Injeções repetidas de psicoestimulantes e/ou compostos opióides desencadeiam respostas comportamentais, bioquímicas, endócrinas e celulares diferentes daquelas induzidas pela exposição aguda. Em particular, a morfina, considerada como o protótipo do estimulante opioidérgico, tem despertado grande interesse devido a seus efeitos múltiplos. Assim, no presente estudo avaliaram-se as consequências dos tratamentos agudo e repetido com morfina (20mg/kg) em camundongos sobre os seguintes aspectos: atividade geral; níveis séricos de corticosterona; concentrações corticais, estriatais e hipotalâmicas de noradrenalina, dopamina e serotonina, de seus metabólitos e as taxas de renovação destes neurotransmissores; atividade funcional ex vivo de neutrófilos sanguíneos e de macrófagos peritoneais, sendo estes desafios realizados in vivo com diferentes estímulos imunológicos, infecciosos ou não; crescimento tumoral e a sobrevida de camundongos portadores de um tumor ascítico de Ehrlich. Os resultados obtidos mostraram que os tratamentos agudo e repetido com morfina modulam diferentemente as repostas comportamental e neuroendócrina, dependendo do tempo de observação pós-desafio. Portanto, conclui-se que camundongos tratados repetidamente com morfina (20mg/kg) apresentam alterações comportamentais e neuroendócrinas que, no entanto, não foram acompanhadas por diferenças nas avaliações de atividade imune inata. / Repeated administrations of psychostimulant and/or opioid compounds trigger different behavioral, biochemical, endocrine and cellular responses as compared with those induced by acute exposure. Morphine, has attracted great interest due to its multiple effects. Thus, this study evaluated the effects of acute and repeated treatment with morphine (20mg/kg) in mice on the following: locomotor activity; serum levels of corticosterone; cortical, striatal and hypothalamic concentration of dopamine, noradrenaline and serotonin, as well as their metabolites and turnover; blood neutrophils and peritoneal macrophage activity ex vivo, which were challenged with different immunological stimulants; tumor growth and survival of mice with Erlich ascitic tumor. The results showed that acute and repeated morphine treatment differently modulated behavioral and neuroendocrine responses, depending on the these period after the injection challenge. Thus, it is concluded that mice repeatedly treated with morphine (20mg/kg) present behavioral and neuroendocrine changes; however, this changes were not accompanied by variation in innate immune activity.

Um aumento da atividade da PLA2 e alterações do sistema dopaminérgico tem sido descrito em esquizofrenia. No presente estudo, foram investigados os efeitos da atividade da PLA2 sobre os receptores D1 e D2 em cérebro post mortem de 10 sujeitos. Foi encontrado que a PLA2GVI é responsável por 85% do total de atividade da PLA2 no cérebro. A estimulação da PLA2GVI (por EDTA) aumentou a afinidade de D1 em estriado e em CPF e diminuiu a afinidade de D2 em estriado. A inibição da PLA2GVI (por BEL) diminuiu a afinidade de D1 em estriado, e em CPF e CT. A estimulação da PLA2GVI resultou em aumento na densidade de D1 em CPF e CT, e de D2 em estriado. Uma elevação da PLA2 em esquizofrenia poderia contribuir para a biologia da doença através de alterações na neurotransmissão dopaminérgica / Increased PLA2 activity and dopaminergic alterations have been described in schizophrenia. In the present study it was investigated the effects of PLA2 activity on D1 and D2 receptors in post mortem brain of 10 subjects. It was found that PLA2GVI corresponds to 85% of all PLA2 activity in the brain. The stimulation of PLA2GVI (by EDTA) increased D1 affinity in striatum and in PFC, and decreased D2 affinity in striatum. Conversely, the inhibition of PLA2GVI (using BEL) decreased D1 affinity in striatum, PFC and TC. The stimulation of PLA2GVI increased D1 density in PFC and TC, as well as the D2 density in striatum. The increased PLA2 activity in schizophrenia may contribute to the biology of the disease through alterations in dopaminergic neurotransmission

