O objetivo desta pesquisa foi avaliar qualitativa e quantitativamente, através de técnicas histológicas e histoquímicas, o córtex cerebral de ratos submetidos a uma subnutrição protéica e posterior recuperação precoce e tardia. Foram utilizados ratos Wistar submetidos às seguintes dietas nutricionais: dieta padrão - normoprotéica - \"AIN-93G\", contendo 20% de proteína (caseína) e dieta hipoprotéica - \"AIN-93G\", contendo 5% desta mesma proteína, durante os períodos experimentais de 42 e 60 dias. Os grupos experimentais foram formados por animais que desde o acasalamento até a eutanásia foram submetidos à dieta normoprotéica (grupos nutridos N42 e N60) e à dieta hipoprotéica (grupos subnutridos S42 e S60). O grupo renutrido precoce (Rp42) foi constituído por animais subnutridos que, a partir do 22º dia passaram a receber a dieta normoprotéica até 42 dias de idade; animais subnutridos que a partir do 43º dia receberam a dieta normoprotéica até 60 dias de idade, constituíram o grupo renutrido tardio (Rt60). Uma vez formado os grupos experimentais no momento do desmame (21 dias) todos os animais foram alojados e assistidos em gaiolas metabólicas. Características comportamentais e físicas foram avaliadas, bem como os seguintes aspectos morfométricos: pesos corporais e encefálicos; comprimento naso-anal, circunferência abdominal, índice de Lee (que avalia o grau de adiposidade) e as dimensões encefálicas. Para as avaliações quantitativas estruturais foram empregadas técnicas histológicas (H.E. e violeta Cresil) e histoquímica (β-Nicotinamide Adenine dinucleotide phosphate - NADPH) para neurônios nitrérgicos. Foram avaliados parâmetros como: número de neurônios, densidade neuronal, área neuronal e o perfil da área dos neurônios corticais e nas camadas corticais. Em linhas gerais, para os parâmetros aqui avaliados, desde o nascimento até o sacrifício inerente a cada grupo experimental (42 e 60 dias), os animais dos grupos subnutridos (S42 e S60) apresentaram valores menores do que os respectivos grupos controle (N42 e N60), exceto no índice de Lee, significativamente maior do que nos animais nutridos. Os grupos renutridos, independentemente se precoce (Rp42) ou tardio (Rt60) conseguem alguma recuperação em alguns parâmetros avaliados como peso encefálico, dimensões encefálicas e espessura cortical; porém outros como o peso corporal, e a área neuronal foram gravemente afetados. Todavia quando se observa a razão peso encefálico/peso corporal, os animais dos grupos subnutridos relativamente aos animais controles nutridos, apesar do peso corporal menor apresentam uma razão maior do que os respectivos controles, sugerindo ser o encéfalo menos afetado pela subnutrição do que o peso corporal. A subnutrição promoveu acentuada diminuição na área dos neurônios corticais e nitrérgicos, não recuperada com a renutrição, independentemente se precoce ou tardia. Portanto, conforme o proposto e com a metodologia utilizada no presente estudo pesquisa, os resultados permitem concluir que frente às condições de subnutrição, o organismo pode sofrer alterações irreparáveis dependendo do momento da vida em que ocorre, e que a renutrição pode promover recuperação ou mesmo adaptações em alguns parâmetros, preferencialmente se ocorrer em fases mais precoces. / The objective of this study was to evaluate qualitative and quantitatively, through histological and histochemical techniques, the cerebral cortex of rats subjected to protein undernutrition subsequent to early and late recovery. Were used Wistar rats fed with standard (normal) diet-\"AIN-93G\" containing 20% of protein (casein), and hypoproteic diet- \"AIN-93G\" containing 5% of casein during experimental periods of 42 and 60 days. The experimental groups were performed by animals fed with normal diet from mating to euthanasia (nourished groups N42 and N60), and to hypoproteic diet (undernourished groups S42 and S60). The early re-nourished group (Rp42) was composed by undernourished animals fed with normal diet from 22nd day, whereas late re-nourished group (Rt60) was composed by undernourished animals fed with normal diet from 43rd to 60 days of age. Once established the experimental groups at weaning (21 days), all animals were housed and assisted in metabolic cages. Physical and behavioral characteristics were examined, as well as, morphometric aspects: body and brain weight; naso-anal length, abdominal circumference, Lee index (evaluation of fat deposit), and brain measures. For quantitative evaluation, histologic techniques (H.E. and Violet Cresyl), and Histochemistry (β-Nicotinamide Adenine dinucleotide phosphate - NADPH) for nitrergic neurons were performed. The number of neurons, neuronal density, neuronal area and profile of cortical neurons in cortical layers area were analyzed. Animals from S42 and S60 groups shown lower average compared to respective controls (N42 and N60), except in the Lee index, that was significantly higher than in nourished animals. Were observed that re-nourished groups either early (Rp42) or late (Rt60) have recovered in some parameters evaluated such as brain weight, brain measures and cortical thickness; however other parameters like body mass, and neuronal area was highly affected. Relatively to the proportion brain weight/body mass, animals from undernourished group although exhibit low body mass, shown higher proportion than respective control nourished, thus suggesting that brain is less affected by undernutrition than body mass. The undernutrition promoted great decreasing of cortical and nitrergic neurons area; the area was not recovered by early and/or late re-nourishment. In the view of the objectives and methodology used in the present study, we can be conclude that in under-nutrition conditions the body can go through irreversible changes depending on period of occurrence; additionally, re-nourishment can promote recovery or adaptations in some parameters preferentially if occur in early phases.

