Efeitos da eliminação de neurônios infragranulares sobre a especificação de neurônios supragranulares do córtex cerebral

The cerebral cortex of mammals is histologically organized into different layers of excitatory neurons that have distinct patterns of connections with cortical or subcortical targets. During development, these cortical layers are established through an intricate combination of neuronal specification and migration in a radial pattern known as "insideout": deep-layer neurons are generated prior to upper-layer neurons. In the last few decades, several genes encoding transcription factors involved in the sequential specification of neurons destined to different cortical layers have been identified. However, the influence of early-generated neurons in the specification of subsequent neuronal cohorts remains unclear. To investigate this possible influence, we induced the selective death of cortical neurons from layer V and VI before the generation of layer II, III and IV neurons. Thus, we can evaluate the effects of ablation of early born neurons on the phenotype of late born neurons. Our data shows that one-day after ablation, layer VI neurons expressing the transcription factor TBR1 are newly generated while virtually no neuron expressing TBR1 was generated in the same age in control animals. This suggests that progenitors involved in the generation of neurons destined for superficial layers suffer interference from the selective death of neurons in deep layers, changing their specification. We also observed that while TBR1-positive neurons are located exclusively in deep cortical layers of control animals, many TBR1-positive neurons are misplaced in superficial layers of ablated animals, suggesting that the migration of cortical neurons could be controlled independently of neuronal phenotypes. Furthermore, we observed an increase in layer V neurons expressing CTIP2 and neurons expressing SATB2 and that these cells have changed their distributions. As a conclusion, our data indicate the existence of a mechanism of control exercised by the early-generated neurons in the cerebral cortex on the fate of the progenitors involved in the generation of the following cortical neurons. This mechanism could help to control the number of neurons in different layers and contribute to the establishment of different cortical areas

Interferindo com oscilações de alta frequência no hipocampo epiléptico: consequências para as crises espontâneas

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Efeitos da sinalização via creb sobre a sobrevivência e diferenciação meuronal

The cortical development requires a precise process of proliferation, migration, survival and differentiation of newly formed neurons to finally achieve the development of a functional network. Different kinases, such as PKA, CaMKII, MAPK and PI3K, phosphorylate the transcription factors CREB, and thus activate it, inducing CREB-dependent gene expression. In order to identify the involvement of such signaling pathways mediated by CREB over neuronal differentiation and survival, in vitro experiments of cell culture were conducted using pharmacological kinase inhibitors and genetic techniques to express different forms of CREB (A-CREB and CREB-FY) in cortical neurons. Inhibition of PKA and CaMKII decreased the length of neuronal processes (neurites); whereas inhibition of MAPK did not affect the length, but increased the number of neurites. Blockade of PI3K do not appear to alter neuronal morphology, nor the soma size changed with the kinase blockades. CREB activation (CREB-FY) along with MAPK and PI3K blockades presented a negative side effect over neuritic growth and the expression of A-CREB leaded to a significant decrease in neuronal survival after 60h in vitro and mimicked some of the effects on neuronal morphology observed with PKA and CaMKII blockade. In summary the signaling through CREB influences the morphology of cortical neurons, particularly when phosphorylated by PKA, and CREB signaling is also important for survival of immature neurons prior to the establishment of fully functional synaptic contacts. Our data contribute to understanding the role of CREB signaling, activated by different routes, on survival and neuronal differentiation and may be valuable in the development of regenerative strategies in different neurological diseases

Indicadores de cálcio e de voltagem codificados geneticamente na detecção de potenciais de ação e inputs sinápticos em cultura de neurônios hipocampais

