O núcleo medial (Me) é parte superficial do complexo amigdaliano e ainda muito pouco se conhece de seus constituintes celulares em seres humanos. Neste estudo desenvolveu-se uma adaptação do método de Golgi do tipo “single-section” para tecido nervoso humano fixado e conservado em formalina por tempo variável. Além disso, descreveu-se a densidade de neurônios e células da glia no Me, sua morfologia geral, incluindo detalhes de espinhos dendríticos e terminações axonais, a imunorreatividade à proteína ácida fibrilar glial (GFAP) e a ultraestrutura sináptica local. Como resultados demonstrou-se que as células da glia são maioria neste núcleo (cerca de 72% do total de células) e que há significativamente mais neurônios no Me do hemisfério esquerdo (1.53 X 105 neurônios/mm3). Os somas neuronais impregnados pelo método de Golgi demonstraram-se redondos/ovais, fusiformes ou poligonais (diâmetros entre 10-30 μm), os dendritos estenderam-se por distâncias variadas e contiveram espinhos pleomórficos, caracterizando neurônios com menos e mais espinhos dendríticos (densidades de 1,5 até 5,2 espinhos/μm), e os axônios revelaram terminações desde simples até muito complexas. Os neurônios multipolares foram classificados em Tipos 1, 2 ou 3 de acordo com trabalhos prévios, ou ainda em tipos morfológicos ainda não classificados Observaram-se astrócitos protoplasmáticos com muitos prolongamentos reativos à GFAP, isolados ou em grupos. Esses, no estudo ultra-estrutural, compuseram sinapses “tripartites” e “tetrapartites”, considerando-se o quarto elemento o da matriz extracelular situada entre os elementos pré- e pós-sinápticos. As sinapses axodendríticas apresentaram-se tanto assimétricas (com vesículas redondas pequenas e elétron-lúcidas) como simétricas (com vesículas pleomórficas pequenas e claras e, adicionalmente, com vesículas redondas grandes ou pequenas de centro escuro). Terminais axonais estabelecendo múltiplas sinapses assimétricas, classificados como de tipo “glomérulo”, também foram observados. A presente tese contribui com dados descritivos e quantitativos inéditos sobre a morfologia das células do Me, o que pode servir de base para o entendimento e novas investigações sobre o funcionamento desse núcleo em situações normais ou patológicas em seres humanos. / The medial nucleus (Me) is a superficial component of the amygdaloid complex. Little is currently known about its cellular composition in humans. Here is reported an adaptation of the “single-section” Golgi method for formalin fixed and stored human brain for diversified periods. Furthermore, the density of neurons and glial cells in the Me, their general morphology including dendritic spines and axonal terminals details, the glial fibrillary acidic protein (GFAP) immunoreactivity, and features of local cells under electron microscopy are described. Our results show that Me had an estimated mean neuronal density around 1.53 X 105 neurons/mm3 (higher in the left hemisphere), more glia (72% of all cells) than neurons, and a nonneuronal/neuronal ratio of 2.7. Golgi-impregnated neurons had cell bodies with a round/ovoid, fusiform or polygonal shape (diameters ranging from 10 to 30 μm), dendrites with varying lengths and pleomorphic spines that characterized neurons more or less spiny (density varying from1.5 to 5.2 spines/μm), and ranging from simple to very complexes terminal axons. Neurons appeared as “bitufted” or stellate multipolar cells, or classified in “Types 1 to 3” according to previous immunohistochemical observations, or other still unclassified morphologies. Protoplasmic astrocytes, either isolated or forming small clusters, were observed and showed multiple branches immunoreactive for GFAP, being equally distributed between right and left hemispheres They were found composing tripartite synapses or, together with an evident extracellular matrix between pre- and postsynaptic elements, tetrapartite ones. Axo-dendritic synapses were both asymmetrical (with various small, round electron-lucent vesicles) and symmetrical (with small pleomorphic vesicles and, occasionally, few intermingled large dense-core vesicles). Terminal axons with a glomerular-like structure were also found forming various asymmetric contacts. The present thesis add novel descriptive and qualitative information about neuronal and glial population of the human Me, which provide a basic contribution to our understanding, and to further research, of the functional implications of the Me in the brain organization both in normal and pathological conditions .