In daily activities, the brain regularly assigns different roles to the hands dependingon task and context. Yet, little is known about the underlying neural processes. Thiscertainly applies to how the brain, where each hemisphere primarily controls onehand, manages the between-hand coordination required in bimanual objectmanipulation. By using behavioral, neurophysiological and functional magneticresonance imaging techniques, the present thesis examines neural mechanisms thatsupport hand coordination during tasks where the two hands apply spatiotemporallycoupled but opposing forces for goal attainment, e.g., as when removing the cap froma bottle. Although the two hands seem to operate symmetrically in such tasks, Study Ishowed that one hand primarily acts while the other assists. Moreover, this roledifferentiation was found to be flexible with the brain appointing either hand asprime actor depending on the spatial congruency between hand forces and desiredmovement consequences. Accordingly, when we remove a cap from a bottle, the handthat grasps the cap, be it left or right depending on overall task constraints, isappointed as prime actor because the twist forces it generates are aligned with thegoal to remove the cap, while the other hand, holding the bottle, applies stabilizingforces in the opposite direction. Changes in hand assignments are caused by amidline shift of lateralized activity throughout the motor system, from distal handmuscles to corticospinal pathways and primary sensorimotor and cerebellar corticalareas (Study I). Although the bimanual actions examined involved both within- andbetween-hand coordination, Study II failed to reveal additional brain activity duringbimanual as compared to matching unimanual actions, except for the primarysensorimotor areas where subpopulations of neurons were preferentially engagedduring either bimanual or unimanual actions. Thus, dedicated neurons in the motorcortices might support critical bimanual coordinative operations. While imagingresults indicated that a mainly left-lateralized parietal-premotor network managedthe task irrespective of prime actor, premotor areas presumably established handassignment by allocating the lead either to the left or the right primary sensorimotorareas (Study I and II). Regarding the process of prime actor selection and hence thecontrol of these premotor networks, imaging results indicate a transitory involvementof prefrontal cortical areas (Study III). The detected areas belong to a networkconsidered critical for cognitive operations such as judgment and decision-making,and for evaluation of utility of actions, including conflict detection. The implicitselection of prime actor during bimanual tasks thus seems to be supported by corticalareas traditionally associated primarily with complex cognitive challenges.

