.2.3 --- Presynaptic GABAb receptors in SNR / Chapter 4.2.4 --- Role of GABA uptake / Chapter 4.3 --- Cannabinoid receptors in SNR neurones --- p.67 / Chapter 4.3.1 --- Postsynaptic cannabinoid receptors in SNR neurones / Chapter 4.3.2 --- Presynaptic cannabinoid receptors in SNR / Chapter 4.4 --- SNR GABAb and cannabinoid receptors - their role in epilepsy --- p.72 / Chapter 4.5 --- Concluding remarks and future direction --- p.75 / REFERENCES --- p.77

Ces dernières années de nombreux marqueurs de l’atteinte nigrostriatale ont été élaborés et testés dans la maladie de Parkinson (MP). Ces marqueurs peuvent détecter et quantifier la neurodégénérescence dans la substance noire (SN) des patients ainsi que dans les formes précoces prémotrices de la maladie. Leur performance diagnostique reste mal connue de même que l’atteinte extra-nigrale.Dans ce travail nous avons étudié l’atteinte de la SN dans les formes précliniques et prémotrices de la MP. La charge en fer était augmentée chez des porteurs des mutations symptomatiques mais également chez des porteurs sains. De plus, nous avons observé une atteinte pré-motrice de la SN chez des sujets touchés par les troubles des comportements en sommeil paradoxal (TCSP) qui n’ont pas encore développé un syndrome parkinsonien. Chez ces sujets, l’atteinte de la SN a été le mieux mise en évidence par l’imagerie sensible à la neuromélanine et le tenseur de diffusion avec la fraction d’anisotropie, suggérant un intérêt dans la caractérisation prodromale de la MP. Ces mêmes marqueurs présentaient également la meilleure performance diagnostique dans la MP. Finalement, nous avons trouvé des anomalies bulbaires en tenseur de diffusion dans la MP, qui étaient corrélées aux symptômes dysautonomiques cardiaques et respiratoires, suggérant l’intérêt de ce marqueur pour étude de l’atteinte bulbaire. Ceci ouvre une possibilité d’étude bulbaire chez des sujets présymptomatiques car une atteinte bulbaire devrait apparaître avant les symptômes moteurs selon le modèle de Braak. Ces marqueurs sont peut-être la première étape vers un diagnostic présymptomatique de la MP dans la pratique clinique. / In recent years numerous biomarkers of nigro-striatal damage were proposed in Parkinson's disease (PD). These markers are able to detect and quantify neurodegenative changes in the substantia nigra (SN) of patients with PD as well as in subjects with premotor conditions. Their diagnostic performances to detect PD as well as the extent of extranigral pathology remain incompletely understood.In this work we observed the damage of the substantia nigra in preclinical and premotor forms of PD. Iron load was increased in both symptomatic and asymptomatic mutations carriers, suggesting the interest of the biomarker in PD related genetic mutations. In addition, we found a pre-clinical impairment of the SN in subjects affected by idiopathic sleep in REM behavioral disorders (iRBD) who have not yet converted to Parkinsonism. In these subjects, the SN damage was best demonstrated by neuromelanin-sensitive imaging and diffusion tensor imaging (DTI) with fractional anisotropy measure, suggesting an interest of these measures in the prodromal characterization of PD. These same markers had the best performance for PD diagnosis.Finally, we found medulla oblongata damage patients with PD using DTI. This damage was correlated with cardiac and respiratory autonomic symptoms, suggesting the importance of this biomarker in medulla oblongata damage exploration. It also opens a possibility of medulla oblongata study in presymptomatic subjects as medulla oblongata damage should appear before motor symptoms based on the Braak model.These biomarkers may be the first step towards a presymptomatic diagnosis of PD in clinical practice.