Recently, genetically encoded optical indicators have emerged as noninvasive tools of high spatial and temporal resolution utilized to monitor the activity of individual neurons and specific neuronal populations. The increasing number of new optogenetic indicators, together with the absence of comparisons under identical conditions, has generated difficulty in choosing the most appropriate protein, depending on the experimental design. Therefore, the purpose of our study was to compare three recently developed reporter proteins: the calcium indicators GCaMP3 and R-GECO1, and the voltage indicator VSFP butterfly1.2. These probes were expressed in hippocampal neurons in culture, which were subjected to patchclamp recordings and optical imaging. The three groups (each one expressing a protein) exhibited similar values of membrane potential (in mV, GCaMP3: -56 ±8.0, R-GECO1: -57 ±2.5; VSFP: -60 ±3.9, p = 0.86); however, the group of neurons expressing VSFP showed a lower average of input resistance than the other groups (in Mohms, GCaMP3: 161 ±18.3; GECO1-R: 128 ±15.3; VSFP: 94 ±14.0, p = 0.02). Each neuron was submitted to current injections at different frequencies (10 Hz, 5 Hz, 3 Hz, 1.5 Hz, and 0.7 Hz) and their fluorescence responses were recorded in time. In our study, only 26.7% (4/15) of the neurons expressing VSFP showed detectable fluorescence signal in response to action potentials (APs). The average signal-to-noise ratio (SNR) obtained in response to five spikes (at 10 Hz) was small (1.3 ± 0.21), however the rapid kinetics of the VSFP allowed discrimination of APs as individual peaks, with detection of 53% of the evoked APs. Frequencies below 5 Hz and subthreshold signals were undetectable due to high noise. On the other hand, calcium indicators showed the greatest change in fluorescence following the same protocol (five APs at 10 Hz). Among the GCaMP3 expressing neurons, 80% (8/10) exhibited signal, with an average SNR value of 21 ±6.69 (soma), while for the R-GECO1 neurons, 50% (2/4) of the neurons had signal, with a mean SNR value of 52 ±19.7 (soma). For protocols at 10 Hz, 54% of the evoked APs were detected with GCaMP3 and 85% with R-GECO1. APs were detectable in all the analyzed frequencies and fluorescence signals were detected from subthreshold depolarizations as well. Because GCaMP3 is the most likely to yield fluorescence signal and with high SNR, some experiments were performed only with this probe. We demonstrate that GCaMP3 is effective in detecting synaptic inputs (involving Ca2+ influx), with high spatial and temporal resolution. Differences were also observed between the SNR values resulting from evoked APs, compared to spontaneous APs. In recordings of groups of cells, GCaMP3 showed clear discrimination between activated and silent cells, and reveals itself as a potential tool in studies of neuronal synchronization. Thus, our results indicate that the presently available calcium indicators allow detailed studies on neuronal communication, ranging from individual dendritic spines to the investigation of events of synchrony in neuronal networks genetically defined. In contrast, studies employing VSFPs represent a promising technology for monitoring neural activity and, although still to be improved, they may become more appropriate than calcium indicators, since neurons work on a time scale faster than events of calcium may foresee

Análise de grafos aplicada a relatos de sonhos: ferramenta diagnóstica objetiva e diferencial para psicose esquizofrênica e bipolar

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Encoding mechanisms based on fast oscillations in the retina of the cat and their dependencies on anesthesia

Processing in the visual system starts in the retina. Its complex network of cells with different properties enables for parallel encoding and transmission of visual information to the lateral geniculate nucleus (LGN) and to the cortex. In the retina, it has been shown that responses are often accompanied by fast synchronous oscillations (30 - 90 Hz) in a stimulus-dependent manner. Studies in the frog, rabbit, cat and monkey, have shown strong oscillatory responses to large stimuli which probably encode global stimulus properties, such as size and continuity (Neuenschwander and Singer, 1996; Ishikane et al., 2005). Moreover, simultaneous recordings from different levels in the visual system have demonstrated that the oscillatory patterning of retinal ganglion cell responses are transmitted to the cortex via the LGN (Castelo-Branco et al., 1998). Overall these results suggest that feedforward synchronous oscillations contribute to visual encoding. In the present study on the LGN of the anesthetized cat, we further investigate the role of retinal oscillations in visual processing by applying complex stimuli, such as natural visual scenes, light spots of varying size and contrast, and flickering checkerboards. This is a necessary step for understanding encoding mechanisms in more naturalistic conditions, as currently most data on retinal oscillations have been limited to simple, flashed and stationary stimuli. Correlation analysis of spiking responses confirmed previous results showing that oscillatory responses in the retina (observed here from the LGN responses) largely depend on the size and stationarity of the stimulus. For natural scenes (gray-level and binary movies) oscillations appeared only for brief moments probably when receptive fields were dominated by large continuous, flat-contrast surfaces. Moreover, oscillatory responses to a circle stimulus could be broken with an annular mask indicating that synchronization arises from relatively local interactions among populations of activated cells in the retina. A surprising finding in this study was that retinal oscillations are highly dependent on halothane anesthesia levels. In the absence of halothane, oscillatory activity vanished independent of the characteristics of the stimuli. The same results were obtained for isoflurane, which has similar pharmacological properties. These new and unexpected findings question whether feedfoward oscillations in the early visual system are simply due to an imbalance between excitation and inhibition in the retinal networks generated by the halogenated anesthetics. Further studies in awake behaving animals are necessary to extend these conclusions