Excitotoxicity, a major hallmark of neurodegeneration associated with cerebral ischemia, is a result of accumulation of extracellular glutamate. This excess glutamate leads to hyperactivation of glutamate receptors such as the N-methyl-D-asparate (NMDA) receptors (NMDARs) following the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPARs). Excessive activation of NMDARs causes an influx of calcium, which can eventually activate apoptotic pathways and lead to death of neurons. Regulation of NMDAR subunit composition, localization, surface expression, and activity can balance cell survival via activation of either pro-death or pro-survival pathways after a course of an ischemic insult. Specifically, phosphorylation of different NMDAR subunits defines their activity and downstream signaling pathways. NMDARs are phosphorylated by multiple kinases and dephosphorylated by different phosphatases. Besides phosphatases and kinases, per se, phosphorylation of synaptic proteins that regulate kinase or phosphatase targeting and activity also mediate NMDAR phosphorylation. Spinophilin, a major synaptic scaffolding and protein phosphatase 1 (PP1) targeting protein, mediates substrate phosphorylation via its ability to bind PP1. Our studies focus on delineating the role of spinophilin in the regulation of phosphorylation and function of the GluN2B subunit of the NMDA receptor as well as the role of spinophilin in modulating glutamate-induced neurotoxicity. Interestingly, our data demonstrate that spinophilin sequesters PP1 away from GluN2B thereby enhancing phosphorylation of GluN2B at Ser-1284. These changes impact GluN2B protein interactions, subcellular localization, and surface expression, leading to alterations in the amount of calcium entering the neuron via GluN2B-containing NMDARs. Our data show that spinophilin biphasically regulates GluN2B function. Specifically, Ser-1284 phosphorylation enhances calcium influx through GluN2B containing NMDA receptors, but spinophilin leads to dramatic decreases in the surface expression of the receptor independent of Ser-1284 phosphorylation. Moreover, in spinophilin knockout mice, we observe less PP1 binding to GluN2B and less phosphorylation of Ser-1284, but more surface expression of GluN2B and greater levels of caspase activity. Together, these observations suggest a potential neuroprotective role for spinophilin by decreasing GluN2B-containing NMDA receptor-dependent surface expression and thereby decreasing intracellular calcium and neuronal cell death.

Indiana University-Purdue University Indianapolis (IUPUI) / Background: Group II metabotropic glutamate receptors (mGluR2/3) are predominately presynaptically located Gi/o coupled receptors that are highly expressed in the cortex, nucleus accumbens, amygdala, and hippocampus. Previous studies suggest that group II mGluRs are involved in regulating ethanol (EtOH) consumption and seeking following extinction (Backstrom and Hyytia, 2005; Kufahl, et al., 2011). The sipper tube model, which allows for procedural separation of seeking and consumption, was used to further clarify the role of mGluR2/3 in EtOH-seeking and consumption. The non-selective group II mGluR agonist LY379268 (LY37) and selective mGluR2 positive allosteric modulator (PAM) BINA were used to determine the relative contribution of mGlu2 and mGlu3 receptors on EtOH seeking and consumption. Following characterization of the agonist and PAM on EtOH reinforcement, a microinjection study was performed examining the effect of blockade of nucleus accumbens core mGluR2/3 on systemic agonist induced suppression of EtOH-seeking. Methods: For the systemic agonist/PAM experiments, separate groups of male Wistar rats [n=8-9 group; LY37 (0-2.0 mg/kg) and BINA (0-20 mg/kg)] were trained to complete a response requirement (RR) of 10 lever presses that resulted in access to 10% EtOH or 2% sucrose (in separate groups) for a 20-minute drinking period. For consummatory testing, animals received weekly drug injections with a RR1. The RR was then increased over sessions to a RR20. For appetitive testing, animals received weekly drug injections followed by a non-reinforced extinction session. To determine effects of blockade of NAc core mGluR2/3 receptors on agonist-induced suppression of EtOH-seeking, a separate group of male Wistar rats (n=15) was trained to complete a RR10 for access to 10% EtOH. Animals were surgically implanted with bilateral guide cannulae terminating 1mm above the NAc core. Following recovery, animals received four sets of microinjections in a balanced design (systemic vehicle + core vehicle, systemic LY37 + core vehicle, systemic LY37 + core LY34, and systemic vehicle + core LY34). A final non-balanced microinjection of LY37 was then performed. Results and Conclusions: Systemic administration of the mGluR2/3 agonist LY37 significantly reduced EtOH- and sucrose- seeking with no systematic effect on locomotion. Systemic administration of the selective mGluR2 PAM BINA had no significant effect on either seeking or consumption. These findings suggest that modulation of glutamatergic neurotransmission by a systemic mGluR2/3 agonist, but not allosteric modulation of mGluR2, significantly reduces reinforcer seeking. Intra- accumbens core administration of LY37 significantly reduced EtOH-seeking, suggesting a role of NAc core mGluR2/3 modulation in EtOH-seeking during maintenance drinking. Systemic administration of LY37 was also found to significantly reduce sucrose consumption and body weight 24-hours following systemic administration, meriting further examination of the role of mGluR2/3 receptors on feeding behavior.

