Yes / Phencyclidine (PCP), used to mimic certain aspects of schizophrenia, induces sexually dimorphic, cognitive deficits in rats. In this study, the effects of sub-chronic PCP on expression of brain-derived neurotrophic factor (BDNF), a neurotrophic factor implicated in the pathogenesis of schizophrenia, have been evaluated in male and female rats. Male and female hooded-Lister rats received vehicle or PCP (n = 8 per group; 2 mg/kg i.p. twice daily for 7 days) and were tested in the attentional set shifting task prior to being sacrificed (6 weeks post-treatment). Levels of BDNF mRNA were measured in specific brain regions using in situ hybridisation. Male rats were less sensitive to PCP-induced deficits in the extra-dimensional shift stage of the attentional set shifting task compared to female rats. Quantitative analysis of brain regions demonstrated reduced BDNF levels in the medial prefrontal cortex (p < 0.05), motor cortex (p < 0.01), orbital cortex (p < 0.01), olfactory bulb (p < 0.05), retrosplenial cortex (p < 0.001), frontal cortex (p < 0.01), parietal cortex (p < 0.01), CA1 (p < 0.05) and polymorphic layer of dentate gyrus (p < 0.05) of the hippocampus and the central (p < 0.01), lateral (p < 0.05) and basolateral (p < 0.05) regions of the amygdaloid nucleus in female PCP-treated rats compared with controls. In contrast, BDNF was significantly reduced only in the orbital cortex and central amygdaloid region of male rats (p < 0.05). Results suggest that blockade of NMDA receptors by sub-chronic PCP administration has a long-lasting down-regulatory effect on BDNF mRNA expression in the female rat brain which may underlie some of the behavioural deficits observed post PCP administration.

Astrocytes are the most abundant glial cell type in the central nervous system (CNS). Most astrocytes are born during the early postnatal period in the rodent brain and mature alongside neurons, demonstrating remarkable morphological structural complexity, and attaining maturity in the second postnatal month. We have shown that astrocyte morphogenesis is regulated in part by brain-derived neurotrophic factor (BDNF) via signaling through the truncated tropomyosin receptor kinase B (TrkB) receptor. TrkB is the primary receptor for BDNF which is broadly expressed and released by neurons in developing and mature brain. TrkB has two predominant isoforms expressed in central nervous system (CNS), the full length TrkB (TrkB.FL) receptor and truncated TrkB (TrkB.T1) receptor. We recently demonstrated in the adult rodent cortex that TrkB.T1 is largely specific to astrocytes and over 90% of all Ntrk2 expression in astrocytes attributed to TrkB.T1. In contrast TrkB.FL is the predominant isoform expressed by neurons. It is not known how astrocytes and neurons regulate their specific TrkB isoform expression, although previous studies in bulk frontal cortical tissue from human postmortem samples indicate that DNA methylation level in promoter region and 3' UTR region of NTRK2 is negatively correlated with TrkB.T1 expression levels, but not with TrkB.FL expression. The mechanism of TrkB.T1 isoform-specific expression and the role of TrkB.T1 in astrocyte developmental process are unknown. In this dissertation, we aimed to determine in the DNA methylation contributes to isoform specific expression of TrkB.T1. We thus profiled the 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in neurons, astrocytes and microglia utilizing nanopore sequencing. We identified robust differences in cell-type specific TrkB isoform expression is associated with significantly different 5mC and 5hmC patterns in neurons and astrocytes. Further, we investigated the role of TrkB.T1 in cortical astrocyte developmental processes and astrocyte function during early postnatal development (postnatal day (P) 8, P14, P28 and P60). RNA sequencing of TrkB.T1 deficient astrocytes isolated at these timepoints revealed aberrant gene expression in astrocyte maturation, while pathway analysis indicated disruptions in synapse organization, neurotransmitter transport and exocytotic processes. Subsequent functional secretory proteomics highlighted disruptions in metabolism and lipid regulation, particularly cholesterol transport, suggesting potential implications for synapse formation. We observed dysregulated spine density in the motor and somatosensory cortices from TrkB.T1-deficient astrocytes relative to control astrocytes. These findings suggest that TrkB.T1 deficiency adversely affects normal astrocyte development, which in turn affects neuronal synapse development. This study provides new insights into the role of BDNF/TrkB.T1 signaling in CNS development and lays the groundwork for evaluating astrocyte BDNF/TrkB.T1 signaling in neurological diseases. / Doctor of Philosophy / Astrocytes are an abundant brain cell type that play crucial roles in maintaining brain health and supporting neuron functions. Astrocytes develop right after birth and reach full maturity by the second month in rodents. Throughout life, astrocytes play crucial roles in stabilizing the brain's environment and supporting most brain functions. the formation of astrocyte complex morphology is regulated by brain-derived neurotrophic factor (BDNF), which interacts with a specific receptor called TrkB.T1 found mainly in astrocytes. Another form of this receptor, TrkB.FL, is primarily found in neurons. Despite the importance of TrkB.T1 in astrocyte development and function, the reason for high expression and its role in astrocytes were not well understood. Previous studies in human postmortem samples indicated that DNA methylation level of the gene NTRK2 is negatively linked to TrkB.T1 expression but not TrkB.FL expression. However, the mechanisms behind TrkB.T1 isoform-specific expression and its role in astrocyte development are unknown. In this study, we used advanced sequencing techniques to analyze DNA methylation patterns, a chemical modification that can control gene activity, in neurons, astrocytes, and microglia, which are three different cell types in the brain. Our study discovered distinct methylation patterns in all three cell types and supported previous research DNA methylation effect on gene regulation and alternative gene splicing. With the data obtain from this study, we also observed a significant difference in DNA methylation may cause that TrkB.T1 is mainly expressed in astrocytes and TrkB.FL is mainly expressed in neurons. The study further investigated TrkB.T1 deficient astrocytes at different developmental stages. By examining RNA from astrocytes, we found abnormal gene expression in astrocyte maturation. Further analysis showed changes in cholesterol transport, suggesting potential implications for synapse formation. Finally, we observed the dysregulated spine density in the animals with astrocyte specific TrkB.T1 deletion. These findings suggest that TrkB.T1 deficiency adversely affects normal astrocyte development, which in turn affects neuronal synapse development. These findings provide new insights into the expression mechanisms and functions of TrkB.T1 in astrocytes, enhancing our understanding of how these cells support brain health and function.