Dans le système nerveux central, les monoamines modulent de nombreuses fonctions essentielles comme la locomotion, la motivation, la cognition, l’humeur et le sommeil. Le niveau extracellulaire de ces neurotransmetteurs est régulé par des transporteurs à haute affinité,cependant d’autres transporteurs, à faible affinité, peuvent contribuer à la recapture des monoamines, comme les transporteurs de cations organiques (OCT) et le transporteur plasmique des monoamines (PMAT). Récemment, l’implication des OCT dans différentes fonctions centrales, notamment le contrôle de l’humeur, la réponse au stress et aux antidépresseurs a été mise en évidence. Le rôle de PMAT dans le cerveau reste quant à lui encore peu caractérisé. Il transporte in vitro les monoamines, avec une préférence pour la dopamine et la sérotonine, avec des affinités submillimolaires. Ce transporteur est exprimé dans de nombreuses régions du cerveau humain et murin et dans différents types neuronaux. Par hybridation in situ fluorescente nous avons déterminé sa distribution cellulaire précise, dans des régions à fort niveau d’expression comme le complexe du cerveau antérieur basal (BFC) et des régions appartenant aux ganglions de la base comme le globus pallidus et la substance noire réticulée (SNr). Nous avons montré qu’il est fortement exprimé dans les neurones GABAergiques exprimant la parvalbumine, dans tous les interneurones cholinergiques dustriatum ainsi qu’une petite fraction des neurones cholinergiques du BFC. Il est également retrouvé dans certains noyaux monoaminergiques comme le locus coeruleus et les noyaux duraphé mais est absent des noyaux dopaminergiques, la substance noire compacte et l’aire tegmentale ventrale.Afin d’étudier sa fonction, nous avons exploité le système Cre-lox, approche couramment utilisée en biologie, en injectant un virus adéno-associé exprimant la recombinase Cre (AAVCre)dans la substance noire (SN) de souris comportant des allèles de PMAT floxés. Cette étude ne nous a pas permis de conclure quant à la fonction de PMAT dans la SN, mais nous a conduit à mettre en évidence une toxicité majeure de cet outil. Nous avons montré que l’injection d’AAV-Cre dans la SN entraine une perturbation anatomique et fonctionnelle des systèmes dopaminergiques et de la SNr, noyau de sortie des ganglions de la base, induisant des altérations comportementales importantes, avec une hyperlocomotion basale robuste et une insensibilité à la cocaïne, potentiellement par une action génotoxique.Nous avons également généré des souris invalidées constitutivement pour PMAT (PMAT-/-). Les tests comportementaux que nous avons commencés récemment nous ont révélé des altérations comportementales significatives chez ces souris de l’activité locomotrice dans un nouvel environnement ainsi que du niveau d’anxiété. Ces altérations pourraient résulter d'une perturbation des voies aminergiques en l’absence de PMAT. Nous poursuivrons cette étude par l'exploration d'autres aspects comportementaux ainsi que par l’évaluation des modifications neurochimiques engendrées par l'invalidation. Ces approches devraient fournir des pistes afin d’identifier les conséquences de l'absence de PMAT sur la signalisation aminergique, que l'on pourra explorer plus précisément par la suite sur le plan fonctionnel / High-affinity reuptake transporters exert a crucial role in the control of synaptic transmissionby ensuring the recycling of the released transmitters into the presynaptic terminals. Other typesof transporters such as Organic Cation Transporters (OCTs) and the Plasma MembranemonoAmine Transporter (PMAT), have been shown to transport, with low-affinity but highcapacity, aminergic neurotransmitters. While the role of OCTs in central nervous system hasbeen partially unraveled, the function of PMAT remains poorly characterized. In vitro, PMATtransports preferentially dopamine and serotonin and its expression is widespread in the brain,encompassing monoamine nuclei but also projection regions. In this study, we determined theprecise neuronal specificity of PMAT in several highly-expressing regions. We show that it isfound mostly in PV+ GABAergic neurons of basal forebrain and basal ganglia, in allcholinergic interneurons of the striatum and in some cholinergic neurons of basal forebraincomplex. These systems, highly regulated by monoamines, are important for locomotion,motivation, learning and wakefulness. Our result show that PMAT is located at a strategicposition to control the aminergic modulation of these integrated functions.To investigate the implication of PMAT in these regions, we used the Cre-lox technology, avalued and widely used approach for the study of gene function in vivo, injecting an adenoassociatedvirus