O diabetes mellitus tipo I é uma enfermidade autoimune relacionada com anormalidades genéticas, influenciada por fatores ambientais, que resultam na destruição de células β-pancreáticas, mediadas por células T. O diabetes durante a gravidez aumenta o risco a alterações no sistema nervoso central. O processo de gliogênese é importante para o desenvolvimento e funcionamento do SNC e começa na segunda semana de gestação, em ratos. A glia é composta por astrócitos, oligodendrócitos, células ependimais e microglia. Os astrócitos são as células mais abundantes e responsáveis pela integração do cérebro com o sistema vascular e imunológico, estando relacionados com as funções cognitivas, além de uma importância física e estrutural na barreira hemato-encefálica. Duas proteínas astrocíticas foram estudadas aqui: a proteína glial fibrilar acida e a S100B, uma proteína ligante de cálcio, produzida e secretada por astrócitos. A S100B está envolvida na comunicação neurônio-glia, possuindo propriedades neurotróficas ou neurotóxicas de acordo com a concentração extracelular. A expressão da proteína glial fibrilar ácida é essencial para a estrutura encefálica e para a integridade da barreira hemato-encefálica. Assim avaliamos essas proteínas gliais na prole oriunda de mães diabéticas (PoD) no desenvolvimento corporal e do sistema nervoso central. Foram utilizadas ratas Wistar-Kyoto diabéticas induzidas por estreptozotocina para obtenção da prole. As fêmeas diabéticas induzidas apresentaram ninhadas menores do que o controle, e os filhotes apresentam menor peso ao nascer A hiperglicemia materna induz aumento da produção insulínica no feto, que leva a um quadro de hipoglicemia pós-natal. Os níveis glicêmicos do PoD foram superiores ao controle (PoC) com 1 dia, e inferiores com 21 dias. A mensuração da proteína S100B no soro de animais PoD14 apresentou diminuição em relação ao controle. A S100B no líquor mostrou valores foram maiores no PoD7. Já a S100B no hipocampo mostrou um crescimento progressivo, com diminuição no PoD14 e um aumento no PoD28. Na quantificação da proteína glial fibrilar acida os animais do grupo PoD7 e PoD14 apresentaram valores menores, em seguida os níveis começaram a subir. No teste de campo aberto os animais PoD apresentaram comportamento ansioso. Já no reconhecimento de objetos os ratos PoD exploraram menos o objeto novo, tanto na memória de curta como de longa duração. As mudanças astrogliais observadas nos animais do grupo PoD estão muito provavelmente relacionadas a alterações da plasticidade neuroglial e ao déficit cognitivo observado. / The type I diabetes mellitus is an autoimmune disease associated with genetic abnormalities, influenced by environmental factors which result in the destruction of pancreatic β-cells, mediated by T cells. Diabetes during pregnancy increases the risk of changes in the central nervous system. The gliogenesis process is important to the development and functioning of the central nervous system and starts the second week of rat gestation. The glia is composed of astrocytes, oligodendrocytes, ependymal cells and microglia. The astrocytes are more abundant cells and responsible for the integration of the brain with vascular and immune systems, being connected with the cognitive functions, as well as a physical and structural importance of the blood-brain barrier. Two astrocity proteins were studied: glial acidic fribrilar protein and S100B, a binding protein of calcium produced and secreted by astrocytes. S100B is involved in neuron-glia communication, having neurotoxic or neurotrophic properties according to the extracellular concentration. Expression of glial acidic fribrilar protein is essential for brain structure and integrity of the blood-brain barrier. So we evaluated this glial proteins the offspring derived from diabetic mothers (PoD) on body development and central nervous system. Wistar-Kyoto rats with diabetes induced by streptozotocin to obtain offspring. Induced diabetic females had smaller litters then the control, and the offsprig have a lower birth weigth Maternal hyperglycemia induces increased insulin production in the fetus. Blood glucose levels were higher in PoD than the control (PoC) with 1 day and less than 21 days. The measurement of S100B protein in PoD14 animal serum showed a decrease compared to the control. The S100B in cerebrospinal fluid showed values were higher in PoD7. Since S100B in the hippocampus showed a progressive increase with decrease in PoD14 and increase in PoD28. Glial acidic fribrilar protein quantification in animals of PoD7 and PoD14 group showed lower values, and then levels began to rise. In the open field test the PoD animals showed anxious behavior. Since the object recognition (RO) least rats explored the new object in both short and long term memory. Induced diabetic females had smaller litters than the control, and the chicks have a lower birth weight. Maternal hyperglycaemia induces increased insulin production in the fetus, which leads to a postnatal hypoglycemia. The variations observed in the animals of group PoD caused biochemical changes related to astroglial plasticity, which caused cognitive impairment.