<p dir="ltr">Neurotrauma encompasses a broad category of injuries affecting the central nervous system (CNS), which includes both the traumatic brain injury (TBI) and spinal cord injury (SCI). These injuries can result from various causes, including accidents, falls, sports-related incidents, and other traumatic events, affecting millions of individuals annually. Traumatic injuries are the leading cause of disability, and moreover are associated with elevated risk of developing cognitive impairments and neurodegenerative diseases (ND) such as Alzheimer’s Disease (AD) and Parkinson’s Disease (PD). The elevated ND risk arising from neurotrauma poses significant burdens on healthcare systems and affect life quality of affected individuals, emphasizing the critical need for research aimed at understanding the underlying mechanisms conferring ND risk from the lesion center to CNS. The goal of my thesis is to understand persistent molecular changes post SCI associated with ND using a combination of a rat animal model and neuronal cultures derived from human induced pluripotent stem cells.</p><p dir="ltr">I started with Sprague-Dawley rats with T10 spinal cord contusive injury; and assessed immediate and persistent changes in transcriptomic and epigenetic markers via next generation sequencing (NGS) at primary lesion site and distal spinal cord tissue. Along with global changes in chromatin arrangements and DNA methylation, we observed significant transcriptomic changes enriched for pathways of inflammatory responses, and synaptogenesis. These changes were further verified using immunohistochemistry and super resolution microscopy. To further understand the long-term brain abnormality linked to SCI, we investigated persistent alterations in the composition and molecular profiles of both the male and female motor cortex 30 days after injury. Immunohistochemistry revealed that SCI leads to neuronal loss and changes in synaptic density and morphology; and significant alterations in the neuron-astrocyte ratio and astrocyte morphology, in male motor cortex supporting our hypothesis that SCI may increase the risk of neurodegeneration by affecting the motor cortex. Comparison of transcriptomic data collected at a sub-acute stage in male rats, namely 7 days post injury, with 30 days post injury, identified persistent and de novo changes that occur primarily after recovery of spinal cord injury, which are enriched for neuronal and synaptic function related pathways. Interestingly, neuroendocrine-related pathways were prominently implicated at the chronic stage of SCI, with Esr1 identified as a major upstream regulator offering protective effects in females that did not exhibit significant alterations in cellular composition or morphology after SCI. Collectively, our study paved the way towards understanding sexual dimorphism in brains after spinal cord injury and provides a plausible connection between spinal cord injury and neurodegeneration later in life that were further investigated using a humanized culture model.</p><p dir="ltr">We established the feasibility of using hiPSC derived neurons to examine long term neurotoxic mechanism using lead (Pb) as a model chemical with strong associations with elevated AD risks later in life. A similar culture system was then used to assess persistent neurotoxicity of acrolein, a chemical that is known to emerge in brains post traumatic injury. We found that acrolein induced alterations in neuronal network morphology, synaptic density, and excitability. Furthermore, acrolein exposure negatively impacted mitochondrial function and persistently altered neuronal resilience towards a secondary stressor of mitochondria, namely MPP+. Acrolein exposure also alters the expression of tau and tau phosphorylation which collectively result in increased cellular vulnerability toward paired helical filament (PHF-tau) seeding, a known neurotoxin associated with ND. These findings collectively provide molecular insights as to how acrolein can partake alterations in neural function and resilience to stressors; and relay ND risks in neurotrauma patients later in life.</p><p dir="ltr">In conclusion, our comprehensive investigation employing both rat and hiPSC models uncovers plausible molecular pathways connecting SCI to neurodegenerative diseases, providing insights into the enduring consequences of these injuries on affected patients.</p>

No description available.