expressing Cre recombinase in substantia nigra (SN) of mice in which PMATgene was floxed. In this study, we could not assess PMAT function in this SN but found thatAAV-CRE expression in this region produces major toxic effects. We showed that AAV-Creinjection in this region engenders a massive decrease of neuronal populations in both parscompacta and reticulata, leading to DA depletion in the nigrostriatal pathway. This wasassociated with a drastic behavioral phenotype with increased basal locomotor activity and lossof locomotor response to cocaine. Several hallmarks of Cre toxicity were found in SN of AAVCreinjected mice, including an increase of the DNA break markers. These observationsunderscore the need for careful control of Cre toxicity in the brain and reassessment of previous studies.To study the role of PMAT, we also generated PMAT knock out mice (PMAT-/-). Behavioralstudies that we just started have revealed significant impairments of locomotor activity in a newenvironment and anxiety level, supporting a possible disruption of monoaminergic systems inthese mice. On-going studies aim to explore other behaviors and search for eventualneurochemical changes provoked by PMAT invalidation. These experiments should providesome cues to understand which monoamines and circuits may be affected, that can beinvestigated functionnally and more specifically in a second step

Le profil d'activité des neurones dopaminergiques semble fortement évoluer au cours des premières semaines post-natales, faisant intervenir des modifications des propriétés intrinsèques et synaptiques, mais la connaissance des mécanismes à l'origine de ces changements et de leur cinétique est encore parcellaire.Dans un premier article, nous avons caractérisé le profil d'expression des sous-unités des principaux canaux ioniques sensibles au potentiel somato-dendritiques des neurones dopaminergiques au cours des premières semaines de développement post-natal. Nous avons pu décrire les principaux changements d'expression de ces canaux entre P6, P21 et P40. Dans un second article, nous avons défini l'évolution du comportement électrophysiologique des neurones dopaminergiques lors des 4 premières semaines postnatales. Nous avons pu montrer que l'acquisition du phénotype électrique "mature" des neurones dopaminergiques implique essentiellement deux transitions développementales, intervenant respectivement entre P3 et P5 puis entre P9 et P11.Enfin dans une troisième étude, nous avons tenté de déterminer les principaux changements morphologiques intervenant au cours des premières semaines post-natales et de définir leur impact sur le profil électrophysiologique des neurones dopaminergiques. Nos résultats suggèrent que la morphologie de l'axone et du segment initial de l'axone changent fortement au cours des trois premières semaines post-natales.Nous avons ainsi pu caractériser les principales transitions développementales intervenant dans l'acquisition du phénotype électrique des neurones dopaminergiques ainsi que les changements morphologiques et biophysiques associés. / The firing pattern of dopaminergic neurons seems to strongly evolve during the first postnatal weeks, involving changes in intrinsic and synaptic properties, but our knowledge of the mechanisms underlying these changes and their precise timecourse is still fragmented.In a first study, we characterized the expression profile of several somato-dendritic voltage-gated ion channels in dopaminergic neurons during postnatal development. Our results describe the major changes in expression of these ion channels occurring between P6, P21 and P40.In a second study, we described the modifications in electrophysiological behavior of dopaminergic neurons across the first four postnatal weeks. We show that the acquisition of the mature electrical phenotype of dopaminergic neurons mainly involves two developmental transitions occurring between P3 and P5 and then between P9 and P11, respectively.Finally, in a third study, we attempted to define the major morphological changes occurring during early postnatal development and to measure their impact on the electrophysiological profile of dopaminergic neurons. Our results suggest that the morphology of the axon and the axon initial segment strongly change during the first three postnatal weeks, even though these changes do not seem to significantly influence the excitability of dopaminergic neurons or the shape of their action potential.We were able to characterize the main developmental transitions leading to the acquisition of the mature electrical phenotype of dopaminergic neurons, and to describe some of the biophysical and morphological changes associated with this electrophysiological maturation.