O Transtorno do Espectro Autista (TEA) é um transtorno do neurodesenvolvimento caracterizado por prejuízos na comunicação e interação social, assim como por comportamentos repetitivos. O TEA pode apresentar diversas comorbidades, como alterações gastrointestinais – incluindo constipação crônica, refluxo gastroesofageal, doença celíaca e cólicas intestinais. A etiologia do TEA ainda é desconhecida, mas pode envolver alterações genéticas e a fatores de risco ambientais durante a gestação, como a exposição ao ácido valproico (VPA). Dessa forma, a exposição embrionária ao VPA tornou-se uma ferramenta confiável para a indução de alterações do tipo autista em roedores. O presente estudo teve como objetivo avaliar alterações no sistema nervoso entérico (SNE) de animais expostos ao VPA durante a gestação. Para isso, ratas Wistar prenhes receberam uma única dose (injeção intraperitoneal) de 600 mg/kg de VPA ou solução fisiológica (salina) no dia embrionário 12,5 (E12,5). A prole de machos, com 30 dias de vida pós-natal, sofreu eutanásia e então removeu-se o íleo, que passou por delaminação. No plexo mioentérico ileal foram avaliados por imunofluorescência, neurônios totais (HuC/D+), neurônios excitatórios (ChAT+), neurônios inibitórios (NOS+) e células gliais (GFAP+). A análise apresentou menor densidade neuronal nos gânglios mioentéricos ileais no grupo VPA (p<0,05); área ganglionar semelhante; mesmo perfil de densidade e corpo celular para neurônios inibitórios; menor densidade de neurônios excitatórios com maior área do corpo celular (p<0,05) e aumento no número de células gliais em 2,3 vezes (p<0,001). Dessa forma, os resultados do trabalho mostram importantes alterações neurogliais no plexo mioentérico ileal mediadas pela exposição pré-natal ao VPA, abrindo novos caminhos ao entendimento da fisiopatologia do TEA. / Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by social interaction and communication deficits and repetitive behavior. The ASD may present several comorbidities, as gastrointestinal alterations – including chronic constipation, gastroesophageal reflux, celiac disease and intestinal cramps. The ASD etiology might be related togenetic alteration and environmental risk factors during pregnancy, as exposure to valproic acid (VPA), which became a reliable tool for induce autism-like alterations in rodents. The present study aimed to evaluate alterations in enteric nervous system (ENS) in an animal model of ASD prenatally exposed to VPA. Pregnant Wistar females received a single intraperitoneal injection of 600 mg/kg VPA or physiological saline (Control) on embryonic day 12.5 (E12.5). The 30 days old male offspring were euthanized by perfusion, and the ileum was removed and delaminated, followed by ileal myenteric ganglia (IMG) evaluation of total neurons (HuC/D+); excitatory neurons(ChAT+); inhibitory neurons(NOS+) and glial cells (GFAP+).The results showed less total neuronal density (p<0.05); no difference in ganglionic area; no difference in the inhibitory neurons population (density and body area); decreased neuronal density and increased body area of excitatory neurons (p<0.05) and 2.3 times increased in the number of glial cells (p<0.001). In conclusion, our results show important neuron-glia alterations in IMG mediated by prenatal exposure to VPA, opening new clues in the understanding of ASD pathophysiology.