<p dir="ltr">Neurodegenerative diseases are progressive, incurable conditions that affect tens of millions of people worldwide and are characterized by the aggregation of misfolded protein in the brain. Though the precise role of these amyloid aggregates in the onset and progression of these diseases is not clear at this time, there is a pressing need to understand how they form and spread in human disease. In service to these aims, I have conducted three small projects to expand knowledge in this regard. I first investigated the use of thioflavin T, a common amyloid stain, as an affinity reagent for the general purification of amyloid filaments from <i>ex vivo </i>samples, observing strong potential using a relatively simple, inexpensive magnetic bead conjugation technique. I next analyzed the formation of filaments of a truncated recombinant amyloid-beta peptide with residues 1-35, observing a new filament type formed at low pH in the wild-type sequence of this truncated peptide. Finally, I conducted structural studies on amyloid-beta(1-42) filaments prepared under different conditions consistent with traumatic brain injury to observe their effect on amyloid folding. While I found no effect of differential conditions on filament type, the low-resolution structures solved were highly consistent with aggregates found in Alzheimer’s disease patients, presenting a promising way forward for <i>in vitro</i> modeling of amyloid filaments that are true to pathology. In sum, the work here presented advances the concepts of both how amyloid aggregates from patient brains can be best prepared for structural analysis, and the factors underpinning their aggregation at the onset of neurodegenerative disease.</p>

Made available in DSpace on 2014-12-17T15:36:57Z (GMT). No. of bitstreams: 1 JanainaSB.pdf: 2453259 bytes, checksum: d4ce3e2bc8b59c2bee9fa61810a98832 (MD5) Previous issue date: 2009-10-29 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The thalamus plays an important role in the sensorial processing information, in this particular case, the visual information. Several neuronal groups have been characterized as conductors and processors of important sensorial information to the cerebral cortex. The lateral geniculate complex is one to them, and appears as a group very studied once it is responsible, in almost all totality, for the processing of visual information. Among the nuclei that constitute the lateral geniculate complex we highlight the dorsal lateral geniculate nucleus of the thalamus (DLG), the main thalamic relay for the visual information. This nucleus is located rostral and lateral to medial geniculate nucleus and ventral to thalamic pulvinar nucleus in most of the mammals. In the primates humans and non-humans, it presents as a laminate structure, arranged in layers, when observed in coronal sections. The objective of this work was to do a mapping of the retinal projections and a citoarchictetonic and neurochemical characterization of DLG in the marmoset (Callithrix jacchus), a New World primate. The retinal projections were traced by anterograde transport of subunit b of cholera toxin (CTb), the citoarchicteture was described by Nissl method, and to