Alzheimer's disease is a progressive neurodegenerative disorder of the central nervous system. One pathological characteristic is excessive neuronal loss in specific regions of the brain. Among the areas most severely affected are the basal forebrain cholinergic neurons and their projection regions, the hippocampus and cortex. Neurotrophic factors, particularly the neurotrophins nerve growth factor and brain-derived neurotrophic factor, play an important role in the development, regulation and survival of basal forebrain cholinergic neurons. Furthermore, brain-derived neurotrophic factor regulates the function of hippocampal and cortical neurons. Neurotrophins are synthesized in hippocampus and cortex and retrogradely transported to the basal forebrain. Decreased levels of neurotrophic factors are suspected to be involved in the neurodegenerative changes observed in Alzheimer's disease. We examined autopsied parietal cortex, hippocampus and nucleus basalis of Meynert samples from age- and gender-matched Alzheimer's diseased and neuro logically non-impaired individuals using the quantitative technique of competitive RT-PCR. We also examined parietal cortex samples by Western blotting. We demonstrate a 3.4-fold decrease in brain-derived neurotrophic factor mRNA levels in the parietal cortex of patients with Alzheimer's disease compared to controls (p < 0.004) but fail to observe changes in BDNF protein levels in that brain region. We also demonstrate, for the first time, BDNF mRNA in the nucleus basalis of Meynert and report an age-related decline in the levels of BDNF mRNA in both control and AD samples. Using the competitive RT-PCR technique we fail to observe differences in BDNF mRNA levels in the hippocampus between AD and control subjects, conflicting with previous in situ hybridization studies and RNase protection assays. A decrease in brain-derived neurotrophic factor synthesis could have detrimental effects on hippocampal, cortical and basal forebrain cholinergic neurons and may account for their selective vulnerability in Alzheimer's disease. / Thesis / Master of Science (MSc)

Estudos recentes associaram fortemente a fisiopatologia da Depressão ao estresse crônico e suas conseqüências, desde o comportamento alterado até a disrupção do eixo HPA e mudanças na expressão gênica. A depressão é uma doença que afeta milhões de pessoas ao redor do mundo, causando um grande impacto na qualidade de vida dos pacientes. Evidências sugerem o papel contributivo do stress de início da vida (ELS) para Depressão Maior (MD). O paradigma do estresse crônico variável é amplamente utilizado, mas seus impactos não foram estudados no início da vida. Portanto, para entender melhor esta condição, os filhotes de ratos Wistar machos (P1-P21) foram expostos ao paradigma Multimodal ELS. Foram avaliados os níveis plasmáticos de corticosterona (cort) e os órgãos relacionados ao eixo HPA. Adicionalmente, estes foram avaliados durante a idade adulta no teste de consumo de sacarose (TCS), no teste de nado forçado (TNF) e no teste da caixa claro-escuro (TCE). Os resultados indicam que os filhotes não se habituaram ao ELS multimodal. Em P21, o peso das glândulas adrenais dos animais ELS é significantivamente maior, e o timo e o peso corporal diminuíram, quando comparados com o grupo controle. O timo também se mantém significativamente reduzido quando comparado ao grupo controle em P90. Além disso, os ratos adultos submetidos ao protocolo ELS apresentaram menor ingestão de sacarose e maior latência para o compartimento claro na TCE, quando comparados ao grupo controle. Um outro modelo animal que apresenta estresse intermitente e tem sido amplamente utilizado é o modelo da restrição do material de ninho. Neste modelo a redução do material do ninho promove uma disrupção no cuidado materno, e gera alterações no desenvolvimento encefálico e comportamental da prole. O ELS leva a um surgimento precoce da inibição do medo contextual e à maturação acelerada do hipocampo. Nós postulamos que a corticosterona desempenha um papel funcional na regulação do tempo de maturação das regiões subjacentes à aprendizagem e expressão de ameaças, incluindo o hipocampo e a amígdala. Nossa hipótese é que alterações nos níveis plasmáticos de corticosterona podem afetar o tempo de processos de maturação cerebral e comportamental. Portanto nosso objetivo foi avaliar como um bloqueador da síntese de corticosterona altera o comportamento de medo condicionado e expressão de BDNF em animais controle ou submetidos ao modelo ELS de redução de material do ninho. Tanto nos grupos controle como ELS utilizamos animais Naïve, ou que receberam Veículo ou Metirapona (MET; 50mg/kg) em P12. Em P18, P21 ou P28 grupos independentes de camundongos foram expostos a uma única sessão de condicionamento do medo, seguido 24 h depois por um único teste de contexto. A análise do comportamento de congelamento no teste de contexto revelou que o tratamento com MET bloqueou a aceleração na inibição do medo contextual em camundongos fêmeas submetidos ao ELS. Curiosamente, em filhotes machos controles tratados com MET, houve um atraso na curva de desenvolvimento de medo contextual. Observamos que houve um correlato do efeito do tratamento com Metirapona na expressão do BDNF em regiões límbicas (hipocampo ventral e amígdala basolateral). Com base nos presentes resultados, a CORT provavelmente desempenha um papel importante no momento do desenvolvimento típico e das mudanças associadas ao ELS no comportamento e na maturação do cérebro. A epilepsia é uma condição neurológica crônica caracterizada pela predisposição