O hipocampo é uma das principais estruturas relacionada aos processos de aprendizado e memória e também ao envelhecimento cerebral. Os astrócitos são células dinâmicas que mantém a homeostase cerebral, regulando sistemas de neurotransmissores, processamento da informação sináptica, metabolismo energético, liberação de fatores tróficos, defesas antioxidantes e resposta inflamatória. Considerando a relevância dessa estrutura cerebral e também a fundamental importância dos astrócitos para manutenção das condições fisiológicas do sistema nervoso central, nesse trabalho nós aprimoramos o estudo de um modelo de cultura de astrócitos hipocampais de ratos Wistar adultos (90 dias) e envelhecidos (180 dias), previamente padronizado pelo grupo. Observamos aqui a alteração da expressão dos principais marcadores gliais, em função da idade: proteína glial fibrilar ácida (GFAP), vimentina, transportadores de glutamato (GLAST e GLT-1) e enzima glutamina sintetase (GS). Além disso, também observamos alterações na liberação de GDNF, BDNF, S100B e TGF-β dependente da idade. Esses astrócitos ainda apresentaram parâmetros de estresse oxidativo/nitrosativo aumentados com o envelhecimento cerebral com simultânea disfunção mitocondrial e da enzima NADPH oxidase (NOX). Ainda em relação à homeostase redox, foram observadas disfunções nas enzimas superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GPx) e glutationa (GSH) em função da idade. Os astrócitos maduros também desencadearam uma resposta inflamatória distinta dos astrócitos de animais neonatos com aumento dos níveis de citocinas proinflamatórias como TNF-α, IL-1β, IL-6, IL-18 e MCP-1. Os principais mecanismos associados a alterações gliais em função da idade em cultura de astrócitos hipocampais envolve as vias de sinalização NFkB, p38, Nrf-2 e HO-1. Essas vias de sinalização estão relacionadas à homeostase redox e resposta inflamatória. Esses resultados reforçam o papel dos astrócitos hipocampais como alvo para o entendimento dos mecanismos envolvendo o envelhecimento cerebral bem como desordens neurológicas relacionadas a ele. / The hippocampus is one of the structures more closely related to behavior, neurochemical and cellular alterations during aging. Astrocytes are dynamic cells that maintain brain homeostasis by regulating neurotransmitter systems, synaptic information processing, energy metabolism, release of trophic factors, antioxidant defense and inflammatory response. Considering the importance of this brain structure and also the astrocyte importance for maintenance physiological conditions of the central nervous system, in this work we have improved the study of hippocampal astrocytes culture model of adult Wistar rats (90 days) and aged (180 days), previously standardized by the group. Here we see the age-dependent changes of expression of the major glial markers: glial fibrillary acidic protein (GFAP), vimentin, glutamate transporters (GLAST and GLT-1) and glutamine synthetase (GS).The GDNF, BDNF, S100B protein and TGF-β also changed their release with age. Astrocytes showed an age-dependent increase in oxidative/nitrosative stress with significant mitochondrial and NADPH oxidase (NOX) dysfunction. Furthermore, alterations related to age in superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities and glutathione (GSH) levels contribute to impaired redox state. Astrocytes also displayed age-dependent inflammatory response with augment of proinflammatory cytokine levels such as TNF-α, IL-1β, IL-6, IL-18 and chemokine MCP-1. The putative mechanisms associated to age-dependent changes in glial functionality in hippocampal astrocyte cultures involve NFκB, p38, Nrf-2 and HO-1 pathways. These signaling pathways are critical to interconnect redox homeostasis and inflammatory response. These results reinforce the role of hippocampal astrocytes as target for understanding mechanisms involved in aging and age-related neurological disorders.

