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<p>Several classes of psychotropic medications are known to produce neurological side effects. It has long been recognized that antipsychotic drugs classically block the D₂ subtype of DA receptors inducing a range of acute and subacute extrapyramidal syndromes (EPS), including parkinsonism and akathisia, as well as chronic syndromes such as tardive dyskinesia. More recently, SSRI-type drugs, which, as the name suggests, inhibit the serotonin transporter (SERT), and have been found to induce a similar profile of EPS. It is unclear how medications with such different pharmacological actions can produce similar neurological side effects. The goal of this thesis was to study the neurochemical alterations induced by antipsychotic and SSRI medications, with a specific focus on the nigrostriatal pathway, the causative location of parkinsonism.</p> <p>Environmental enrichment and exercise (EE) has been shown to have protective effects in various neurological settings. In the first experiment, we studied the changes induced by SERT inhibition compared to those induced by a non-pharmacological form of therapy, namely, environmental enrichment with exercise. The SSRI, fluoxetine (FLX) significantly reduced the levels of tyrosine hydroxylase (TH) and phosphorylated glycogen synthase kinase-3β (pGSK-3β-inactive), while increasing phosphorylated TH (pTH) in the striatum (STR). EE also reduced TH and increased pTH, but contrary to FLX, it significantly increased striatal pGSK-3β protein expression.</p> <p>Microglia, the brain’s primary immune cells, have been implicated in several neuroinflammatory conditions, including Parkinson’s disease. The purpose of the second experiment was to explore the modulatory effects of microglia on neuroleptic-induced changes in the nigrostriatal system. The typical antipsychotic, haloperidol (HAL), did not affect the overall levels of TH, though it did induce a robust increase in pTH. The microglial NADPH oxidase inhibitor, apocynin (APO), significantly attenuated this increase in pTH. HAL also induced a significant increase in striatal pGSK-3β, while apocynin, rather surprisingly, induced a stark decrease in pGSK-3β protein expression.</p> <p>The results of this thesis indicate that both pTH and pGSK-3β are intriguing markers to study in the context of dopamine neurotransmission. In addition, EE proved to be a valuable modality in which to compare the downstream effects of pharmacological treatment. It is also clear that microglia fulfill an undefined, but fascinating role as modulators of neural transmission.</p> / Master of Science in Medical Sciences (MSMS)

Traumatic brain injury (TBI) is a major medical concern that has demonstrated to be particularly challenging to treat because of the disparity amongst injury modes and severities. Increased use of explosive devices during combat has caused blast TBI (bTBI) to become a widespread consequence in military and Veteran populations, and impact-related trauma from contact-related sports or motor vehicle accidents has made mild impact-induced TBIs (concussion) a major health problem. There is a high risk for those who have sustained a TBI to develop behavioral and cognitive disorders following injury, and these symptoms can present as delayed onset, causing diagnosis to be a major feat when planning for treatment and long-term healthcare. Both preclinical and clinical studies report the neuropathological changes following TBI, yet investigating the distinct mechanistic changes in blast and impact trauma that contribute to pathological disparities has yet to be elucidated. Microglia dynamics play a key role in initiating the inflammatory response after injury, as microglia become activated by undergoing morphological changes that influence their function in the injured brain, and unique signaling pathways influence their functional inflammatory states. While previous literature report on the unique responses of microglia, their mediated-inflammatory responses are still not well defined. This work aimed to investigate the acute and subacute responses of microglia to injury through their diverse activation states following blast and impact trauma. The work herein employed rodent models to investigate these changes, finding that microglia activation was spatially and temporally heterogeneous within and across injury paradigms. Three days following bTBI, activated microglia in the cortex displayed morphologies similar to microglia that are known to increase their interactions with dysfunctional synapses, while dystrophic microglia were prevalent in the hippocampus seven days following injury. Moreover, transhemispheric changes in microglia activation were noted following impact TBI, with stressed/primed microglia responding to immune challenges of the cortex at three days, whereas a unique morphological state that was markedly different from those traditionally reported in CNS injury and disease was present within the hippocampus three- and seven-days following injury. State-of-the-art cell sorting techniques were used for in vivo analysis of microglia, which also exhibited that functional changes of microglia vary between injury paradigms, providing insight into how differences in primary insult may elicit distinct signaling pathways involved in microglia-mediated inflammatory responses. These in vivo studies were then crucial in understanding the malleable responses of microglia to complex injuries such as "blast plus impact" TBI, indicating that phenotypic