persistente a gerar crises epilépticas, e pelas consequências neurobiológicas, cognitivas, psicológicas e sociais desta condição. A depressão é uma comorbidade psiquiátrica muito comum em pacientes com epilepsia. Cada vez mais dados sugerem que a epilepsia, depressão e outros possíveis distúrbios psiquiátricos como a ansiedade compartilham mecanismos patogênicos. Nesse sentido, foram encontradas anormalidades na linhagem WAR (Wistar Audiogenic Rat) que a tornam um modelo interessante para o estudo do estresse, a Epilepsia e as comorbidades neuropsiquiátricas envolvidas. Com base nisso, o presente estudo teve comoxiv Godoy, L.D. objetivo avaliar na linhagem WAR: 1. Cuidados maternos na linhagem WAR nas condições controle e sob estresse 2. Comportamentos de tipo depressivo basal e após o kindling audiogênico (crises convulsivas crônicas- KAu). Não houve diferença no tempo em postura de amamentação ativa, licking/grooming ou tempo da mãe no ninho, e no número de ataques e comportamentos agressivos. Observamos um aumento na latência de ratas WAR para recuperar os filhotes após separação materna, e enquanto 100% das ratas Wistar agruparam toda a ninhada, nas fêmeas WAR foi observado apenas 40%. 2. Para avaliar os comportamentos de tipo depressivo, os ratos foram submetidos a 20 estímulos acústicos duas vezes ao dia (KAu) (Wistar-KAu, WAR-KAu), enquanto os respectivos grupos controle permaneceram sem estímulo (Wistar, WAR). Posteriormente, os grupos foram submetidos ao TCS e TNF Não houve diferença entre o grupo WAR e Wistar no TCS , porém o grupo WAR-KAu grupo apresentou um aumento significativo em relação aos grupos controles. No TNF ambos os grupos WAR e WAR-KAu apresentaram redução significativa de escalada na sessão teste quando comparados ao Wistar. Tomados em conjunto, esses achados indicam que o ELS pode gerar suceptibilidade às comorbidades psiquiátricas associadas ao estresse, e que os modelos experimentais em questão permitem investigar como os efeitos dos glicocorticoies se relacionam com o neurodesenvolvimento, especificamente na maturação do comportamento de medo e de estruturas límbicas. Além disso, dados preliminares indicam que, embora a linhagem WAR não apresente diferenças comportamentais maternas em condições basais, pode apresentar alterações sob o efeito de eventos estressantes. Também, com base nos achados, a susceptibilidade de crises convulsivas pode estar relacionada às alterações nas estratégias comportamentais em situações estressantes na vida adulta, que também pode constituir vulnerabilidade às comorbidades psiquiátricas. / Recent studies have strongly associated the pathophysiology of depression with chronic stress and its consequences, from altered behavior to HPA axis disruption and changes in gene expression. Depression is a disease that affects millions of people around the world, causing a major impact on the quality of life of patients. Evidence suggests the contributory role of early life stress (ELS) for Major Depression (MD). The variable chronic stress paradigm is widely used, but its impacts were not studied early in life. Therefore, to better understand this condition, male Wistar rat pups (P1-P21) were exposed to the Multimodal ELS paradigm. Plasma levels of corticosterone (CORT) and organs related to the HPA axis were evaluated. Additionally, these were evaluated during adulthood in the sucrose consumption test (SCT), the forced swimming test (FST) and the light-dark box test (LDT). The results indicate that pups did not habituate to multimodal ELS. In P21, the weight of the adrenal glands of the ELS animals is significantly greater, and the thymus and body weight decreased, when compared with the control group. The thymus also remains significantly reduced when compared to the control group in P90. In addition, adult rats submitted to the ELS protocol presented lower intake of sucrose and greater latency to light compartment in the LDT when compared to the control group. Another animal model that presents intermittent stress and has been widely used is the constraint model of nest material. In this model, the reduction of nest material promotes disruption in maternal care and causes changes in the encephalic and behavioral development of offspring. ELS leads to an early onset of contextual fear inhibition and accelerated maturation of the hippocampus. We postulate that CORT plays a functional role in regulating the maturation time of the regions underlying the learning and expression of threats, including the hippocampus and amygdala. Our hypothesis is that changes in plasma CORT levels may affect the timed brain and behavioral maturation processes. Therefore, our objective was to evaluate how a CORT synthesis blocker (Metyrapone; MET) alters conditioned fear behavior and BDNF expression in animals reared under control conditions or submitted to the ELS model of limited nesting bed. Both in control and ELS groups included Naïve animals or animals that received vehicle or MET; 50 mg/kg in P12. In P18, P21 or P28 independent groups of mice were exposed to a single session of fear conditioning, followed 24 hours later by a single context test. Analysis of the freezing behavior in the context test revealed that MET treatment blocked the acceleration of contextual fear suppression at P22 induced by ELS, with no difference between the P19 or P29 groups. This effect was observed mainly in female mice. Interestingly, in control male pups treated with MET, there was an altered contextual fear developmental curve. MET showed significant reduction in freezing in PND22, increase in freezing in PND29 followed