Denna avhandling i datalogi föreslår modeller för hur vissa beräkningsmässiga uppgifter kan utföras av hjärnbarken. Utgångspunkten är dels kända fakta om hur en area i hjärnbarken är uppbyggd och fungerar, dels etablerade modellklasser inom beräkningsneurobiologi, såsom attraktorminnen och system för gles kodning. Ett neuralt nätverk som producerar en effektiv gles kod i binär mening för sensoriska, särskilt visuella, intryck presenteras. Jag visar att detta nätverk, när det har tränats med naturliga bilder, reproducerar vissa egenskaper (receptiva fält) hos nervceller i lager IV i den primära synbarken och att de koder som det producerar är lämpliga för lagring i associativa minnesmodeller. Vidare visar jag hur ett enkelt autoassociativt minne kan modifieras till att fungera som ett generellt sekvenslärande system genom att utrustas med synapsdynamik. Jag undersöker hur ett abstrakt attraktorminnessystem kan implementeras i en detaljerad modell baserad på data om hjärnbarken. Denna modell kan sedan analyseras med verktyg som simulerar experiment som kan utföras på en riktig hjärnbark. Hypotesen att hjärnbarken till avsevärd del fungerar som ett attraktorminne undersöks och visar sig leda till prediktioner för dess kopplingsstruktur. Jag diskuterar också metodologiska aspekter på beräkningsneurobiologin idag. / In this thesis I take a modular approach to cortical function. I investigate how the cerebral cortex may realise a number of basic computational tasks, within the framework of its generic architecture. I present novel mechanisms for certain assumed computational capabilities of the cerebral cortex, building on the established notions of attractor memory and sparse coding. A sparse binary coding network for generating efficient representations of sensory input is presented. It is demonstrated that this network model well reproduces the simple cell receptive field shapes seen in the primary visual cortex and that its representations are efficient with respect to storage in associative memory. I show how an autoassociative memory, augmented with dynamical synapses, can function as a general sequence learning network. I demonstrate how an abstract attractor memory system may be realised on the microcircuit level -- and how it may be analysed using tools similar to those used experimentally. I outline some predictions from the hypothesis that the macroscopic connectivity of the cortex is optimised for attractor memory function. I also discuss methodological aspects of modelling in computational neuroscience. / QC 20100916

The developing brain goes through a number of developmental periods during which it displays an increased sensitivity to exogenous disturbances. On such period is the so called “Brain growth spurt” (BGS) which in humans takes place starting from the third trimester of pregnancy and throughout the first few years of life. The corresponding period in rats and mice is the first postnatal weeks. Exposure to relatively modest concentrations of the brominated flame retardant PBDE-99 during the second week of life in mice causes a more or less permanent impairment in the ability of the animals to adjust properly to environmental changes at adulthood. This “late response on early exposure” reflects the long-term consequences of disrupting the developing brain during a sensitive time period. The cellular mechanisms underlying the behavioral effects are far from clear. To address the initial damage occurring around the time of exposure, the approach used in this thesis is to use proteomics to analyze the effects of PBDE-99 on protein expression soon (24 hours) after exposure of the neonatal mouse on postnatal day (PND) 10.The thesis comprises the effects on the proteome in three distinct brain parts: cerebral cortex, striatum and the hippocampus. In addition, an in vitro model was developed and used to evaluate the PBDE-99 effects on cultured cerebral cortex cells from embryonic rat brains. Gel-based proteomics (2D-DIGE) coupled to MALDI- or ESI-MS has been used throughout for the proteomics experiments, but other techniques aimed at analyzing both proteins and mRNA have also been used to better characterize the effects. Even if the protein complements expressed by the different brain parts and separated with 2D-DIGE are seemingly similar, the effects are apparently specific for the different brain regions. In hippocampus, PBDE induces effects on proteins involved in metabolism and energy production, while the effects in striatum point towards effects on neuroplasticity. PBDE-99 changes the expression of cytoskeletal proteins in the cerebral cortex 24 hours after exposure. Interestingly, in vitro exposure of cerebral cortex cells to a PBDE-99 concentration in the same order of magnitude as in the in vivo neonatal brain also induces cytoskeletal effects, in the absence of cytotoxicity. This may suggest effects on regulatory aspects of cytoskeletal dynamics such as those involved in neurite sprouting. This thesis also addresses the problems involved in presenting proteomics data. Many of the available methods and approaches for presenting transcriptomics data are not suitable for isoform rich protein data. Modifications of existing methods and the development of a new approach (DEPPS) is also presented. Most importantly, the thesis presents the application and usefulness of proteomics as hypothesis generating techniques in neurotoxicology.