Tanto a proteína glial fibrilar ácida (GFAP) quanto a proteína S100B têm sido utilizadas como marcadores de plasticidade astroglial, particularmente em danos encefálicos; entretanto, elas não necessariamente sofrem alterações ao mesmo tempo ou no mesmo sentido. Neste trabalho, nós induzimos um modelo da doença de Parkinson (DP) através de injeções intraestriatais de 6-OHDA em ratos e investigamos as alterações em GFAP e S100B, por meio de ELISA, na substância negra (SN), estriado e líquido cefalorraquidiano (LCR), um, sete e 21 dias após a cirurgia. O modelo experimental foi validado por meio da medida do comportamento rotacional induzido por metilfenidato e conteúdo da enzima tirosina-hidroxilase (TH) na SN e estriado. Até onde sabemos, esta é a primeira vez que se mostra a dosagem de S100B e GFAP no LCR no modelo de 6-OHDA. Foi identificada uma gliose no estriado (com base no aumento de GFAP), mas tal alteração não foi identificada na SN. Identificamos um aumento transitório de S100B e GFAP, no 1° e 7° dias após a cirurgia, respectivamente. Esta alteração inicial de S100B no LCR aparentemente está relacionada à lesão mecânica. Entretanto, em culturas de astrócitos, confirmamos a indução da secreção de S100B provocada por 6-OHDA. A toxina dopaminérgica MPTP também foi capaz de aumentar a secreção. Estes dados apontam para um efeito direto das toxinas 6-OHDA e MPTP nas células gliais. Também testamos o efeito destas toxinas em fatias estriatais frescas. Porém, neste tipo de preparação, não foram capazes de induzir a secreção de S100B, em 1h. O conteúdo intracelular de S100B e GFAP não foi alterado. Os dados apresentados reforçam a utilização destas toxinas em modelos da DP, além de indicar a importância da proteína S100B como um marcador útil nesta doença. / Both glial fibrillary acidic protein (GFAP) and S100B have been used as markers of astroglial plasticity, particularly in brain injury; however, they do not necessarily change in the same time frame or direction. Herein, we induced a Parkinson’s disease (PD) model via a 6-OHDA intrastriatal injection in rats and investigated the changes in GFAP and S100B using ELISA in the substantia nigra (SN), striatum, and cerebrospinal fluid on the 1st, 7th, and 21st days following the injection. The model was validated using measurements of rotational behaviour induced by methylphenidate and tyrosine hydroxylase in the dopaminergic pathway. To our knowledge, this is the first measurement of cerebrospinal fluid S100B and GFAP in the 6-OHDA model of PD. Gliosis (based on a GFAP increase) was identified in the striatum, but not in the SN. We identified a transitory increment of cerebrospinal fluid S100B and GFAP on the 1st and 7th days, respectively. This initial change in cerebrospinal fluid S100B was apparently related to the mechanical lesion. However, the 6-OHDA-induced S100B secretion was confirmed in astrocyte cultures. MPTP also increased S100B secretion in astrocyte cultures. These data point to a direct effect of the toxins 6-OHDA and MPTP on glial cells evaluated by S100B secretion. Under these conditions acute striatal slices did not secrete S100B in response to these toxins. No direct changes were observed in the cellular content of S100B or GFAP. In summary, these data reinforce the use of these toxins in PD models, as well as indicate the importance of the glial-derived protein S100B as useful marker in PD.

O hipocampo é uma das principais estruturas relacionada aos processos de aprendizado e memória e também ao envelhecimento cerebral. Os astrócitos são células dinâmicas que mantém a homeostase cerebral, regulando sistemas de neurotransmissores, processamento da informação sináptica, metabolismo energético, liberação de fatores tróficos, defesas antioxidantes e resposta inflamatória. Considerando a relevância dessa estrutura cerebral e também a fundamental importância dos astrócitos para manutenção das condições fisiológicas do sistema nervoso central, nesse trabalho nós aprimoramos o estudo de um modelo de cultura de astrócitos hipocampais de ratos Wistar adultos (90 dias) e envelhecidos (180 dias), previamente padronizado pelo grupo. Observamos aqui a alteração da expressão dos principais marcadores gliais, em função da idade: proteína glial fibrilar ácida (GFAP), vimentina, transportadores de glutamato (GLAST e GLT-1) e enzima glutamina sintetase (GS). Além disso, também observamos alterações na liberação de GDNF, BDNF, S100B e TGF-β dependente da idade. Esses astrócitos ainda apresentaram parâmetros de estresse oxidativo/nitrosativo aumentados com o envelhecimento cerebral com simultânea disfunção mitocondrial e da enzima NADPH oxidase (NOX). Ainda em relação à homeostase redox, foram observadas disfunções nas enzimas superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GPx) e glutationa (GSH) em função da idade. Os astrócitos maduros também desencadearam uma resposta inflamatória distinta dos astrócitos de animais neonatos com aumento dos níveis de citocinas proinflamatórias como TNF-α, IL-1β, IL-6, IL-18 e MCP-1. Os principais mecanismos associados a alterações gliais em função da idade em cultura de astrócitos hipocampais envolve as vias de sinalização NFkB, p38, Nrf-2 e HO-1. Essas vias de sinalização estão relacionadas à homeostase redox e resposta inflamatória. Esses resultados reforçam o papel dos astrócitos hipocampais como alvo