changes in microglia following this injury are also unique and spatially heterogeneous. To date, therapeutic efforts for TBI are limited due to the lack of understanding the underlying mechanisms that influence TBI pathology. This work also investigated novel therapeutic targets, noting that administration of polyester nanoparticles restored microglia to baseline levels following impact. The fundamental research presented in this study is innovative and advantageous as it can provide essential data into targeted and personalized treatments that can improve long-term healthcare and ultimately, the quality of life for those suffering from a TBI. / Doctor of Philosophy / Traumatic brain injury (TBI) is a major medical concern that has demonstrated to be particularly challenging to treat because of the differences in injury modes and severities. Increased use of explosive devices during combat has caused blast TBI (bTBI) to become a widespread result in military and Veteran populations, and impact-related trauma from contact sports or motor vehicle accidents has made mild impact-induced TBIs (concussion) a major health problem. There is a high risk for those who have sustained a TBI to develop behavioral and cognitive disorders following injury, and these symptoms can present later on, causing diagnosis to be a major feat when planning for treatment and long-term healthcare. Microglia play a key role in inducing the inflammatory response after injury, as they change shape and size, which then influences their function in the injured brain. Although prior research reports on the unique responses of microglia, their effects on inflammation following TBI are still not well defined. This work aimed to investigate the early responses of microglia to injury through their diverse activation states following blast and impact trauma. The experiments in this study used animal models, finding that microglia activation can be distinct across time and brain regions, which may be injury-type-specific. To date, therapeutic efforts of TBI are limited due to the lack of understanding the underlying mechanisms that influence TBI pathology. This work also investigated beneficial treatments for TBI, noting that administration of nanoparticles helped restore microglia to levels similar to the control group. The fundamental research presented in this study is innovative and important as it can provide essential data into targeted and personalized treatments that can improve long-term healthcare and ultimately the quality of life for those suffering from a TBI.

The central nervous system (CNS) is protected by a vascular blood-brain barrier that prevents many types of pathogens from entering the brain. Still, some pathogens have evolved mechanisms to traverse this barrier and establish a long-term infection. The apicomplexan parasite, Toxoplasma gondii, is one such pathogen with the ability to infect the CNS in virtually all warm-blooded animals, including humans. Across the globe, an estimated 30% of the human population is infected with Toxoplasma, an infection for which mounting evidence suggests increases the risk for developing neurological and neuropsychiatric disorders, like seizures and schizophrenia. In my dissertation, I investigate the telencephalic neural circuit changes induced by long-term Toxoplasma infection in the mouse brain and the neuroimmune signaling role of the complement system in mediating microglial remodeling of neural circuits following parasitic infection. While there has been keen interest in investigating neural circuit changes in the amygdala – a region of the brain involved in fear response and which Toxoplasma infection alters in many species of infected hosts – the hippocampus and cortex have been less explored. These are brain regions for which Toxoplasma also has tropism, and moreover, are rich with fast-spiking parvalbumin perisomatic synapses, a type of GABAergic synapse whose dysfunction has been implicated in epilepsy and schizophrenia. By employing a range of visualization techniques to assess cell-to-cell connectivity and neuron-glia interactions (including immunohistochemistry, ultrastructural microscopy, and microglia-specific reporter mouse lines), I discovered that longterm Toxoplasma infection causes microglia to target and ensheath neuronal somata in these regions and subsequently phagocytose their perisomatic inhibitory synapses. These findings provide a novel model by which Toxoplasma infection within the brain can lead to seizure susceptibility and a wider range of behavioral and cognitive changes unrelated to fear response. In the Toxoplasma infected brain, microglia, along with monocytes recruited to the brain from the periphery, coordinate a neuroinflammatory response against pathogenic invasion. This is characterized by a widespread activation of these cells and their increased interaction with neurons and their synaptic inputs. Yet, whether T. gondii infection triggers microglia and monocytes (i.e. phagocytes) to target, ensheath, and remove perisomatic inhibitory synapses on neuronal somata indiscriminately, or whether specificity exists in this type of circuit remodeling, remained unclear. Through a comprehensive assessment of phagocyte interactions with cortical neuron subtypes, I demonstrate that phagocytes selectively target and ensheath excitatory pyramidal cells in long-term infection. Moreover, coupling of in situ hybridization with transgenic reporter lines and immunolabeling revealed that in addition to phagocytes, excitatory neurons