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Le néocortex est une région du cerveau qui traite toutes les entrées sensorielles et créé des réponses comportementales. Il est subdivisé en zones fonctionnelles, chacune ayant une cytoarchitecture, un motif d’expression génique et un profil de connectivité spécifiques. L'organisation en zones est pré-modelée par des gènes organisateurs, et ensuite affinée par l’activité sensorielle. Dans cette étude, j'ai étudié d'abord si ce pré-modelage est établi dans les progéniteurs et/ou les cellules post-mitotiques, et si l'activité neuronale spontanée est nécessaire pour l’établissement de la connectivité correcte entre néocortex et thalamus, station relais principale des données sensorielles. Avec l'aide d'une série de souris transgéniques, j’ai montré que la fonction du gène organisateur COUP-TFI est suffisante et nécessaire pour organiser l'identité sensorielle dans les cellules post-mitotiques, et que COUP-TFI régule l'activité intrinsèque des neurones corticaux, influençant la bonne intégration des entrées thalamiques dans le cortex somatosensoriel. J’ai montré que COUP-TFI contrôle directement l'expression du gène Egr1, qui dépend fortement de l'activité neuronale. COUP-TFI et Egr1 agissent sur l'acquisition de la morphologie des cellules étoilées dans les neurones de la couche 4, cibles principales des axones thalamiques et trait typique des zones somatosensoriels primaires. En conclusion, ce travail montre que le pré-modelage cortical dépend primordialement d’un programme génétique établi dans les cellules post-mitotiques et que l'activité intrinsèque et les propriétés génétiques agissent ensemble pour façonner l'organisation des premiers circuits dans le néocortex. / The neocortex is a region of the brain that processes all sensory inputs creating appropriate behavioral responses. It is subdivided into functional areas, each with a specific cytoarchitecture, gene expression pattern and connectivity profile. The organization into areas is pre-patterned by the action of areal patterning genes, and subsequently refined by sensory evoked activity. In this study, I have first investigated whether early areal patterning is committed in progenitor and/or post-mitotic cells, and then assessed whether spontaneous neuronal activity is required in establishing correct connectivity between the neocortex and the thalamus, the principal relay station of peripheral sensory inputs. With the help of a series of transgenic mice, my work showed that the function of the areal patterning gene COUP-TFI is sufficient and necessary to organize sensory identity in post-mitotic cells, and that COUP-TFI regulates intrinsic activity properties of cortical neurons, and thus proper integration of thalamic inputs into the somatosensory cortex. In particular, I found that COUP-TFI directly controls the expression of the immediate early gene Egr1, which expression levels strongly depend on neuronal activity. Both COUP-TFI and Egr1 act on the acquisition of the stellate cell morphology of layer 4 neurons, the main targets of thalamic axons and a typical trait of primary somatosensory areas. In conclusion, this work demonstrates that cortical area patterning primordially depends on a genetic program established in post-mitotic cells and that intrinsic genetic and activity properties act together to shape the organization of early circuits in the neocortex.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Methylmalonic acidemia is an inborn error of metabolism characterized clinically and biochemically by tissue accumulation of methylmalonic acid (MMA) and neurological dysfunction, including convulsion. Furthermore, clinical data suggest that infections conditions can precipitate metabolic crisis and cause neurological changes observed in patients of acidemia. Provided that the MMA cause neurological complications, and that the inflammation can contribute to the occurrence of convulsions and cognitive deficit in several animal models, it is possible to suggest that inflammatory mediators, such as inducible nitric oxide synthase (iNOS), facilitate MMA-induced convulsions. The iNOS is one of three isoforms of nitric oxide synthase (NOS), which generates nitric oxide (NO), a simple gaseous signaling molecule and free radical. The iNOS is induced at injury/inflammation sites, but is also constitutively expressed on some cells, such as in neurons. Studies in experimental models have already demonstrated that NO generated in the central nervous system (CNS), by endothelial and neuronal isoforms of NOS, is involved in MMA-induced convulsions. However, until the present moment are scarce the data in the literature evaluating