neurochemical characterization immunohistochemicals technical were used to examine the main neurotransmitters and neuroatives substances present in this neural center. In DGL of marmoset thalamus, in coronal sections labeled by Nissl method, was possible to visualize the division of this nucleus in four layers divided in two portions: magnocellular and parvocellular. The retinal projections were present being visualized fibers and terminals immunorreactives to CTb (IR-CTb) in the DLG ipsilateral and contralateral. And through the immunohistochemicals techniques was observed that DLG contain cells, fibers and/or terminals immunoreactives against neuronal nuclear protein, subunits of AMPA 15 glutamate receptors (GluR1, GluR2/3, GluR4), choline acetyltransferase, serotonin, glutamic acid decarboxylase, binding calcium proteins (calbindin, parvalbumin and calretinin), vasopressin, vasoactive intestinal polypeptide, and an astrocyte protein, glial fibrillary acidic protein. / O t?lamo exerce um importante papel no processamento de informa??es sensoriais, em particular, a informa??o visual. V?rios grupos neuronais j? foram caracterizados como condutores e processadores de informa??es sensoriais importantes para o c?rtex cerebral. O complexo geniculado lateral ? um deles e aparece como um grupo muito estudado uma vez que ? respons?vel, em quase toda sua totalidade, pelo processamento de informa??o visual. Entre os n?cleos que constituem o complexo geniculado lateral destacamos o n?cleo geniculado lateral dorsal do t?lamo (GLD), o principal rel? tal?mico para as informa??es visuais. Este n?cleo se localiza rostral e lateral ao n?cleo geniculado medial e ventral ao n?cleo pulvinar do t?lamo na maioria dos mam?feros. Nos primatas humanos e n?o humanos, apresenta-se como uma estrutura laminar, disposto em camadas, quando observada em sec??es coronais. O objetivo neste trabalho foi fazer um mapeamento da proje??o retiniana e uma caracteriza??o citoarquitet?nica e neuroqu?mica do GLD no Callithrix jacchus (sag?i), um primata do Novo Mundo. As proje??es retinianas foram tra?adas por transporte anter?grado da subunidade B da toxina col?rica (CTb), a citoarquitetura foi descrita atrav?s do m?todo de Nissl, e para a caracteriza??o neuroqu?mica t?cnicas imunoistoqu?micas foram utilizadas para examinar os principais neurotransmissores e subst?ncias neuroativas presentes neste centro neural. No GLD do t?lamo do sag?i, nas sec??es coronais coradas pelo m?todo de Nissl, foi poss?vel visualizar a divis?o desse n?cleo em quatro camadas dividas em duas por??es: magnocelular e parvocelular. As proje??es retinianas estavam presentes visualizando-se fibras e terminais imunorreativos a CTb (CTb- IR) no GLD ipsolateral e contralateral. E atrav?s das t?cnicas imunoistoqu?micas observou-se que o GLD cont?m c?lulas, fibras e/ou terminais 13 imunorreativos a prote?na nuclear neuronal, subunidades dos receptores AMPA de glutamato (GluR1, GluR2/3, GluR4), colina acetiltransferase, serotonina, descarboxilase do ?cido glut?mico, prote?nas ligantes de c?lcio (calbindina, calretinina e parvalbumina), vasopressina, polipept?deo intestinal vasoativo, e uma prote?na astrocit?ria, prote?na ac?dica fibrilar glial.