<p>Dopamine regulates movement, cognition and the rewarding effects of addictive drugs. Sex differences mediated by gonadal hormones affect each of these processes. An extensive literature suggests that estrogen augments dopaminergic function. Our laboratory found that female rats exhibit increased locomotor stimulation in response to cocaine and greater cocaine-induced dopamine overflow compared to males, sex differences that emerge in early adulthood. Currently, the underlying mechanisms for these differences are poorly understood. I hypothesized that female rats would have more dopamine neurons in midbrain regions and that ovarian hormones would exert trophic effects on dopamine neurons. Immunohistochemical and stereological techniques were used to quantitate the number of cells in the SNpc and VTA of male and female rats and mice to assess: (1) if sex differences in dopamine neuron number exist and when they emerge, (2) how gonadal hormones influence dopaminergic cell number and dopamine-mediated behaviors (3) the role of specific hormone receptors in the effects on cell number (4) the possibility that dopamine neuron number is directly linked to cocaine-stimulated behavior and electrically-stimulated dopamine release and that these responses to cocaine are mediated through gonadal hormone modulation of midbrain dopamine neuron number. I discovered sex differences in midbrain dopamine neuron number; adult female rodents have more neurons in the SNpc and VTA. We also found that gonadectomy in adulthood reduced midbrain dopamine neuron number in females and increased neuron number in males, establishing the trophic effects of estrogen in the intact midbrain and possible suppressive effects of androgens. Treatment with agonists for estrogen receptor subtypes alpha and beta and androgen receptor reversed the effects of gonadectomy on cell number in females and males, respectively. In an effort to bridge cocaine-stimulated behavior and cell number in sham ovariectomized and ovariectomized females, we discovered cocaine-stimulated behavior, dopamine release and SNpc cell density were positively correlated in intact female rats, an effect that is lost with ovariectomy. This dissertation demonstrates that estrogen is critical for the maintenance of dopaminergic cell populations that enhance behavioral responses to psychostimulants in females, thereby contributing to the observed sex differences.</p> / Dissertation

Daugelis neurodegeneracinių ligų klinikinių žymenų yra nespecifiniai ir nustatomi tik progresavus ligai. Neurodegeneracinio parkinsonizmo diagnostika remiasi klinikiniais kriterijais, tačiau idiopatinės Parkinsono ligos (PL), kuri yra pati dažniausia neurodegeneracinio parkinsonizmo priežastis, klinikiniai požymiai dažnai pasireiškia ir esant kitiems parkinsonizmo sindromams. Patikimas, neinvazinis, nebrangus ir saugus diagnostinis testas PL, galbūt ir demencijų diagnostikai bei diferencinei diagnostikai yra aktualus dėl kelių pagrindinių priežasčių: skirtingų parkinsonizmo sindromų medikamentinis gydymas ir ligos prognozė labai skiriasi; tiksli diagnozė svarbi imantis neatidėliotinų priemonių užkertant kelią kai kurioms sindromams būdingoms gyvybei grėsmingoms komplikacijoms; PL ankstyvoji diagnostika ikiklinikinėje stadijoje leistų anksčiau parinkti ir pradėti patogenezinį gydymą; atsiradus efektyvios neuroprotekcijos galimybėms, atsirastų ir tam tikro amžiaus populiacijos atrankinės patikros įrankio poreikis. Disertacinio darbo tikslas buvo nustatyti transkranijinės sonografijos vertę neurodegeneracinių ekstrapiramidinės nervų sistemos ligų diagnostikai, diferencinei diagnostikai ir atrankinei patikrai. Užsienio mokslininko dalyvavimas doktorantūros procese finansuojamas Lietuvos mokslo tarybos (konkurso paraiškos Nr. VIZ-DOK-124, doktorantūros tarybos narė doc. dr. Pille Taba). / The most symptoms of neurodegenerative disorders are non-specific and come to clinical attention only when the disease has progressed. The diagnostics of neurodegenerative parkinsonism is based on clinical criteria. However, the symptoms of idiopathic Parkinson‘s disease (PD), which is the most common cause of neurodegenerative parkinsonism, are often observed in patients with other parkinsonian syndromes. A reliable, non-invasive, safe and affordable test for the diagnosis and differential diagnosis of PD and, perhaps, dementia is relevant for multiple reasons: the disease prognosis and treatment of parkinsonian syndromes differs greatly and an unbiased disease marker would facilitate the development of neuroprotective drugs; the exact diagnosis is crucial in order to take urgent measures to avoid life-threatening complications; early diagnosis of PD would enable pathogenetic treatment; with the arrival of neuroprotective options, the need for selective screening of population according to certain age and other risk factors would arise. The aim of the doctoral study was to investigate the value of transcranial sonography for the diagnosis, differential diagnosis and screening of neurodegenerative extrapyramidal disorders. Participation of the Scientist from foreign institution in the Process of Doctoral Studies is financed by the Research Council of Lithuania (Tender-based financing application No VIZ-DOK-124, the Defence Council Member Assoc. Prof. Dr. Pille Taba).