para o entendimento dos mecanismos envolvendo o envelhecimento cerebral bem como desordens neurológicas relacionadas a ele. / The hippocampus is one of the structures more closely related to behavior, neurochemical and cellular alterations during aging. Astrocytes are dynamic cells that maintain brain homeostasis by regulating neurotransmitter systems, synaptic information processing, energy metabolism, release of trophic factors, antioxidant defense and inflammatory response. Considering the importance of this brain structure and also the astrocyte importance for maintenance physiological conditions of the central nervous system, in this work we have improved the study of hippocampal astrocytes culture model of adult Wistar rats (90 days) and aged (180 days), previously standardized by the group. Here we see the age-dependent changes of expression of the major glial markers: glial fibrillary acidic protein (GFAP), vimentin, glutamate transporters (GLAST and GLT-1) and glutamine synthetase (GS).The GDNF, BDNF, S100B protein and TGF-β also changed their release with age. Astrocytes showed an age-dependent increase in oxidative/nitrosative stress with significant mitochondrial and NADPH oxidase (NOX) dysfunction. Furthermore, alterations related to age in superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities and glutathione (GSH) levels contribute to impaired redox state. Astrocytes also displayed age-dependent inflammatory response with augment of proinflammatory cytokine levels such as TNF-α, IL-1β, IL-6, IL-18 and chemokine MCP-1. The putative mechanisms associated to age-dependent changes in glial functionality in hippocampal astrocyte cultures involve NFκB, p38, Nrf-2 and HO-1 pathways. These signaling pathways are critical to interconnect redox homeostasis and inflammatory response. These results reinforce the role of hippocampal astrocytes as target for understanding mechanisms involved in aging and age-related neurological disorders.

Tanto a proteína glial fibrilar ácida (GFAP) quanto a proteína S100B têm sido utilizadas como marcadores de plasticidade astroglial, particularmente em danos encefálicos; entretanto, elas não necessariamente sofrem alterações ao mesmo tempo ou no mesmo sentido. Neste trabalho, nós induzimos um modelo da doença de Parkinson (DP) através de injeções intraestriatais de 6-OHDA em ratos e investigamos as alterações em GFAP e S100B, por meio de ELISA, na substância negra (SN), estriado e líquido cefalorraquidiano (LCR), um, sete e 21 dias após a cirurgia. O modelo experimental foi validado por meio da medida do comportamento rotacional induzido por metilfenidato e conteúdo da enzima tirosina-hidroxilase (TH) na SN e estriado. Até onde sabemos, esta é a primeira vez que se mostra a dosagem de S100B e GFAP no LCR no modelo de 6-OHDA. Foi identificada uma gliose no estriado (com base no aumento de GFAP), mas tal alteração não foi identificada na SN. Identificamos um aumento transitório de S100B e GFAP, no 1° e 7° dias após a cirurgia, respectivamente. Esta alteração inicial de S100B no LCR aparentemente está relacionada à lesão mecânica. Entretanto, em culturas de astrócitos, confirmamos a indução da secreção de S100B provocada por 6-OHDA. A toxina dopaminérgica MPTP também foi capaz de aumentar a secreção. Estes dados apontam para um efeito direto das toxinas 6-OHDA e MPTP nas células gliais. Também testamos o efeito destas toxinas em fatias estriatais frescas. Porém, neste tipo de preparação, não foram capazes de induzir a secreção de S100B, em 1h. O conteúdo intracelular de S100B e GFAP não foi alterado. Os dados apresentados reforçam a utilização destas toxinas em modelos da DP, além de indicar a importância da proteína S100B como um marcador útil nesta doença. / Both glial fibrillary acidic protein (GFAP) and S100B have been used as markers of astroglial plasticity, particularly in brain injury; however, they do not necessarily change in the same time frame or direction. Herein, we induced a Parkinson’s disease (PD) model via a 6-OHDA intrastriatal injection in rats and investigated the changes in GFAP and S100B using ELISA in the substantia nigra (SN), striatum, and cerebrospinal fluid on the 1st, 7th, and 21st days following the injection. The model was validated using measurements of rotational behaviour induced by methylphenidate and tyrosine hydroxylase in the dopaminergic pathway. To our knowledge, this is the first measurement of cerebrospinal fluid S100B and GFAP in the 6-OHDA model of PD. Gliosis (based on a GFAP increase) was identified in the striatum, but not in the SN. We identified a transitory increment of cerebrospinal fluid S100B and GFAP on the 1st and 7th days, respectively. This initial change in cerebrospinal fluid S100B was apparently related to the mechanical lesion. However, the 6-OHDA-induced S100B secretion was confirmed in astrocyte cultures. MPTP also increased S100B secretion in astrocyte cultures. These data point to a direct effect of the toxins 6-OHDA and MPTP on glial cells evaluated by S100B secretion. Under these conditions acute striatal slices did not secrete S100B in response to these toxins. No direct changes were observed in the cellular content of S100B or GFAP. In summary, these data reinforce the use of these toxins in PD models, as well as indicate the importance of the glial-derived protein S100B as useful marker in PD.