the relationship of iNOS in experimental models of Methylmalonic Acidemia. The results published in the article has shown that iNOS knock-out C57BL/6 mice, when injected acutely with MMA (2 μmols/2 μl, intracerebroventricularly), have a shorter duration of seizures, no significant change in the mean amplitude of electroencephalographic waves (EEG); not increase the levels of nitrite and nitrate (NOx) compared to animals injected with saline, but have a partial reduction in the levels of 3-nitrotyrosine (3-NT) compared to wild animals that were also treated with MMA; similarly, show a partially lower inhibition of Na+,K+-ATPase, but exhibit no difference in succinate dehydrogenase (SDH) inhibition on cerebral cortex compared to wild mice which also received MMA. The results submitted in the manuscript has shown that Wistar rats, after being injected chronically with MMA (from 5th to 28th day of life, twice daily, with doses ranging from 0.76 to 1.67 mmol/g depending on the age of the animal, via subcutaneous) showed a reduced index of recognition in spatial learning/memory test, but show no anxiety at elevated plus maze test; they have a reduction in neutrophils, but an increase in the number of mononuclear leukocytes in the blood; and in addition show increased levels of interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), iNOS and 3-NT in the cerebral cortex. Considering the data presented in both studies, it was concluded that the MMA can cause seizures, nitrosative stress and inhibition of Na+,K+-ATPase activity in cerebral cortex of mice by mechanisms related to NO production via iNOS; and that the MMA can also cause neurocognitive deficits, altered immune system in blood and increase of pro-inflammatory cytokines, leading to increased expression of iNOS and nitrosative stress. / A Acidemia Metilmalônica é um erro inato do metabolismo caracterizado bioquimicamente e clinicamente pelo acúmulo tecidual de ácido metilmalônico (MMA) e disfunção neurológica, incluindo convulsões e déficit cognitivo. Além disso, dados clínicos sugerem que quadros infecciosos podem precipitar crises metabólicas e causar as alterações neurológicas observadas nos pacientes com essa acidemia. Desde que o MMA causa complicações neurológicas, e que a inflamação pode contribuir para a ocorrência de convulsões e déficits cognitivos em vários modelos animais, é possível sugerir que mediadores inflamatórios, como a enzima Óxido Nítrico Sintase Induzível (iNOS), facilitem as convulsões induzidas por MMA. A iNOS é uma das três isoformas da enzima Óxido Nítrico Sintase (NOS), que gera o óxido nítrico (NO), uma molécula gasosa simples, sinalizadora e um radical livre. A iNOS é induzida em sítios de lesão/inflamação, mas também se expressa constitutivamente em algumas células, como nos neurônios. Estudos em modelos experimentais já demonstraram que o NO gerado no sistema nervos central (SNC), pelas isoformas endotelial e neuronal da NOS, tem envolvimento nas convulsões induzidas por MMA. Contudo, até o presente momento são escassos os dados na literatura avaliando a relação da iNOS em modelos experimentais da Acidemia Metilmalônica. Os resultados publicados no artigo mostraram que camundongos C57BL/6 nocaute para iNOS, ao serem injetados agudamente com MMA (2 μmols/2 μL, via intracerebroventricular), apresentam uma duração menor das convulsões, sem alteração significativa na amplitude média das ondas eletroencefalográficas (EEG); não aumentam os níveis de nitrito e nitrato (NOx) comparado aos animais injetados com solução salina, mas têm uma redução parcial nos níveis de 3-nitrotirosina (3-NT) comparado aos animais selvagens que também foram tratados com MMA; semelhantemente, mostram uma inibição parcialmente menor na atividade da enzima Na+,K+-ATPase; mas não exibem diferença na inibição da atividade da succinato desidrogenase (SDH) no córtex cerebral quando comparados aos camundongos selvagens que também receberam MMA. Os resultados apresentados no manuscrito submetido mostram que ratos Wistar, após serem injetados cronicamente com MMA (do 5º ao 28º dia de vida, duas vezes ao dia, com doses variando de 0,76 à 1,67 mmol/g em função da idade do animal, via subcutânea), apresentam um reduzido índice de reconhecimento em teste de memória/aprendizado espacial, mas não demonstram ansiedade no teste do labirinto em cruz elevado; têm uma redução no número de neutrófilos, mas um aumento no número de leucócitos mononucleares no sangue; e além disso mostram aumento nos níveis de interleucina-1beta (IL-1β), do fator de necrose tumoral-alfa (TNF-α), de iNOS e de 3-NT no córtex cerebral. Considerando os dados apresentados nos dois estudos, concluiu-se que o MMA pode causar convulsões, estresse nitrosativo e inibição da enzima Na+,K+-ATPase no córtex cerebral de camundongos por mecanismos relacionados à produção de NO via iNOS; e que o MMA também pode causar déficit neurocognitivo, alteração do sistema imunológico no sangue, e aumento de citocinas pró-inflamatórias, levando ao aumento na expressão da iNOS e estresse nitrosativo.