by delayed suppression in P39. We observed that MET behavioural data were further supported by BDNF expression in limbic regions (ventral hippocampal and basolateral amygdala). Based on the current results, CORT probably plays an important role at the time of typical development and changes associated with ELS in the behavior and maturation of the brain. Epilepsy is a chronic neurological condition characterized by a persistent predisposition to epileptic seizures, and by neurobiological, cognitive, psychological and social consequences. Depression is a very common psychiatric comorbidity in patients with epilepsy. Increasingly data suggest that epilepsy, depression and other possible psychiatric disorders such as anxiety share pathogenic mechanisms. In this sense, abnormalities in the WAR strain (Wistar Audiogenic Rat) have been found that make it an interesting model for the study of stress, Epilepsy and neuropsychiatric comorbidities involved. Based on this, the present study aimed to evaluate in the WAR strain: 1. Maternal care under control and under stress conditions 2. Basal depressive type behaviors and after audiogenic kindling (chronic seizures-KAu).xvi Godoy, L.D. There was no difference for active breastfeeding posture, licking/grooming or time of mother in the nest and in the number of attacks and aggressive behaviors in maternal aggression test. WAR rats showed a higher latency to recover pup in pup retrieval test, whereas 100% of the Wistar rats grouped the entire litter, and in WAR it was only 40%. 2. In order to evaluate the depressive type behaviors, the rats were submitted to 20 acoustic stimuli twice a day (AuK) (Wistar-AuK, WAR-AuK), while the respective control groups remained without stimulus (Wistar, WAR). Subsequently, the groups were submitted to sucrose consumption test (SCT) and forced swim test (FST). There was no difference between the WAR and Wistar groups in the SCT, however the WAR-KAu group presented an increase in sucrose consumption in relation to the control groups 3 or 13 dasy after the AuK. In FST, both WAR and WARKAu groups showed a significant decrease in the test session when compared to Wistar. Taken together, these findings indicate that ELS may induce susceptibility to psychiatric comorbidities associated with stress, and that the current experimental models allow investigating how the effects of glucocorticoids are related to neurodevelopment, specifically in the maturation of fear behavior and limbic structures. In addition, preliminary data indicate that, although the WAR strain does not show maternal behavioral differences at baseline, it may present changes under stressful events. Also, based on the findings, the susceptibility of seizures may be related to changes in behavioral strategies in stressful situations in adult life, which may also constitute vulnerability to psychiatric comorbidities.

Le PACAP38 est un polypeptide endogène secrété par le thalamus exerçant différents rôles physiologiques tels que vasodilatateur, immunomodulateur ou encore, analgésique. Selon certaines études, il aurait, également, un impact sur les processus cognitifs au niveau de l’hippocampe. Cependant, son mécanisme d’action est assez peu connu. Son analogue, récemment synthétisé, a, quant à lui, été très peu étudié. Il s'agit d'une version beaucoup plus stable et avec une meilleure affinité aux récepteurs au PACAP que le peptide natif.Le travail effectué et présenté dans ce mémoire a permis de comparer les effets du PACAP38 et de son analogue sur le processus de mémorisation chez des rats ayant subit des expériences de navigation spatiale hippocampo-dépendante.Lors du test de l'objet déplacé (OLT), nous avons montré qu'une dose de 30 µg/kg de PACAP38 injecté en intraveineuse améliore la consolidation de la mémoire chez des rats souffrant de troubles cognitifs induits par une injection en intrapéritonéale de lipopolysaccharides ou d'ifenprodil. L'analogue n'a, quant à lui, pas permis de réduire ces troubles.Lors du test de la piscine de Morris, le PACAP38 a exercé un effet promnésique mais pas l'analogue.Il s'est avéré que le PACAP38 contribue à l'amélioration des processus cognitifs par son activité antioxydante, en régulant les taux de cytokines pro et anti-inflammatoires et les taux de BDNF centraux d'une manière plus efficace que celle de l'analogue. / PACAP38 is an endogenous polypeptide secreted by the thalamus. It is exerting different physiological roles such as vasodilator, immunomodulator or analgesic. According to some studies, it would also have an impact on cognitive processes in the hippocampus. However, its action mechanism is relatively unknown. Its analog, recently synthesized, has, for its part, been studied very little. It is a much more stable version with better affinity to PACAP receptors than the native peptide.The work performed compares the effects of PACAP38 and its analog on memory process in rats that had undergone hippocampo-dependent spatial navigation experiments.In the object location test (OLT), we have shown that a 30 µg/kg dose of PACAP38 injected intravenously improves memory consolidation in rats with cognitive impairment induced by intraperitoneal injection of lipopolysaccharides or ifenprodil. The analog was not able to reduce these disorders.In the Morris water maze test, PACAP38 exerted a promnesic effect but not the analog.It has been found that PACAP38 contributes to the improvement of cognitive processes by its antioxidant activity, by regulating pro and anti-inflammatory cytokine levels and central BDNF levels in a more efficient manner than the analog. .