Caracterização comportamental e distribuição de neurônios inibitórios em um modelo animal de autismo induzido por ácido valpróico

Autism comprises a heterogeneous group of neurodevelopmental disorders that affects the brain maturation and produces sensorial, motor, language and social interaction deficits in early childhood. Several studies have shown a major involvement of genetic factors leading to a predisposition to autism, which are possibly affected by environmental modulators during embryonic and post-natal life. Recent studies in animal models indicate that alterations in epigenetic control during development can generate neuronal maturation disturbances and produce a hyper-excitable circuit, resulting in typical symptoms of autism. In the animal model of autism induced by valproic acid (VPA) during rat pregnancy, behavioral, electrophysiological and cellular alterations have been reported which can also be observed in patients with autism. However, only a few studies have correlated behavioral alterations with the supposed neuronal hyper-excitability in this model. The aim of this project was to generate an animal model of autism by pre-natal exposure to VPA and evaluate the early post-natal development and pre-puberal (PND30) behavior in the offspring. Furthermore, we quantified the parvalbumin-positive neuronal distribution in the medial prefrontal cortex and Purkinje cells in the cerebellum of VPA animals. Our results show that VPA treatment induced developmental alterations, which were observed in behavioral changes as compared to vehicle-treated controls. VPA animals showed clear behavioral abnormalities such as hyperlocomotion, prolonged stereotipies and reduced social interaction with an unfamiliar mate. Cellular quantification revealed a decrease in the number of parvalbumin-positive interneurons in the anterior cingulate cortex and in the prelimbic cortex of the mPFC, suggesting an excitatory/inhibitory unbalance in this animal model of autism. Moreover, we also observed that the neuronal reduction occurred mainly in the cortical layers II/III and V/VI. We did not detect any change in the density of Purkinje neurons in the Crus I region of the cerebellar cortex. Together, our results strengthens the face validity of the VPA model in rats and shed light on specific changes in the inhibitory circuitry of the prefrontal cortex in this autism model. Further studies should address the challenges to clarify particular electrophysiological correlates of the cellular alterations in order to better understand the behavioral dysfunctions

Revisitando o eletrocorticograma intra-operatório na epilepsia mesial do lobo temporal: relevância das oscilações de alta frequência

Epilepsies are neurological disorders characterized by recurrent and spontaneous seizures due to an abnormal electric activity in a brain network. The mesial temporal lobe epilepsy (MTLE) is the most prevalent type of epilepsy in adulthood, and it occurs frequently in association with hippocampal sclerosis. Unfortunately, not all patients benefit from pharmacological treatment (drug-resistant patients), and therefore become candidates for surgery, a procedure of high complexity and cost. Nowadays, the most common surgery is the anterior temporal lobectomy with selective amygdalohippocampectomy, a procedure standardized by anatomical markers. However, part of patients still present seizure after the procedure. Then, to increase the efficiency of this kind of procedure, it is fundamental to know the epileptic human brain in order to create new tools for auxiliary an individualized surgery procedure. The aim of this work was to identify and quantify the occurrence of epilepticform activity -such as interictal spikes (IS) and high frequency oscillations (HFO) - in electrocorticographic (ECoG) signals acutely recorded during the surgery procedure in drug-resistant patients with MTLE. The ECoG recording (32 channels at sample rate of 1 kHz) was performed in the surface of temporal lobe in three moments: without any cortical resection, after anterior temporal lobectomy and after amygdalohippocampectomy (mean duration of each record: 10 min; N = 17 patients; ethic approval #1038/03 in Research Ethic Committee of Federal University of São Paulo). The occurrence of IS and HFO was quantified automatically by MATLAB routines and validated manually. The events rate (number of events/channels) in each recording time was correlated with seizure control outcome. In 8 hours and 40 minutes of record, we identified 36,858 IS and 1.756 HFO. We observed that seizure-free outcome patients had more HFO rate before the resection than non-seizure free, however do not differentiate in relation of frequency, morphology and distribution of IS. The HFO rate in the first record was better than IS rate on prediction of seizure-free patients (IS: AUC = 57%, Sens = 70%, Spec = 71% vs HFO: AUC = 77%, Sens = 100%, Spec = 70%). We observed the same for the difference of the rate of pre and post-resection (IS: AUC = 54%, Sens = 60%, Spec = 71%; vs HFO: AUC = 84%, Sens = 100%, Spec = 80%). In this case, the algorithm identifies all seizure-free patients (N = 7) with two false positives. To conclude, we observed that the IS and HFO can be found in intra-operative ECoG record, despite the anesthesia and the short time of record. The possibility to classify the patients before any cortical resection suggest that ECoG can be important to decide the use of adjuvant pharmacological treatment or to change for tailored resection procedure. The mechanism responsible for this effect is still unknown, thus more studies are necessary to clarify the processes related to it