The methylmalonic acidemia is an inborn error of metabolism (IEM) characterized by methylmalonic acid (MMA) accumulation in body fluids and tissues, causing neurological dysfunction, mitochondrial failure, oxidative stress and neuroinflammation. Although neurological evidence demonstrate that infection and/or inflammation mediators facilitate metabolic crises in patients, the involvement of neuroinflammatory processes in the neuropathology of this organic acidemia is not yet established. In this experimental study, a single intracerebroventricular dose of MMA (MMA 2.5μmol /g, 12 hs after birth; at dose that raise its concentration in blood and in the brain from affected) was administered to mice pups at postnatal day 0 (P0) to induce an acute, transient rise of MMA levels in the central nervous system (CNS). In the following days (21st 33th or 40th 52th ) animal behavior was assessed in the radial maze test and elevated plus maze. It was measured tumor necrosis factor-alpha (TNF-α), DCFH, Ache activity and caspase levels in the cerebral cortex, striatum and hipoccampus from mice with 21 e 40 days of life. Behavioral tests showed that animals injected with MMA have a reduction in the working memory test, but no in the reference test. The animals did not exhibit anxiety-like behaviors. Furthermore, MMA increased levels of TNF-α, AchE activity and activation of caspases 1, 3 and 8 in the cerebral cortex, hippocampus and striatum of mice with 21 and 40 days of life. The overall results indicate that a simple administration of MMA increased pro-inflammatory markers in the structure studied, increased apoptotic markers, and coincide with the behavioral changes found in young mice. This leads to speculate that, through mechanisms not yet elucidated, the transient metabolic insult with MMA may cause a neuroinflammatory processes during critical periods of development, contributing to the progression of cognitive impairment in patients with methylmalonic acidemia. / A acidemia metilmalônica (AM) é um erro inato do metabolismo (IEM) caracterizado pelo acúmulo do ácido metilmalônico (MMA) nos fluidos corporais e tecidos, causando disfunção mitocondrial, estresse oxidativo e neuroinflamação, resultando principalmente em disfunções neurológicas. Embora, estudos sugerem que a infecção ou mediadores da inflamação facilitem as crises metabólicas nos pacientes afetados, o envolvimento de processos neuroinflamatórios na patologia dessas acidemia orgânica ainda não foi estabelecido. Neste estudo, uma dose única intracerebroventricular de MMA (MMA 2.5μmol/g; dose encontrada no cérebro e fluidos corporais de pacientes afetados) foi administrada a filhotes de camundongos logo após o nascimento (P0). Além disso, o peso dos animais foi aferido até o dia dos testes comportamentais. A partir do 21° até 33° ou 40º até 52° dias de vida, os animais foram avaliados em tarefas comportamentais como o teste de labirinto radial e em cruz elevado. Os níveis de fator de necrose tumoral-alfa (TNF-α), DCFH, atividade da acetilcolinesterase (AchE) e os níveis de caspases foram determinados no córtex cerebral, estriado e hipocampo de camundongos com 21 e 40 dias de vida. Os testes do labirinto radial mostrou que os animais injetados com o MMA apresentaram um pior desempenho no teste de memória de trabalho, mas não no teste de memória de referência com 21 e 40 dias de vida. Os animais não apresentaram comportamento de ansiedade. Além disso, o MMA aumentou os níveis de TNF- α, a atividade AchE e a ativação das caspases 1, 3 e 8 no córtex cerebral, hipocampo e estriado dos camundongos com 21 e 40 dias de vida. Entretanto, a administração de MMA não causou alterações histológicas nas estruturas analisadas. Dessa forma, os resultados sugerem que uma administração única de MMA aumentou os níveis de mediadores pró-inflamatórios e a expressão de marcadores apoptóticos. Esses eventos podem estar associados com as mudanças comportamentais encontradas nos camundongos jovens. Assim, pode-se sugerir que, devido a mecanismos ainda não totalmente esclarecidos, o acúmulo de MMA durante períodos críticos de desenvolvimento pode causar processos neuroinflamatórios, que contribuem para a progressão da piora de memória nos pacientes com acidemia metilmalônica.

O complexo da esclerose tuberosa (TSC) é um transtorno clínico, com expressividade variável, caracterizado por hamartomas que podem ocorrer em diferentes órgãos. Tem herança autossômica dominante e é devido a mutações em um de dois genes supressores de tumor, TSC1 ou TSC2. Estes codificam para as proteínas hamartina e tuberina, respectivamente, que se associam formando um complexo macromolecular que regula funções como proliferação, diferenciação, crescimento e migração celular. As lesões cerebrais podem ser muito graves em pacientes com TSC e caracterizam-se por nódulos subependimários (SEN), astrocitomas subependimários de células gigantes (SEGA), tuberosidades corticais e heterotopias neuronais, podendo relacionar-se clinicamente à epilepsia refratária à terapia medicamentosa, deficiência intelectual, desordens do comportamento e hidrocefalia. O potencial de crescimento de SEGA até os 21 anos de idade dos pacientes exige acompanhamento periódico por exame de imagem e condutas clínicas ou cirúrgicas, conforme indicação