also express complement component C3 following infection (while inhibitory interneurons do not). Lastly, by employing targeted deletion of complement components, C1q and C3, I show that complement is required for phagocyte ensheathment of excitatory cells and the subsequent removal of perisomatic inhibitory synapses on these cells (albeit not through the classical pathway). Together, these studies highlight a novel role for complement in mediating synapse-type and cell-type specific circuit remodeling in the Toxoplasma infected brain. / Doctor of Philosophy / Parasites are microorganisms that rely on other living organisms (called hosts) for their survival. Although some parasites only live on their hosts, others have developed ways to establish infections and obtain the nutrients that keep them alive from host cells. My Ph.D. research has focused on studying one of these parasites, Toxoplasma gondii (commonly referred to as Toxo), that has evolved the unique ability to establish brain infections in almost all animals around the world, from rodents to humans. Recent discoveries show that brain infection with this parasite can cause seizures, an imbalance in the way that specialized cells of the brain (called neurons) communicate with each other, causing harmful hyperactivity within the brain. Toxo infection can also cause behavioral and cognitive changes in infected animals, making them more susceptible to predation. In humans, infection with Toxo increases their risk for developing different types of mental illness, such as schizophrenia. The focus of my Ph.D. research has been in trying to understand, at the cellular and molecular level, how infection with this parasite can lead to seizures and behavioral changes, by using mice as a model. Mice have a similar brain structure to humans, and over the years, scientists have developed many tools that allow us to visualize and study the connections between neurons (called synapses). I'm interested in understanding how changes in these connections affect how neurons communicate with each other, and ultimately, how we behave and think. I have been studying a type of connection that, if lost or damaged, can lead to seizures and some types of mental illness. These connections are called 'perisomatic inhibitory synapses', and they form on many distinct types of neurons, but specifically on the cell bodies of these neurons. They act as a traffic light, informing neurons when and for how long to 'slow down' their activity. I discovered that after the parasite enters the brain, it causes another type of cell in the brain, called microglia, to extensively interact with neurons in the cortex and hippocampus (areas of your brain important for thinking, executing behavior, and learning). Microglia are immune cells of the brain that inspect the brain for anything damaged or that doesn't belong (like parasites) and removes them from the brain. By performing experiments where I delete individual immune molecules from mice, I found that one immune molecule, called 'complement component C3' acts as cue for microglia to find these cells, wrap around them, and permanently remove these important connections. Surprisingly, however, microglia don't remove these connections from all neurons, indiscriminately, they do so only on one specific cell type called 'excitatory pyramidal neurons,' and as the name implies, they're the ones who drive activity in the brain. My half-a-decade's worth of research helps us understand parasitic infections in the brain in a couple of ways: First, I have discovered one of the mechanisms by which neuronal connections are lost in the Toxo-infected brain (which is a mechanism that leads to loss of neuronal connections in the injured and aging brain as well). This is significant because it might provide insight into why some people who are infected with Toxo develop seizures or mental illness, while others don't. More importantly, Toxo-infection causes changes in the brain that are very specific, in terms of both the type of neuronal connection that is affected and the type of cell that is affected. Why these changes are so specific remain to be uncovered, but it suggests that Toxo can either a) trigger a unique immune response in the brain that leads to very precise changes in neuron-toneuron connections and signaling or b) the parasite, while hiding inside of neurons, may hijack the machinery of certain cell types in a way that helps them survive longer.

O envelhecimento, processo iminente a todo ser vivo, no SNC é caracterizado por alterações como, por exemplo, a neuroinflamação crônica, que estão associadas a processos de neurodegeneração e ao aumento da incidência de doenças neurológicas ligadas ao surgimento de demência. A insulina, o hormônio anabólico mais importante descoberto até hoje, tem sua sinalização como processo vital que está presente desde bactérias até a espécie humana e desde os tecidos periféricos até o SNC. Mesmo a sinalização cerebral de insulina sendo um tema bem definido na literatura, pouco se sabe sobre a sua função em células da glia, principalmente astrócitos e microglia, componentes chaves do processo de neuroinflamação. A neuroinflamação foi considerada, por muitos anos, tóxica ao SNC, mas atualmente evidências importantes têm sido encontradas sugerindo que processos pró-inflamatórios são primariamente benéficos ao cérebro ou encéfalo e podem assumir papel tóxico à medida que se tornam crônico. Assim, considerando o papel da insulina no SNC, bem como o aumento da expectativa de vida da