Efeitos da administração aguda de quetamina sobre as oscilações eletrofisiológicas da região CA1 hipocampal

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

The influence of visual inter-hemispheric connections on spiking, assembly and LFP activities, and their phase relationship during figure-ground stimulation

Desde os descobrimentos pioneiros de Hubel e Wiesel acumulou-se uma vasta literatura descrevendo as respostas neuronais do córtex visual primário (V1) a diferentes estímulos visuais. Estes estímulos consistem principalmente em barras em movimento, pontos ou grades, que são úteis para explorar as respostas dentro do campo receptivo clássico (CRF do inglês classical receptive field) a características básicas dos estímulos visuais como a orientação, direção de movimento, contraste, entre outras. Entretanto, nas últimas duas décadas, tornou-se cada vez mais evidente que a atividade de neurônios em V1 pode ser modulada por estímulos fora do CRF. Desta forma, áreas visuais primárias poderiam estar envolvidas em funções visuais mais complexas como, por exemplo, a separação de um objeto ou figura do seu fundo (segregação figura-fundo) e assume-se que as conexões intrínsecas de longo alcance em V1, assim como as conexões de áreas visuais superiores, estão ativamente envolvidas neste processo. Sua possível função foi inferida a partir da análise das variações das respostas induzidas por um estímulo localizado fora do CRF de neurônios individuais. Mesmo sendo muito provável que estas conexões tenham também um impacto tanto na atividade conjunta de neurônios envolvidos no processamento da figura quanto no potencial de campo, estas questões permanecem pouco estudadas. Visando examinar a modulação do contexto visual nessas atividades, coletamos potenciais de ação e potenciais de campo em paralelo de até 48 eletrodos implantados na área visual primária de gatos anestesiados. Estimulamos com grades compostas e cenas naturais, focando-nos na atividade de neurônios cujo CRF estava situado na figura. Da mesma forma, visando examinar a influência das conexões laterais, o sinal proveniente da área visual isotópica e contralateral foi removido através da desativação reversível por resfriamento. Fizemos isso devido a: i) as conexões laterais intrínsecas não podem ser facilmente manipuladas sem afetar diretamente os sinais que estão sendo medidos, ii) as conexões inter-hemisféricas compartilham as principais características anatômicas com a rede lateral intrínseca e podem ser vistas como uma continuação funcional das mesmas entre os dois hemisférios e iii) o resfriamento desativa as conexões de forma causal e reversível, silenciando temporariamente seu sinal, permitindo conclusões diretas a respeito da sua contribuição. Nossos resultados demonstram que o mecanismo de segmentação figurafundo se reflete nas taxas de disparo de neurônios individuais, assim como na potência do potencial de campo e na relação entre sua fase e os padrões de disparo produzidos pela população. Além disso, as conexões laterais inter-hemisféricas modulam estas variáveis dependendo da estimulação feita fora do CRF. Observamos também uma influência deste circuito lateral na coerência entre potenciais de campo entre eletrodos distantes. Em conclusão, nossos resultados dão suporte à ideia de um mecanismo complexo de segmentação figura-fundo atuando desde as áreas visuais primárias em diferentes escalas de frequência. Esse mecanismo parece envolver grupos de neurônios ativos sincronicamente e dependentes da fase do potencial de campo. Nossos resultados também são compatíveis com a hipótese que conexões laterais de longo alcance também fazem parte deste mecanismo

Sonhos antecipatórios: influência de um evento significativo da vigília na atividade onírica