Healthy brain development requires coordinated synapse growth and synapse elimination, with disruptions to these processes often resulting in neurodevelopmental disorder. While glia, the non-neuronal cells of the brain, are increasingly recognized as important regulators of both of these processes, the extent of this regulation and, in the case of disorder, dysregulation is still unknown. In this dissertation, I made classic use of the mouse visual system to outline the contours of glial regulation of synapse development in both synapse growth and synapse elimination. First, I examined astrocytes and microglia in the context of normal brain development, characterizing their spatiotemporal expression patterns in and around the mouse optic tract throughout late embryonic and early postnatal development, as RGC axons are growing into their synaptic target, the dLGN (Chapter 2). Next, I examined astrocyte and microglia in the context of disorder. Here, I found that synapses are reduced in size and eye-specific RGC synapse segregation is enhanced in a mouse model of Fragile X Syndrome, the most common single-gene cause of autism and intellectual disability, (Fmr1 KO mouse) during brain development. I identified glial phagocytic genes as disrupted within the developing Fmr1 KO dLGN and demonstrated that both microglial and astrocytic engulfment of synapses were aberrantly increased during this period of enhanced segregation, providing evidence that over-active glial engulfment may drive aberrant synapse refinement during development in a model of Fragile X Syndrome (Chapter 3).

Glia, whose name derives from the original Greek word, meaning “glue,” have long been understood to be cells that play an important functional role in the nutritive and structural support of the central nervous system, yet their full involvement has been historically undervalued. Despite the strong evidence that glial reactions to cellular debris govern the health of the nervous system, the specific properties of damaged axonal debris and the mechanisms by which glia sense them, morphologically adapt to their presence, and initiate phagocytosis for clearance, have remained poorly understood. The work presented in this thesis was aimed at addressing this fundamental gap in our understanding of the role for glia in neurodegenerative processes. I demonstrate that the cellular machinery responsible for the phagocytosis of apoptotic cell corpses is well conserved from worms to mammals. Draper is a key component of the glial response machinery and I am able to show here, for the first time, that it signals through Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70. Shark binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. I show that Shark activity is essential for Draper-mediated signaling events in vivo, including the recruitment of glial membranes to axons undergoing Wallerian degeneration. I further show that the Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. Therefore I propose that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the subsequent downstream activation of the Draper pathway. I observed that these Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signaling events in myeloid and lymphoid cells. Thus, Draper appears to be an ancient immunoreceptor with an extracellular domain tuned to modified-self antigens and an intracellular domain that promotes phagocytosis through an ITAM domain-SFK-Syk-mediated signaling cascade. I have further identified the Drosophila guanine-nucleotide exchange factor (GEF) complex Crk/Mbc/dCed-12, and the small GTPase Rac1 as novel modulators of glial clearance of axonal debris. I am able to demonstrate that Crk/Mbc/dCed-12 and Rac1 function in a non-redundant fashion with the Draper pathway to promote a distinct step in the clearance of axonal debris. Whereas Draper signaling is required early during glial responses, promoting glial activation and extension of glial membranes to degenerating axons, the Crk/Mbc/dCed-12 complex functions at later stages of glial response, promoting the actual phagocytosis of axonal debris. Finally, many interesting mutants have been identified in primary screens for genes active in neurons that are required for axon fragmentation or clearance by glia, and genes potentially active in glia that orchestrate clearance of fragmented axons. The further characterization of these genes will likely unlock the mystery surrounding “eat me” and “find me” cues hypothesized to be released or exposed by neurons undergoing degeneration. Illuminating these important glial pathways could lead to a novel therapeutic approach to brain trauma or other neurodegenerative conditions by providing a druggable means of inducing early attenuation of the glial response to injury down to levels less damaging to the brain. Taken together, my combined work identifies new components of the glial engulfment machinery and shows that glial activation, phagocytosis of axonal debris, and the termination of glial responses to injury are genetically separable events mediated by distinct signaling pathways.