população mundial que acarreta o aumento dramático da incidência de doenças neurodegenerativas, foi investigada, na presente tese a relação da sinalização fisiológica de insulina com processos cognitivos, neurotróficos e neuroinflamatórios e também a resistência na sinalização da mesma causada pelo envelhecimento cerebral. Foi demonstrado que a administração intracerebroventricular de insulina melhora a cognição de animais jovens, mas o mesmo não ocorre no envelhecimento. A nível celular e molecular, foi visto um distúrbio na conexão da sinalização de insulina e BDNF, bem como na ativação microglial e sinalização pró-inflamatória da insulina que parecem estar comprometidos no envelhecimento. Além disso, foi observado que a microglia é sensível à sinalização direta de insulina via PI3K e que essa sinalização microglial é adaptada e sofre mudanças na vida adulta. Em conjunto com a literatura, foi demostrado por esta tese que existe uma ruptura de paradigmas na interpretação dos processos neuroinflamatórios, que deixam de ser vistos somente como um fator tóxico ao cérebro, mas também como um artifício elementar de adaptação do SNC aos diversos estímulos que as células nervosas recebem durante o curso da vida, desde o nascimento até o envelhecimento. / Aging is a process that is found in all living being in the CNS. It is characterized by modifications, such as chronic neuroinflammation, which are associated with neurodegeneration and represent a risk factor for neurological diseases. Insulin is the most important anabolic hormone ever discovered. Insulin signaling represents an essential process that is present from bacteria to humans and from the periphery to the brain. Insulin signaling in the CNS is a well-defined topic in the literature. Most of the knowledge regarding brain insulin signaling still report findings in neurons and little is known about insulin function in glia, especially astrocytes and microglia that are key players of neuroinflammation. Neuroinflammation has been considered a toxic factor to the CNS, however, in the last few years, important evidences have been found that proinflammatory processes are primarily beneficial and may play a toxic role as soon as they become chronic. Thus, considering the role of insulin in the CNS, as well as the increased populational life spam worldwide, the present thesis investigated the relation of physiological insulin signaling and the brain insulin signaling caused by aging in cognition, neurochemistry and neuroinflammation. We showed that insulin intracerebroventricular administration improved the cognition of young animals, but the same was not observed in aging. At the cellular and molecular level, we found a disruption in the connection of insulin and BDNF signaling. We also show that a microglial activation and pro-inflammation triggered by insulin in young brain appear to be lost during aging. In addition, it was observed that microglia is sensitive to direct insulin signaling via PI3K and that this microglial signaling suffers adaptations and changes during life. Together with the recently changes in the literature, the findings of this work demonstrate that there is a rupture of paradigms in the interpretation of neuroinflammatory processes, which are no longer seen only as a toxic factor to the brain, but also as an smart adaptation of the CNS to the various stimuli that brain cells receive during the course of life, from birth to aging.

A disfunção temporomandibular (DTM) afeta os músculos da mastigação, a articulação temporomandibular e estruturas associadas, induzindo dor por mecanismos pouco compreendidos. A inflamação tem papel relevante nessas disfunções crônicas pela produção de mediadores químicos. A fractalquina (FK) é uma quimiocina expressa na membrana de neurônios, e seu receptor esta presente na microglia, sugerindo que a FK induz a ativação da microglia, promovendo mudanças funcionais e liberação de mediadores inflamatórios, envolvidas na manutenção de processos nociceptivos. As terapias medicamentosas possuem efeitos indesejáveis. O laser de baixa intensidade (LBI) é uma alternativa promissora para o alívio da dor. O efeito do LBI foi avaliado sobre a nocicepção de animais em modelo de dor persistente e foi capaz de reverter a hipersensibilidade mecânica por uma inibição da resposta inflamatória local e inibição central de fractalquina reforçando papel do LBI como uma alternativa terapêutica importante no alívio dor e modulação da resposta inflamatória em nível central. / Temporomandibular dysfunction (TMD) affects the chewing muscles, the temporomandibular joint and associated structures, inducing pain by poorly understood mechanisms. Inflammation has a relevant role in these chronic dysfunctions by the production of chemical mediators. Fractalkine (FK) is a chemokine expressed on the membrane of neurons, and its receptor is present in the microglia, suggesting that FK induces the activation of microglia, promoting functional changes and release of inflammatory mediators involved in the maintenance of nociceptive processes. Drug therapies have undesirable effects. Low-intensity laser (LILT) is a promising alternative for pain relief. The effect of LILT was evaluated on the nociception of animals in a persistent pain model and was able to reverse mechanical hypersensitivity by an inhibition of the local inflammatory response and central inhibition of fractalkine enhancing LIL\'s role as an important therapeutic alternative in pain relief and modulation Inflammatory response at the central level.