Le BDNF (brain-derived neurotrophic factor) a été découvert dans le cerveau et est largement impliqué dans la neuroplasticité, la mémoire et la cognition via l’activation des récepteurs TrkB (tropomyosin receptor kinase B) neuronaux. Nous avons récemment montré que le système cardiovasculaire contenait autant de BDNF que le cerveau et que le BDNF exogène était capable d’induire une relaxation vasculaire dépendante de l’endothélium. D’autres études ont suggéré que l’activation des récepteurs TrkB endothéliaux par le BDNF était impliquée dans les processus athérosclérotiques. Notre laboratoire soupçonne une interaction étroite entre NO et BDNF endothélial et a même envisagé la possibilité d’une implication du BDNF secrété par l’endothélium des microvaisseaux cérébraux dans la cognition. Les patients souffrant de polyarthrite rhumatoïde (PR), une maladie inflammatoire d’origine auto-immune, sont à risque cardiovasculaire et présentent une altération de la cognition avec notamment un risque plus élevé de dépression. Etonnamment, l’effet de la PR sur le BDNF est peu documenté. Les seules études disponibles rapportent une élévation du BDNF dans le sang et le liquide synovial en cas de PR. Notre hypothèse est qu’une réduction de l’expression endothéliale du BDNF pourrait contribuer au risque cardiovasculaire et au déficit cognitif associés à la PR. Ainsi, dans notre travail, nous avons étudié le BDNF vasculaire et cérébral et son récepteur TrkB sur le modèle rat d’arthrite induite à l’adjuvant.Nos principaux résultats montrent que l’arthrite conduit à 1) une réduction des taux aortiques de BDNF indépendamment de la sévérité des symptômes inflammatoires mais dépendante de la fonction endothéliale, 2) une diminution des taux cérébraux en BDNF indépendamment de la sévérité des symptômes inflammatoires mais en lien avec la fonction endothéliale, 3) une diminution de l’expression du BDNF et de son récepteur TrkB activé au niveau tant neuronal qu’endothélial dans les régions cérébrales impliquées dans la cognition, 4) une corrélation positive entre l’expression du BDNF par l’endothélium vasculaire et l’expression neuronale des TrkB activés, 5) une absence de corrélation entre les taux sériques de BDNF et ses taux cérébraux ou vasculaires mais en revanche l’existence d’une corrélation positive entre les taux sériques de BDNF et l’inflammation qu’elle soit clinique ou biologique.L’ensemble de ces données est en faveur de l’hypothèse selon laquelle le BDNF endothélial pourrait être impliqué dans le risque athérosclérotique et le déficit cognitif associé à l’arthrite. L’inflammation doit être considérée comme un facteur confondant lorsque les taux circulants de BDNF sont utilisés comme un reflet des taux présents dans le cerveau. / BDNF (brain-derived neurotrophic factor) has been discovered in the brain and is widely implicated in neuroplasticity, memory and cognition through the activation of neuronal TrkB (tropomyosin receptor kinase B) receptors. We have recently shown that the cardiovascular system contained as much BDNF as the brain and that exogenous BDNF was able to induce endothelium-dependent vascular relaxation. Other studies have suggested that activation of endothelial TrkB receptors by BDNF is involved in atherosclerotic processes. Our laboratory suspects a close interaction between endothelial NO and BDNF and has even considered the possibility of involvement of BDNF secreted by cerebral microvessel endothelium in cognition. Patients suffering from rheumatoid arthritis (RA), an inflammatory disease of autoimmune origin, are at risk of cardiovascular disease and have an impairment of cognition, including a higher risk of depression. Surprisingly, the effect of RA on BDNF is poorly documented. The only available studies report an increase of BDNF in the blood and synovial fluid in RA. Our hypothesis is that a reduction in endothelial expression of BDNF may contribute to the cardiovascular risk and cognitive deficit associated with RA. Thus, in our work, we studied vascular and cerebral BDNF and its TrkB receptor on the rat model of adjuvant-induced arthritis.Our main findings show that arthritis leads to 1) a decrease in BDNF levels in aortas independently of the severity of inflammatory symptoms but dependent on endothelial function, 2) a decrease in brain BDNF levels independent of the severity of inflammatory symptoms, but link with endothelial function, 3) a decreased expression of BDNF and its activated TrkB receptor at both neuronal and endothelial levels in the brain regions involved in cognition, 4) a positive correlation between endothelial expression of BDNF and neuronal expression of activated TrkB, 5) a lack of correlation between serum BDNF levels and its cerebral or vascular levels, but on the other hand the existence of a positive correlation between serum BDNF levels and inflammation, whether clinical or biological.All of these data support the hypothesis that endothelial BDNF may be involved in atherosclerotic risk and cognitive impairment associated with arthritis. Inflammation should be considered as a confounding factor when circulating levels of BDNF are used as a reflection of levels present in the brain.