médica. As lesões subependimárias têm sido explicadas por déficits de controle da proliferação, crescimento e diferenciação de precursores neuro-gliais na zona subventricular telencefálica. Embora a capacidade da tuberina em inibir a proliferação celular pela repressão do alvo da rapamicina em mamíferos (mTOR) esteja bem documentada, outros aspectos celulares do desenvolvimento de SEGA ainda não foram examinados. Assim, é importante estabelecer um sistema in vitro para o estudo de células da zona subventricular e testá-lo na análise das proteínas hamartina e tuberina. Neste sentido, o cultivo de neuroesferas em suspensão é muito apropriado. Neste estudo, buscamos relacionar a expressão e distribuição subcelular da hamartina e tuberina à capacidade proliferativa e de diferenciação das células de neuroesferas cultivadas in vitro a partir da dissociação da vesícula telencefálica de embriões de ratos normais. Analisamos a expressão e distribuição subcelular da hamartina e tuberina por imunofluorescência indireta em células entre a primeira e a quarta passagens das neuroesferas, sincronizadas nas fases G1 ou S do ciclo celular e após a reentrada no ciclo celular, através da incorporação de 5-bromo-2\'-desoxiuridina (BrdU) e imunofluorescência com anticorpo anti-BrdU. Em geral, células de neuroesferas apresentaram baixa colocalização entre hamartina e tuberina in vitro. A expressão da tuberina foi elevada em basicamente todas as células das esferas e fases do ciclo celular; ao contrário, a hamartina apresentou-se principalmente nas células da periferia das esferas. A colocalização entre hamartina e tuberina foi observada em células mais periféricas das esferas, sobretudo no citoplasma e, em G1, no núcleo celular. A proteína rheb, que conhecidamente interage diretamente com a tuberina, apresentou distribuição subcelular muito semelhante à desta. Ao carenciamento das células visando à parada do ciclo celular na transição G1/S, tuberina distribuiu-se ao núcleo celular em quase todas as células avaliadas e, de forma menos frequente, a hamartina também. À reentrada no ciclo celular pelo reacréscimo dos fatores de crescimento, avaliaram-se células com incorporação de BrdU ao seu núcleo celular, após 72 e 96 horas. Nestas, tuberina mostrou-se novamente no citoplasma de forma preponderante e hamartina manteve-se citoplasmática, em geral subjacente à membrana plasmática, em níveis mais baixos. Os grupos cujas células reciclaram por 72 ou 96 horas diferiram quanto ao aumento significativo da expressão da hamartina em células proliferativas no último. À diferenciação neuronal, aumentaram-se os níveis de expressão de hamartina observáveis à imunofluorescência indireta, tornando-se equivalentes àqueles da tuberina. Concluímos que as células de neuroesferas cultivadas em suspensão apresentam-se como um sistema apropriado ao estudo da distribuição das proteínas hamartina e tuberina e sua relação com o ciclo celular / The tuberous sclerosis complex (TSC) is a clinical disorder with variable expressivity, characterized by hamartomas that can occur in different organs. It has autosomal dominant inheritance and is due to mutations in one of two tumor suppressor genes, TSC1 or TSC2. These encode for the proteins hamartin and tuberin, respectively, which are associated in a macromolecular complex which functions as a regulator of cell proliferation, differentiation, growth and migration. TSC brain lesions may be severe and are characterized by subependymal nodules (SEN), subependymal giant cell astrocytomas (SEGA), neuronal heterotopias and cortical tubers, and may be clinically related to refractory epilepsy, intellectual disability, behavioral disorders and hydrocephaly. The growth potential of SEGA up to 21 years of age in TSC patients requires regular monitoring by imaging. Clinical and surgical interventions may be medically indicated. Subependymal lesions have been explained by deficient control of proliferation, growth and differentiation of neuro-glial progenitors from the telencephalic subventricular zone. While tuberin ability to inhibit cell proliferation by repressing the mammalian target of rapamycin (mTOR) has been well documented, other cell aspects of SEGA development have not been thoroughly examined. Therefore, it is important to establish conditions for an in vitro system to study the cells from the subventricular zone and to test its suitability for the study of the TSC proteins. In this regard, the neurosphere suspension culture is very appropriate. We evaluated the expression and subcellular distribution of hamartin and tuberin in relation to the proliferation and differentiation capability of neurosphere cells derived in vitro from the dissociation of the telencephalic vesicle of normal E14 rat embryos. These analyses were performed by indirect immunofluorescence in cells from first through fourth passages of neurospheres, synchronized in G1 or S phases of the cell cycle, and after reentry into the cell cycle by the addition of 5-brome-2\'-desoxyuridine (BrdU) and immunolabeling with anti-BrdU antibody. In general, neurosphere cells presented low colocalization between hamartin and tuberin in vitro. Tuberin expression was relatively high in basically all neurosphere cells and cell cycle phases, whereas hamartin distributed