Interactions between neurons and glia occupy a central role in many aspects of development, maintenance, and function of the central nervous system (CNS). A fundamental event in CNS development is the elaboration of two distinct neuronal processes, axons and dendrites. The overall aim of this research was to characterize the interactions between central nervous system neurons and astroglial cells that regulate dendrite growth from cerebral cortical neurons. Embryonic (E18) mouse cerebral cortical neurons were cocultured with early postnatal (P4) rat astroglia derived from cerebral cortex, retina, olfactory bulb, mesencephalon, striatum and spinal cord. Axon and dendrite outgrowth from isolated neurons was quantified using morphological and double-labeling immunohistochemical techniques at 18 hours and 1, 3 and 5 days in vitro. Neurons initially extended the same number of neurites, regardless of the source of glial monolayer; however, astroglial cells differed in their ability to maintain primary dendrites. Homotypic cortical astroglia maintained the greatest number of primary dendrites. Astroglia derived from the olfactory bulb and retina maintained intermediate numbers of dendrites, whereas only a small number of primary dendrites were maintained by astroglia derived from striatum, spinal cord or mesencephalon. Initially longer axons were observed from neurons grown on astroglia that did not maintain dendrite number. After 5 days in vitro, axon growth was similar on the various monolayers, total primary dendrite outgrowth, however, was nearly threefold greater on astroglia derived from the cortex, retina and olfactory bulb than on astroglia derived from mesencephalon, striatum or spinal cord. This effect was principally on the number of primary dendrites rather than the elongation of individual dendrites and was independent of neuron survival. Similar morphological differences were observed after 5 days in vitro when cortical neurons were grown on polylysine in either a noncontact coculture system where astroglia continuously conditioned the culture medium or in astroglial conditioned medium. Preliminary biochemical analysis of the medium conditioned by cortical astroglia using heat and trypsin degradation, ultracentrifugation, dialysis, and heparin affinity chromatography suggested that a heparin binding protein with a molecular weight between 10 and 100kDa may be responsible for astroglial mediated dendrite growth. Neurons that were grown in medium conditioned by either mesencephalic or cortical astroglia for the first 24 hours followed by culture medium from astroglia of the alternate source for 4 days in vitro, confirmed that astroglia maintained, rather than initiated, the outgrowth of the primary dendritic arbor. In the next series of experiments, E18 mouse cortical neurons were cocultured with neonatal (P4) or mature (P12) rat astroglia derived from cortex and mesencephalon or astroglia derived from P4 and P12 lesioned cortex. After 5 days in vitro, the maturational age of astroglia did not appear to alter the extent of primary dendrite growth; instead dendrite growth reflected the region of the CNS from which the astroglia were derived. By contrast, a reduced ability to support axon growth from mouse cortical neurons in culture was observed on astroglia derived from mature rat cortex or mesencephalon. Reactive astroglia demonstrated similar neurite supporting characteristics to mature astroglia and were able to maintain dendrite growth, principally primary dendrite number. Axon elongation, however, was reduced on both neonatal and mature reactive astroglia. Neuron survival did not correlate with the ability of the various astroglia to support process outgrowth. Collectively these results indicate: 1) neuron-glial interactions are critical for the regulation of process outgrowth from embryonic cortical neurons in vitro, 2) axon and dendrite growth appear to be differently controlled by astroglia, 3) CNS astroglia demonstrate regional differences in maintaining, but not initiating growth of the primary dendritic arbor, 4) this effect may be due, in part, to release of a diffusible heparin binding protein factor, and 5) mature and reactive astroglia support primary dendrite, but limited axon growth. We propose therefore that the local astroglial environment maintains primary dendrite growth from neurons until synaptic contacts can be established. A mechanism that maintains the primary dendritic arbor and allows separate regulation of axon and dendrite growth, prior to the arrival of afferents, may be critical for establishing appropriate and specific synaptic connections. These findings have important implications in understanding development and function of the mammalian central nervous system and may lead to novel strategies for intervention in acute and chronic neurological disorders.