Introdução: Penfigoide bolhoso (PB) resulta da produção de autoanticorpos contra proteínas hemidesmossomais BP (Bullous Pemphigoid) 180 e/ou 230, acomete os idosos, e está associado com doenças neurológicas (DN), especialmente com a demência (DEM). BP180/230 foram identificadas no Sistema Nervoso Central (SNC), aventando-se possível mimetismo antigênico entre moléculas da pele e do SNC. O fator neurotrófico derivado do cérebro (BDNF) participa da neurogênese, sinaptogênese e sobrevivência neuronal, e a sua diminuição sérica tem sido relacionada com DN. Objetivo: Quantificar o peptídeo BDNF e os anticorpos anti-BP180/230 na relação PB com DEM. Material e Métodos: Em estudo comparativo, 50 pacientes com PB, 50 com demência e 50 controles foram avaliados. A detecção dos anticorpos anti-BP180/P230 e do peptídeo BDNF foi determinada por ensaios ELISA. Imunofluorescência indireta (IFI) foi conduzida nas amostras de soro dos pacientes do grupo DEM e dos controles que apresentaram positividade para anti-BP180/230. Resultados: No grupo PB, a frequência de DN foi de 26%: DEM 16%, acidente vascular cerebral 6%, e epilepsia 4% - 5/8 (63%) pacientes apresentaram demência vascular e 3/8 (38%) demência por doença de Alzheimer. Positividade para anti-BP180/230 foi observada no grupo PB (74% e 40%, respectivamente), no grupo DEM (10% e 10%) e nos controles (14% e 0%). No grupo DEM, em 2/10 pacientes que apresentaram positividade para antiBP180/230, a IFI evidenciou depósito de IgG e C3 no lado epidérmico da clivagem, configurando quadro subclínico de PB. A mediana do BDNF resultou menor no grupo DEM (25,41 pg/ml) comparado aos controles (38,21 pg/mL), e o grupo PB apresentou os menores valores de BDNF (16,88 pg/mL). Não houve correlação dos títulos de anticorpos antiPB180/230 com a concentração do peptídeo BDNF no grupo PB. Os pacientes do grupo DEM foram alocados de acordo com a escala de demência - CDR1, CDR2 e CDR3; a mediana de BDNF do subgrupo CDR3 (23,37 pg/mL) foi inferior ao CDR1 (30,17 pg/ mL). Não houve diferença na concentração do BDNF segundo o tipo de demência. O grupo PB, quando estratificado em - com DEM, outras DN e sem DN, aqueles com associação com DEM apresentaram menores níveis de BDNF (9,1 pg/mL), comparados ao grupo sem DN e ao subgrupo CDR3 do grupo DEM. Conclusão: Marcadores para PB não são úteis para o diagnóstico de DEM. Valores sorológicos baixos de BDNF no grupo PB podem sugerir associação com DEM. BDNF pode ser utilizado como biomarcador de gravidade da DEM. / Introduction: Bullous pemphigoid (BP) is characterized by autoantibodies against the hemidesmossomal proteins BP180 and/or BP230, affects the elderly people and has been strongly associated with neurological disorders (ND), especially dementia. A possible antigenic mimicry hypothesis between the skin and the nervous system molecules is strong reasonable because BP peptides have also been identified in the central nervous system (CNS). Brain-derived neurotrophic factor (BDNF) plays a role in the synaptogenesis, neurogenesis, and neuronal survival, and some studies have been correlated the decreased serum BDNF levels with ND. The aim of this study was to quantify the BDNF peptide and the anti-BP180 and anti-BP230 antibodies in the BP and DEM relationship. Methods: AntiBP180/230 and BDNF quantification were analyzed in three groups: 50 patients with BP, 50 patients with dementia and 50 elderly individuals comprised a case-control study. Serum IgG anti-180/230, and the BDNF peptide were evaluated by using ELISA commercial kits; and immunofluorescence allied to salt split skin technique (SSS) was conducted in serum samples from patients of the dementia group and from controls who showed positive anti-BP180/230. Results: In BP group, 26% were associated with ND, and dementia was the most frequent (16%), followed by stroke (6%) and epilepsy (4%) - 5/8 (63%) patients showed vascular dementia and 3/8 (38%) patients presented dementia due to Alzheimer\'s disease. AntiBP180/230 positivity was observed in BP group (74%, 40%, respectively), in dementia group (10%, 10%) and in controls (14%, 0%). In 2/10 patients of the dementia group with positive anti-BP180/230, IIF showed IgG and C3 deposition in the epidermal side of cleavage, configuring a subclinical BP dermatosis. The medians of BDNF resulted lower in the patients of the dementia group (25.41 pg/mL) compared with controls (38.21 pg/mL), and the BP group presented the lowest BDNF values (16.88 pg/mL). There was no correlation between the anti-BP180/230 antibodies titres and the BDNF levels in BP group. There was no difference of BDNF levels accordingly with the clinical type of dementia in the dementia group. Patients of the dementia group were sub grouped accordingly with the clinical dementia severity - CDR1, CDR2 and CDR3 - being the median of BDNF in CDR3 (23.37 pg/mL) lower than in CDR1 (30.17 pg/mL) subgroup. BP group had lower levels of BDNF compared to CDR3 subgroup. BP patients when stratified with dementia, other ND and without ND, those with association with dementia presented the lowest levels of BDNF (9.1 pg/mL) compared to the PB patients without DN and to the CDR3 subgroup. Conclusion: BP biomarkers are not useful for the diagnosis of dementia. Low BDNF levels seen in BP patients may suggest an association with dementia. BDNF may be used as a biomarker of severity of dementia.

O fator neurotrófico derivado do cérebro (BDNF) tem sido associado com a angiogênese por meio do seu receptor específico tropomiosina quinase B (TrkB). Uma vez que ambos são expressos em células endoteliais vasculares e o treinamento físico aeróbio (TF) pode aumentar os níveis séricos de BDNF, este estudo teve como objetivo testar as hipóteses de que: 1) Os níveis séricos de BDNF modulam o fluxo de sangue periférico; e 2) A presença do alelo Met no polimorfismo BDNF Val66Met prejudica o ganho de vasodilatação por TF. Foram genotipados para o polimorfismo do gene BDNF 304 voluntários saudáveis do sexo masculino (Val66Val, n = 221; Val66Met, n = 83) que foram submetidos a intenso TF em uma pista de corrida 3 vezes/semana durante 4 meses. Foram avaliados pré e pós-TF as concentrações circulantes de BDNF e pró-BDNF (ELISA), frequência cardíaca (FC), pressão arterial média (PAM), o fluxo de sangue do antebraço (FSA) e resistência vascular periférica (RVP). No período pré-TF, BDNF, pró-BDNF, a razão BDNF/pró-BDNF, FSA