<p>Microglia are a population of specialized, tissue-resident immune cells that make up around 10% of total cells in our brain. They actively prune neuronal synapses, engulf cellular debris, and misfolded protein aggregates such as the Alzheimer’s Disease (AD)-associated amyloid-beta (Aβ) by the process of phagocytosis. During AD, microglia are unable to phagocytose Aβ, perhaps due to the several disease-associated changes affecting their normal function. Functional molecules such as lipids and metabolites also influence microglial behavior but have primarily remained uncharacterized to date. The overarching question of this work is, <i>How do microglia become dysfunctional in chronic inflammation</i>? To this end, we developed new chemical tools to better understand and investigate the microglial response to Aβ <i>in vitro</i> and <i>in vivo</i>. Specifically, we introduce three new tools. (1) Recombinant human Aβ was developed via a rapid, refined, and robust method for expressing, purifying, and characterizing the protein. (2) A pH-sensitive fluorophore conjugate of Aβ (called Aβ^pH) was developed to identify and separate Aβ-specific phagocytic and non-phagocytic glial cells <i>ex vivo</i> and <i>in vivo</i>. (3) New lysosomal, mitochondrial, and nuclei-targeting pH-activable fluorescent probes (called LysoShine, MitoShine, and NucShine, respectively) to visualize subcellular organelles in live microglia. Next, we asked, <i>What changes occur to the global lipid and metabolite profiles of microglia in the presence of Aβ in vitro and in vivo</i>? We screened 1500 lipids comprising 10 lipid classes and 700 metabolites in microglia exposed to Aβ. We found significant changes in specific lipid classes with acute and prolonged Aβ exposure. We also identified a lipid-related protein that was differentially regulated due to Aβ <i>in vivo</i>. This new lipid reprogramming mechanism “turned on” in the presence of cellular stress was also present in microglia in the brains of the 5xFAD mouse model, suggesting a generic response to inflammation and toxicity. It is well known that activated microglia induce reactive astrocytes during inflammation. Therefore, we asked, <i>What changes in proteins, lipids, and metabolites occur in astrocytes due to their reactive state? </i>We provide a comprehensive characterization of reactive astrocytes comprising 3660 proteins, 1500 lipids, and 700 metabolites. These microglia and astrocytes datasets will be available to the scientific community as a web application. We propose a final model wherein the molecules secreted by reactive astrocytes may also induce lipid-related changes to the microglial cell state in inflammation. In conclusion, this thesis highlights chemical neuroimmunology as the new frontier of neuroscience propelled by the development of new chemical tools and techniques to characterize glial cell states and function in neurodegeneration.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that has proven to be an important signaling molecule both as an extracellular primary messenger and as an intracellular second messenger. Extracellular S1P acts through a family of five S1P receptors, S1PR1-5, all of which are G protein-coupled receptors associated with different G proteins. Previous work from our laboratory shows that externally applied S1P increases the excitability of small-diameter sensory neurons by enhancing the action potential firing. The increased neuronal excitability is mediated primarily, but not exclusively, through S1PR1. This raises the question as to which other S1PRs mediate the enhanced excitability in sensory neurons. To address this question, the expression of different S1PR subtypes in small-diameter sensory neurons was examined by single-cell quantitative PCR. The results show that sensory neurons express the mRNAs for all five S1PRs, with S1PR1 mRNA level significantly greater than the other subtypes. To investigate the functional contribution of other S1PRs in augmenting excitability, sensory neurons were treated with a pool of three individual siRNAs targeted to S1PR1, R2 and R3. This treatment prevented S1P from augmenting excitability, indicating that S1PR1, R2 and/or R3 are essential in mediating S1P-induced sensitization. To study the role of S1PR2 in S1P-induced sensitization, JTE-013, a selective antagonist at S1PR2, was used. Surprisingly, JTE-013 by itself enhanced neuronal excitability. Alternatively, sensory neurons were pretreated with FTY720, which is an agonist at S1PR1/R3/R4/R5 and presumably downregulates these receptors. FTY720 pretreatment prevented S1P from increasing neuronal excitability, suggesting that S1PR2 does not mediate the S1P-induced sensitization. To test the hypothesis that S1PR1 and R3 mediate S1P-induced sensitization, sensory neurons were pretreated with specific antagonists for S1PR1 and R3, or with siRNAs targeted to S1PR1 and R3. Both treatments blocked the capacity of S1P to enhance neuronal excitability. Therefore my results demonstrate that the enhanced excitability produced by S1P is mediated by S1PR1 and/or S1PR3. Additionally, my results indicate that S1P/S1PR1 elevates neuronal excitability through the activation of mitogen-activated protein kinase kinase. The data from antagonism at S1PR1 to regulate neuronal excitability provides insight into the importance of S1P/S1PR1 axis in modulating pain signal transduction.