mainly to cells from the periphery of the spheres. In these cells, hamartin and tuberin colocalization was evident mostly in the cytoplasm and, in G1, also in the cell nucleus. Rheb, which is known to interact directly with tuberin, had subcellular distribution very similar to tuberin. Cell starvation indicating cell cycle arrest at G1/S redistributed tuberin to the cell nucleus in virtually all cells examined, what was accompanied by nuclear location of hamartin in a small subset of cells. When cells were allowed to reenter cell cycle by adding growth factors, we evaluated BrdU-labeled nuclei 72 and 96 hours later. In the two groups, tuberin was shown to move back to the cytoplasm as well as hamartin, which apparently maintained its lower expression levels distribution underneath the plasma membrane. Group of cells that recycled for 96 hours had significantly more expression of hamartin than those cells that cycled for only 72 hours. After neuronal differentiation, hamartin expression levels observed by immunofluorescence were similar to those of tuberin. We conclude that neurosphere cells cultured in suspension showed to be an appropriate cell system to study hamartin and tuberin distribution in respect to the cell cycle

INTRODUÇÃO: O transtorno afetivo bipolar (TAB) e o transtorno obsessivocompulsivo (TOC) estão entre as dez condições médicas mais incapacitantes. No entanto, suas bases neurobiológicas são ainda desconhecidas. Os estudos postmortem podem dar uma boa contribuição para o entendimento da fisiopatologia desses transtornos, pois permitem a comparação das alterações celulares, citoarquitetônicas e moleculares com as manifestações clínicas, além de auxiliarem numa melhor compreensão dos achados dos estudos de neuroimagem. OBJETIVOS: Comparar a densidade neuronal, volume e número total de neurônios do córtex orbitofrontal (COF) e suas sub-regiões: antero-medial (AM), médio-orbitofrontal (MO) e antero-lateral (AL) entre casos psiquiátricos e controles; Verificar se há diferenças de densidade neuronal, volume e número total de neurônios do COF entre os casos de TAB e casos de TOC e controles. MÉTODOS E CASUÍSTICA: 17 encéfalos de indivíduos acima de 50 anos foram coletados, diagnosticados e submetidos a análises estereológicas, sendo três indivíduos acometidos por TAB, sete indivíduos acometidos por TOC e sete controles saudáveis pareados por idade, gênero e hemisfério cerebral analisado. Um hemisfério foi fixado por perfusão com formalina 20% e processado para estudos neuroestereológicos, enquanto o outro teve as 45 regiões de interesse dissecadas e congeladas a -80ºC para futuros estudos moleculares. RESULTADOS: O COF e suas sub-regiões apresentaram menor densidade neuronal no grupo total de casos vs. controles (p < 0,05). No entanto, não houve diferença em relação ao volume. A subregião MO apresentou um número menor de neurônios nos casos que em controles (p < 0,05). Curiosamente, na análise de densidade neuronal das camadas corticais, apenas a camada IV não apresentou diferença estatisticamente significante entre casos e controles. CONCLUSÕES: Nossos achados mostram que alterações no COF podem estar envolvidas com a fisiopatologia do TOC ou TAB, e indicam que podem haver interações entre elas e, além disso, concordam com estudos de imagem funcional e de atividades cerebrais na região MO que nos faz refletir que além de uma perda neuronal, há também uma hipoativação e uma redução funcional. Estudos com um número maior de amostras e com diferenciação celular poderão trazer novas contribuições para o entendimento da fisiopatologia desses transtornos neuropsiquiátricos / INTRODUCTION: Bipolar disorder (BD) and obsessive compulsive disorder (OCD) are within the ten medical condition promoving incapacity worldwide. To date, their neuropathological substrates are yet to be disclosed. Postmortem studies designed to estimate cytoarchitectonic and molecular changes for clinical and imaging correlation, have the potencial to undercover pathophysiological aspects of these conditions. OBJECTIVE: Objective: To compare neuronal density, volume and total neuron number of orbitofrontal cortex (OFC) as a whole and divided by sub-regions: anteromedial (AM), medio-orbitofrontal (MO) and antero-lateral (AL) among BD, OCD and matched controls. METHODS: We used 17 postmortem brains sourced from the Psy- BBBABSG. All the subject were older than 50 years and were classified based on clinical evaluation in BD, OCD and healthy control. Subjects were matched by age, gender and brain hemisphere. One hemisphere were perfusion fixed with 20% formalin and used for neuroestereological studies. The second hemisphere had ROIs dissected and snap frozen for future molecular studies. OUTCOMES: Neuron density in OFC and the sub-regions were decreased in cases vs. controls (p < 0,05). This result was observed in cortical layers analyses with exception of layer IV. We did not observed significant changes in volume. The MO sub-region had reduced total neuron number in cases than in controls (p < 0,05). CONCLUSION: Ours results suggest that OFC changes may be part of BD and OCD pathogenesis. These results go in line with functional imaging findings. Further studies with a higher number of cases and adressing specific neuron types are needed