O Acidente Vascular Encefálico (AVE) Hemorrágico representa mais de 10% de todos os casos de AVE e possui altas taxas de morbidade e de mortalidade. Os pacientes que sobrevivem a este evento permanecem com alguma disfunção motora, que algumas vezes é incapacitante. O extravasamento de sangue em um AVE hemorrágico ocorre, geralmente, em regiões onde há bifurcação de pequenas arteríolas penetrantes, como na região dos núcleos da base. O estriado, importante componente dessa região, está relacionado a funções motoras superiores, como o planejamento e a execução do movimento. Alguns estudos demonstram que o pré-condicionamento (PC) isquêmico pode gerar a tolerância a outros eventos que acometem o sistema nervoso. O PC é definido como fenômeno decorrente da exposição de um tecido ou órgão a um insulto sub-letal capaz de resultar em adaptações determinantes para a tolerância tecidual. Isso ocorre mesmo quando esses dois estímulos são de origens diferentes; neste caso diz-se que o PC desenvolveu tolerância cruzada. Desta forma, o presente estudo dedicou-se ao estudo de efeitos celulares e funcionais do pré-condicionamento isquêmico, por oclusão bilateral das artérias carótidas durante 10 minutos, sobre o modelo de hemorragia intracerebral (HIC), por administração intraestriatal de colagenase do tipo IV-S em ratos. A hipótese de trabalho era de que o PC causaria tolerância cruzada para a HIC, e consequente neuroproteção avaliada por testes motores, volume de lesão, com envolvimento de astrocitose e de micróglia reativa Foram usados 67 ratos machos Wistar adultos, divididos em 4 grupos: Sham (controle cirúrgico), PC, HIC, PC+HIC. Assim, os animais dos grupos PC e PC+HIC foram submetidos ao pré-condicionamento e 24 horas depois os animais HIC e PC+HIC receberam a injeção de colagenase, enquanto os animais Sham e PC receberam uma injeção de salina. A avaliação motora dos animais foi realizada a partir dos testes do cilindro e do Staircase. Trinta e quatro dias após a HIC os animais foram perfundidos e o estriado ipsilateral à injeção foi dissecada para obtenção de amostras teciduais necessárias à avaliação da perda tecidual e quantificação de intensidade de fluorescência de GFAP (proteína glial fibrilar ácida) e OX-42, importantes marcadores de astrócitos e microglia, respectivamente. Os resultados demonstram que: a) a HIC causa deficits motores em ambos os testes realizados, e que o PC reverte este efeito; b) a HIC causa lesão estriatal que não é revertido pelo pré-condicionamento; c) a HIC causa aumento da intensidade de fluorescência para GFAP e para OX-42, e o PC reverte apenas a reatividade da micróglia. Em conjunto, sugere-se que o pré-condicionamento isquêmico causa tolerância cruzada com a hemorragia intracerebral experimental, resultando em proteção funcional, mas não morfológica, possivelmente associada a uma diminuição da reatividade da microglia após o evento hemorrágico. / Hemorrhagic Vascular Stroke (EVA) represents more than 10% of all stroke cases with high rates of morbidity and mortality. Patients who survive this event, remain with some motor dysfunction, which is sometimes disabling. The extravasation of blood in a hemorrhagic stroke occurs, generally, in regions where there is bifurcation of small vessels, as in the region of striatum. The striatum is related to the higher motor functions, such as the planning and execution of the movement. Some studies have shown that preconditioning (PC) can generate a tolerance to other events that accompany the nervous system. The PC is presented as the source of the exposure of a sub-lethal, resulting in an adaptation of determinants to a tissue tolerance. Thus, the present study aimed shows the ischemic preconditioning effects, by bilateral occlusion of the carotid arteries for 10 minutes, on the intracerebral hemorrhage (ICH) model, by intra- striatum administration of type IV S collagenase in rats. The working hypothesis was tolerance to HIC, and consequent neuroprotection by motor function, lesion volume, astrocytosis and reactive microglia. A total of 84 male Wistar adult rats were divided into 4 groups: Sham (surgical control), PC, HIC, PC + HIC Thus, the animals of the PC and PC + HIC groups were introduced to the preconditioning and 24 hours later, the HIC and PC + HIC animals received a collagenase injection, while the Sham and PC animals received a saline injection. The evaluation of the animal function was performed from cylinder and Staircase tests. Thirty-four days after the surgery, the striatum was dissected and prepared to lesion volume analysis and fluorescence intensity of GFAP quantification (acid glial fibrillary protein) and OX-42, important astrocyte and microglia markers respectively. The results demonstrate that: a) an HIC causes motor deficits in both tests performed, and that the PC reverses this effect; b) an ICH causes a striatal lesion that is not reversed by preconditioning; c) an HIC promoted high fluorescence intensity for GFAP and OX-42, and PC reverses the microglia reactivity. Taken together, we suggest that ischemic preconditioning combined with experimental intracerebral hemorrhage, promotes functional but not morphological protection, being associated with the microglial reactivity decrease after the hemorrhagic event.