e RVP foram semelhantes entre todos os genótipos. Depois do TF, a capacidade funcional (VO2pico) aumentou e houve a diminuição da FC de repouso de forma semelhante em ambos os grupos. O grupo Val66Val, mas não o Val66Met, aumentou os níveis de BDNF (interação, p=0,04) e aumentou a razão BDNF/pró-BDNF (interação, p < 0,001). Curiosamente, as respostas do FSA (Interação, p=0,04) e da RVP (Interação, p=0,01), durante o exercício handgrip, do grupo Val66Val, apresentou melhoras quando comparado com o grupo Val66Met, mesmo com respostas similares de FC e PAM. Outras análises mostraram associação entre a razão BDNF/pró-BDNF e FSA (R=0,64; p < 0,001) e RVP (R=-0,58; p < 0,001). Estes resultados mostram que, em resposta ao TF, tanto a reatividade vascular periférica quanto o BDNF circulante são prejudicados pela presença do polimorfismo Val66Met do gene BDNF e esta responsividade está associada às concentrações de BDNF sérico em indivíduos saudáveis / The neurotrophin Brain-derived neurotrophic factor (BDNF) has been associated with angiogenesis through its specific receptor tropomyosin-related kinase B (TrkB). Since both are expressed in vascular endothelial cells and aerobic training (ET) can increase serum BDNF levels, this study aimed to test the hypotheses that: 1) Serum BDNF levels modulate peripheral blood flow; and 2) The presence of the allele Met in the BDNF Val66Met polymorphism impairs vasodilation gain by ET. We genotyped for BDNF polymorphism 304 healthy male volunteers (Val66Val, n= 221; Val66Met, n=83) which underwent to intense aerobic ET on a running track 3 times/week for 4 months. We evaluated pre and post ET serum BDNF and proBDNF concentration (ELISA), heart rate (HR), mean blood pressure (MBP), forearm blood flow (FBF) and forearm vascular resistance (FVR). In the pre ET, BDNF, proBDNF, BDNF/proBDNF ratio, FBF and FVR were similar between all genotypes. After ET, functional capacity (VO2peak) increased and HR decreased similarly in both groups. Val66Val, but not Val66Met, increased BDNF (Interaction, p= 0.04) and BDNF/proBDNF ratio (Interaction, p < 0.001). Interestingly, FBF (Interaction, p=0.04) and the FVR (Interaction, p=0.01) responses during handgrip exercise improved in Val66Val compared with Val66Met, even with similar responses of HR and MBP. Further analyses showed association between BDNF/proBDNF ratio and FBF (R=0.64, p < 0.001) and FVR (R=-0.56, p < 0001). These results show that peripheral vascular reactivity and serum BDNF responses to ET are impaired by the BDNF Val66Met polymorphism and such responsiveness is associated to the serum BDNF concentrations in healthy subjects

Das Ziel dieser Arbeit war es, die Modulation der GABAergen synaptischen Transmission in den visuellen Schichten des Colliculus superior der Maus durch das Neurotrophin BDNF zu charakterisieren. Hierzu wurden bdnf+/+ und -/- Mäuse kurz vor und nach der Augenöffnung in einer die Morphologie erhaltenden Schnittpräparation elektrophysiologisch und molekularbiologisch untersucht. Das Fehlen von BDNF veränderte das Präparat hinsichtlich Neurondichte und -größe nicht. Ebenso blieben der Membranwiderstand und die Ganzzellkapazität unbeeinflusst von der chronischen Abwesenheit von BDNF. Im Gegensatz dazu zeigten sich deutliche funktionelle Defizite im Entladungsverhalten und in der GABAergen Hemmung. Durch Registrierung von Aktionspotentialen wurde demonstriert, dass BDNF für die Aufrechterhaltung der Netzwerkaktivität erforderlich ist. Durch Applikation eines GABAA-Rezeptor-Inhibitors konnte die Suppression der GABAergen Hemmung durch BDNF als zugrunde liegender Mechanismus aufgedeckt werden. Daraufhin durchgeführte Ganzzellableitungen bestätigten dies und legten einen postsynaptischen, TrkB-vermittelten Mechanismus der BDNF-Wirkung nahe. Es war möglich, den Einfluss der chronischen Abwesenheit von BDNF durch akute lokale Superfusion von BDNF vollständig aufzuheben. Die ausschließlich postsynaptische Blockade der PKC reichte aus, dies zu verhindern. Hierdurch wird unterstrichen, dass in diesem Präparat der Angriffspunkt von BDNF an der GABAergen Synapse auf der postsynaptischen Seite liegt. Um den genauen Wirkungsmechanismus von BDNF an der GABAergen Synapse zu beleuchten, wurde die mRNA-Expression der GABAA-Rezeptor-Untereinheiten alpha 1-3 untersucht. Diese ist in Anwesenheit von BDNF höher. Demzufolge sollte eine reduzierte Expression dieser Untereinheiten in bdnf-/- Tieren zu einer verringerten Rezeptoranzahl und somit zur Sättigung postsynaptischer Rezeptoren führen. Durch die Analyse von Amplitude und Kinetik GABAerger IPSC und die Applikation von Zolpidem wurde dies bestätigt. Demnach führt die Abwesenheit von BDNF zur Aufregulation der GABAergen Inhibition, obwohl die Rezeptorzahl in der Postsynapse wahrscheinlich niedriger ist. Als zentraler Mechanismus der akuten BDNF-Wirkung kommt deshalb am ehesten eine PKC-vermittelte Phosphorylierung und nachfolgende Veränderung des Desensitisierungsverhaltens in Betracht. Außerdem muss an eine Reduktion der Öffnungswahrscheinlichkeit oder der Leitfähigkeit des Rezeptors gedacht werden. Unmittelbar vor der Augenöffnung hatte die BDNF-Defizienz keinerlei Einfluss auf die GABAerge Hemmung. Es ist also davon auszugehen, dass BDNF im CS erst nach der Augenöffnung eine wesentliche Rolle in der Modulation der GABAergen Synapsen und damit in der Kontrolle der Netzwerkaktivität spielt. Dies steht in Einklang mit der Vorstellung, dass sowohl Translation und Freisetzung als auch Transport von BDNF durch neuronale Aktivität reguliert werden. Der Ablauf der neurotrophinergen Regulation im Colliculus superior stellt sich wie folgt dar: Durch Aktivation colliculärer Afferenzen wird BDNF vermehrt freigesetzt. BDNF reduziert nun zunächst über die Modulation von Rezeptoreigenschaften die GABAerge