In most cultures, dreams are believed to predict the future on occasion. Several neurophysiological studies indicate that the function of sleep and dreams is to consolidate and transform memories, in a cyclical process of creation, selection and generalization of conjectures about the reality. The aim of the research presented here was to investigate the possible adaptative role of anticipatory dreams. We sought to determine the relationship between dream and waking in a context in which the adaptive success of the individual was really at risk, in order to mobilize more strongly the oneiric activity. We used the entrance examination of the Federal University of Rio Grande do Norte (UFRN) as a significant waking event in which performance could be independently quantified. Through a partnership with UFRN, we contacted by e-mail 3000 candidates to the 2009 examination. In addition, 150 candidates were approached personally. Candidates who agreed to participate in the study (n = 94) completed questionnaires specific to the examination and were asked to describe their dreams during the examinaton period. The examination performance of each candidate in the entrance examination was provided by the UFRN to the researcher. A total of 45 participants reported dreams related to the examination. Our results show a positive correlation between performance on the examination and anticipatory dreams with the event, both in the comparison of performance on objective and discursive, and in final approval (in the group that not dreamed with the exam the rate of general approval, 22,45%, was similar to that found in the selection process as a whole, 22.19%, while for the group that dreamed with the examination that rate was 35.56%). The occurrence of anticipatory dreams reflectes increased concern during waking (psychobiological mobilization) related to the future event, as indicated by higher scores of fear and apprehension, and major changes in daily life, in patterns of mood and sleep, in the group that reported testrelated dreams. Furthermore, the data suggest a role of dreams in the determination of environmentally relevant behavior of the vigil, simulating possible scenarios of success (dream with approval) and failure (nightmares) to maximize the adaptive success of the individual

Estimação de variáveis físico-químicas de solo por espectroscopia no visível e no infravermelho próximo através de sistemas inteligentes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Uma discussão do problema mente-corpo em Descartes e Espinosa, a partir da neurofilosofia de Antonio Damásio

Pós-graduação em Filosofia - FFC

Atividade dos neurônios noradrenérgicos do Locus coeruleus e o conteúdo de GnRH em ratas Wistar acíclicas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Envelhecimento, linguagem e memória visuo-espacial: um estudo comparativo exploratório do desempenho humano em testes neuropsicológicos selecionados

O reconhecimento dos limites entre o envelhecimento normal e o patológico é essencial para a adoção de políticas de saúde baseadas em evidências para o grupo etário acima de 65 anos. Este estudo é parte de um esforço sistemático que o grupo de pesquisa do Laboratório de Investigações em Neurodegeneração e Infecção da Universidade Federal do Pará tem feito para fornecer informações translacionais sobre a neurobiologia do envelhecimento normal e alterada. A meta principal em longo prazo é permitir políticas públicas para o envelhecimento saudável na Região Amazônica. No presente trabalho investigamos os efeitos do envelhecimento sobre os desempenhos em testes neuropsicológicos selecionados para avaliar aprendizagem, memória e alterações de linguagem. 29 adultos jovens (29,9 ± 1,06 anos) e 31 idosos (74,1 ± 1,15 anos) saudáveis foram submetidos aos testes e os resultados de seus desempenhos foram comparados por testes paramétricos e estatística multivariada. Uma anamnese e uma variedade de testes cognitivos, incluindo Mini Exame do Estado Mental, tarefas visuo-espaciais de aprendizagem e de memória da bateria de testes neuropsicológicos automatizados - CANTAB e testes de linguagem incluindo fluência verbal semântica e fonológica, teste de nomeação de Boston reduzido, performance narrativa utilizando a figura “o roubo dos biscoitos” e alguns testes da Bateria Montreal de Comunicação - MAC. O programa BioEstat versão 5.0 e o pacote estatístico SPSS foram utilizados para a análise. O teste paramétrico t de Student ou o não paramétrico de Mann-Whitney foram aplicados para detectar diferenças significativas (fixadas em valores de p <0,05). Os grupos foram pareados por escolaridade e incluiu homens e mulheres. A análise das subcategorias que compõem o MEEM mostrou diferença significativa apenas na recuperação da memória de evocação de lista de palavras, em que o grupo de idosos apresentou pior desempenho em comparação com o grupo de jovens. Foram encontradas diferenças estatisticamente significativas entre os desempenhos de idosos e jovens adultos nos seguintes testes de linguagem: 1) Nomeação de Boston, 2) Testes de Narrativa, 3) Metáforas; 4) Prosódia Emocional e Linguística. Em comparação com os adultos jovens, indivíduos idosos apresentaram pior desempenho em 10 medidas diferentes nos testes de memória visuo-espacial e de aprendizagem do CANTAB. Distâncias Euclidianas e análise discriminante obtidas a partir do CANTAB e dos testes de linguagem demonstraram que os primeiros distinguem os grupos com maior resolução. Os efeitos do envelhecimento sobre o desempenho nos testes neuropsicológicos selecionados revelam que a Bateria CANTAB, empregada para testar a memória visuo-espacial, é mais sensível e discrimina melhor a formação de subgrupos tanto no grupo de adultos jovens quanto no grupo de idosos. Por essa razão sugerimos que a aplicação em larga escala de testes selecionados da bateria CANTAB, tanto em estudos transversais como em longitudinais, vai aumentar nossa capacidade de resolução na distinção dos limites entre o envelhecimento normal e o patológico.