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Diss. (sammanfattning) Göteborg : Univ., 2009. / Härtill 3 uppsatser.

Submitted by Ramon Santana (ramon.souza@ufpe.br) on 2015-03-13T14:42:13Z No. of bitstreams: 2 Dissertação completa(David).pdf: 16509548 bytes, checksum: 44c6bbf3d82089ebc82817764572ff34 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-13T14:42:13Z (GMT). No. of bitstreams: 2 Dissertação completa(David).pdf: 16509548 bytes, checksum: 44c6bbf3d82089ebc82817764572ff34 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013 / Fundação de Amparo a Ciências e Tecnologia do Estado de Pernambuco (FACEPE) / Os ácidos graxos essenciais exercem um papel crucial para o desenvolvimento e manutenção do sistema nervoso. Estudos recentes do nosso laboratório têm demonstrado que a restrição dietética crônica destes macronutrientes é capaz de induzir sinais de degeneração e perda de neurônios dopaminérgicos na substantia nigra (SN) de ratos. O presente estudo visa testar a hipótese de que esta restrição dietética é capaz de alterar a distribuição de neurônios GABAérgicos nigrais bem como aumentar a funcionalidade e reatividade de astrócitos na SN e corpo estriado (CE). Ratos Wistar foram divididos em dois grupos de acordo com a dieta materna, a qual foi fornecida a partir do acasalamento e mantida por duas gerações. O grupo controle recebeu dieta balanceada contendo óleo de soja como fonte lipídica; grupo deficiente em ácidos graxos essenciais (DAGEs) recebeu dieta semelhante, mas tendo o óleo de coco como fonte lipídica. A capacidade funcional da glia foi analisada em homogenados do CE e SN a partir da atividade da enzima Glutamina Sintetase (GS). A distribuição dos astrócitos e neurônios GABAérgicos foi analisada a partir de imunohistoquímica utilizando-se anticorpos monoclonais para a Proteína Ácida Fibrilar Glial (GFAP) e parvalbumina, respectivamente. Os níveis protéicos de GFAP foram analisados por Western blot. O perfil de ácidos graxos essenciais em fosfolipídios da SN ou CE foi analisado por cromatografia gasosa. Análise do padrão de complexidade morfológica dos astrócitos foi realizada avaliando-se índices como dimensão fractal, área de arborização e lacunaridade. Os resultados obtidos evidenciaram um aumento significativo (p < 0.01) na atividade da GS no CE de animais jovens (30-40 dias) e adultos (90-110 dias) do grupo DAGES, comparado ao controle. Na SN, um aumento na atividade da GS do grupo DAGES (p<0.001) só foi observado aos 90 dias. Um aumento na dimensão fractal dos astrócitos (p=0.009) foi observado apenas nos animais jovens do grupo DAGES, comparado ao controle. No entanto um aumento da área de arborização dos astrócitos foi detectado no grupo DAGES tanto em animais jovens (p=0.004) como nos adultos (p=0.005). Não houve diferença na lacunaridade tanto nos animais jovens quanto nos animais adultos. Modificações na expressão das isoformas da proteína GFAP foram também induzidas pela dieta experimental nos animais jovens, observando-se um aumento na isoforma de 42 KDa no CE, SN assim como no córtex cerebral. Análise dos astrócitos marcados para GFAP sugere ausência de astrogliose na SN do grupo DAGEs tanto em animais jovens como adultos. Quantificação preliminar realizada em 4 animais adultos do grupo DAGES e 3 animais do grupo controle sugere que o número de neurônios imunorreativos a parvalbumina da SN não difere entre os grupos. Os resultados obtidos sugerem que a restrição dietética em ácidos graxos essenciais por duas gerações induz efeitos adversos sobre a diferenciação e atividade funcional dos astrócitos, evidenciando também uma reatividade diferenciada entre as células da glia da SN e CE. Contudo, a restrição dietética por duas gerações não é capaz de causar astrogliose na SN dos animais jovens e adultos.