Hemmung und disinhibiert die Netzwerkaktivität. Längerfristig kommt es über eine vermehrte Expression von GABAA-Rezeptor-Untereinheiten zum Anstieg der Rezeptorzahl, damit zur Wiederherstellung der GABAergen Hemmung und letztlich zu einer Reduktion der Netzwerkaktivität. BDNF ist also in der kritischen Zeitperiode der Augenöffnung, wenn das Mustersehen einsetzt, ein wichtiger Faktor in der Regulation neuronaler Aktivität. / The aim of the study was to characterise the influence of the neurotrophin BDNF on the GABAergic synaptic transmission in the visual layers of the mouse superior colliculus. Acute slices prepared from bdnf+/+ and -/- mice shortly before and after eye opening were employed in the experiments. The absence of BDNF altered neither the density or size of neurons nor their membrane resistance or whole cell capacity. However, registration of action potentials revealed a decreased firing rate in the absence of BDNF. Stronger disinhibition induced by application of a GABAA receptor blocker suggested an enhanced GABAergic inhibition as an underlying mechanism. This assumption was confirmed by performing whole cell experiments. Further analysis indicated a postsynaptic enhancement of GABAergic synaptic transmission in the absence of BDNF. In bdnf+/+ slices, blockade of BDNF signalling through the TrkB receptor strengthened GABAergic synaptic transmission. Contrariwise, superfusion of exogenous BDNF in bdnf-/- suppressed GABAergic synaptic transmission. An exclusively postsynaptic block of the PKC abolished the effect of BDNF application. Therefore, a BDNF induced, TrkB mediated, PKC dependent suppression of the GABAergic synaptic transmission at the postsynaptic site can be assumed. To further elucidate the mechanism of BDNF action the expression of the GABAA receptor subunits alpha 1-3 mRNA was studied. In the presence of BDNF an elevated expression was observed. A lower expression of these subunits in bdnf-/- slices could result in a reduced number of GABAA receptors. During synaptic transmission they may become saturated. Two observations support this idea: In bdnf-/- slices application of Zolpidem does not induce an increase of GABAergic IPSC amplitude as present in bdnf+/+ slices. Amplitude and decay kinetics of GABAergic IPSCs correlate in bdnf-/- but not +/+ slices. Therefore, absence of BDNF may strengthen GABAergic synaptic transmission although mediated by a reduced number of postsynaptic receptors. A BDNF induced PKC dependent receptor phosphorylation followed by a change in receptor desensitisation is most likely the underlying mechanism. Nevertheless, a reduction of the receptor opening probability or a reduced receptor conductance have to be considered as well. Shortly before eye opening BDNF deficiency had no impact on GABAergic inhibition. In accordance with the general idea that expression and release of BDNF is activity dependent, this finding suggests that BDNF controls network activity by modulating GABAergic synaptic transmission only after eye opening. Taken together, the following sequence of BDNF action in the superior colliculus during eye opening can be proposed: Activation of collicular inputs triggers an increased BDNF release. BDNF suppresses GABAergic synaptic transmission leading to a disinhibition of network activity. Later, the increased expression of GABAA receptor subunits by prolonged BDNF release results in an increased GABAA receptor number, the recovery of the GABAergic inhibition and finally a gently tuned network activity. In summary, BDNF constitutes a major regulator of neuronal activity in the critical period of eye opening marking the transition to pattern vision.

O enriquecimento ambiental (EA) reverte a sensibilização comportamental (SC) ao etanol em camundongos e diminui os níveis de BDNF no córtex pré-frontal (CPF). O objetivo deste trabalho foi estudar os efeitos do EA sobre parâmetros comportamentais, resposta de corticosterona ao estresse, expressão de microRNAS e a participação da via BDNF-TrkB no CPF frente à SC ao etanol. Camundongos mantidos em EA foram submetidos a testes comportamentais (ansiedade, depressão, anedonia e comportamentos repetitivos). Também foram tratados repetidamente com etanol e expostos ao EA para a avaliação da corticosterona plasmática e da expressão de microRNAs no CPF. A via BDNF-TrkB foi inibida no CPF de camundongos que desenvolveram a SC ao etanol. O EA diminuiu a atividade locomotora e exploratória, a ativação do eixo HPA após estresse agudo, aumentou o peso do baço e diminuiu a expressão de miR-132 e let-7d. Os resultados sugerem o envolvimento de microRNAs na reversão da SC ao etanol; contudo, a via BDNF-TrkB no CPF não parece estar diretamente envolvida neste processo. / The environmental enrichment (EE) reverts the ethanol-induced behavioral sensitization (EIBS) in mice and decreases BDNF levels in the prefrontal cortex (PFC). The aim of this work was to evaluate the effects of EE on behavioral parameters, on the corticosterone response to stress and on microRNA expression and to assess the involvement of the BDNF-TrkB pathway in the PFC on the EIBS. Mice kept in EE were submitted to behavioral tests (anxiety, depression, anhedonia and repetitive behaviors). Mice were also treated repeatedly with ethanol and exposed to EE for the assessment of plasma corticosterone levels and microRNA expression in the PFC. The BDNF-TrkB pathway was blocked in the PFC of mice that developed EIBS. EE decreased locomotion, exploratory activity and HPA axis activation after acute stress, increased spleen weight and decreased miR-132 and let-7d expression. The results suggest the involvement of microRNAs in the reversal of EIBS promoted by EE; however, the BDNF-TrkB pathway in the PFC does not seem to be directly involved in this process.