Sustained synchronized neuronal network activity in a human astrocyte co-culture system

Impaired neuronal network function is a hallmark of neurodevelopmental and neurodegenerative disorders such as autism, schizophrenia, and Alzheimer's disease and is typically studied using genetically modified cellular and animal models. Weak predictive capacity and poor translational value of these models urge for better human derived in vitro models. The implementation of human induced pluripotent stem cells (hiPSCs) allows studying pathologies in differentiated disease-relevant and patient-derived neuronal cells. However, the differentiation process and growth conditions of hiPSC-derived neurons are non-trivial. In order to study neuronal network formation and (mal) function in a fully humanized system, we have established an in vitro co-culture model of hiPSC-derived cortical neurons and human primary astrocytes that recapitulates neuronal network synchronization and connectivity within three to four weeks after final plating. Live cell calcium imaging, electrophysiology and high content image analyses revealed an increased maturation of network functionality and synchronicity over time for co-cultures compared to neuronal monocultures. The cells express GABAergic and glutamatergic markers and respond to inhibitors of both neurotransmitter pathways in a functional assay. The combination of this co-culture model with quantitative imaging of network morphofunction is amenable to high throughput screening for lead discovery and drug optimization for neurological diseases

2D vs 3D morphological analysis of dorsal root ganglia in health and painful neuropathy

Dorsal root ganglia (DRGs) are clusters of sensory neurons that transmit the sensory information from the periphery to the central nervous system, and satellite glial cells (SGCs), their supporting trophic cells. Sensory neurons are pseudounipolar neurons with a heterogeneous neurochemistry reflecting their functional features. DRGs, not protected by the blood brain barrier, are vulnerable to stress and damage of different origin (i.e., toxic, mechanical, metabolic, genetic) that can involve sensory neurons, SGCs or, considering their intimate intercommunication, both cell populations. DRG damage, primary or secondary to nerve damage, produces a sensory peripheral neuropathy, characterized by neurophysiological abnormalities, numbness, paraesthesia and dysesthesia, tingling and burning sensations and neuropathic pain. DRG stress can be morphologically detected by light and electron microscope analysis with alterations in cell size (swelling/atrophy) and in different sub-cellular compartments (i.e., mitochondria, endoplasmic reticulum, and nucleus) of neurons and/or SGCs. In addition, neurochemical changes can be used to portray abnormalities of neurons and SGC. Conventional immunostaining, i.e., immunohistochemical detection of specific molecules in tissue slices can be employed to detect, localize and quantify particular markers of damage in neurons (i.e., nuclear expression ATF3) or SGCs (i.e., increased expression of GFAP), markers of apoptosis (i.e., caspases), markers of mitochondrial suffering and oxidative stress (i.e., 8-OHdG), markers of tissue inflammation (i.e., CD68 for macrophage infiltration), etc. However classical (2D) methods of immunostaining disrupt the overall organization of the DRG, thus resulting in the loss of some crucial information. Whole-mount (3D) methods have been recently developed to investigate DRG morphology and neurochemistry without tissue slicing, giving the opportunity to study the intimate relationship between SGCs and sensory neurons in health and disease. Here, we aim to compare classical (2D) vs whole-mount (3D) approaches to highlight “pros” and “cons” of the two methodologies when analysing neuropathy-induced alterations in DRGs

Nervous system and computation.

none5Computational systems are useful in neuroscience in many ways. For instance, they may be used to construct maps of brain structure and activation, or to describe brain processes mathematically.Furthermore, they inspired a powerful theory of brain function, in which the brain is viewed as a system characterized by intrinsic computational activities or as a "computational information processor." Although many neuroscientists believe that neural systems really perform computations, some are more cautious about computationalism or reject it. Thus, does the brain really compute? Answering this question requires getting clear on a definition of computation that is able to draw a line between physical systems that compute and systems that do not, so that we can discern on which side of the line the brain (or parts of it) could fall. In order to shed some light on the role of computational processes in brain function, available neurobiological data will be summarized from the standpoint of a recently proposed taxonomy of notions of computation, with the aim of identifying which brain processes can be considered computational. The emerging picture shows the brain as a very peculiar system, in which genuine computational features act in concert with noncomputational dynamical processes, leading to continuous self-organization and remodeling under the action of external stimuli from the environment and from the rest of the organism.openD. Guidolin; G. Albertin; M. Guescini; K. Fuxe; L.F. Agnati LF.D., Guidolin; G., Albertin; Guescini, Michele; K., Fuxe; L. F. Agnati L., F

Predicting the internal model of a robotic system from its morphology

The estimation of the internal model of a robotic system results from the interaction of its morphology, sensors and actuators, with a particular environment. Model learning techniques, based on supervised machine learning, are widespread for determining the internal model. An important limitation of such approaches is that once a model has been learnt, it does not behave properly when the robot morphology is changed. From this it follows that there must exist a relationship between them. We propose a model for this correlation between the morphology and the internal model parameters, so that a new internal model can be predicted when the morphological parameters are modified. Di erent neural network architectures are proposed to address this high dimensional regression problem. A case study is analyzed in detail to illustrate and evaluate the performance of the approach, namely, a pan-tilt robot head executing saccadic movements. The best results are obtained for an architecture with parallel neural networks due to the independence of its outputs. Theses results can have a great significance since the predicted parameters can dramatically speed up the adaptation process following a change in morpholog

Comparative study on the physiological dynamics evoked by different profiles of lead exposure

Tese de mestrado, Neurociências, Universidade de Lisboa, Faculdade de Medicina, 2018Lead is a toxic metal which widespread use has resulted in environmental contamination and significant health problems. It is a cumulative toxicant that affects multiple body systems, including the cardiovascular, hematopoietic, reproductive, and renal systems. Lead is also a well-known neurotoxin, inducing changes in neurogenesis, neurodegeneration and changes on glial cells. These changes in the molecular and cellular processes lead to cognitive and behaviour alterations, particularly during developmental phases, persisting throughout the lifetime. Most of the studies that have been performed in both humans and animals were focused in a continuous chronic exposure to lead. This lead exposure causes behavioural changes, cognitive impairment and hypertension associated with sympathoexcitation, baroreceptor reflex hyposensitivity and increased chemoreceptor reflex sensitivity. But the effects of an intermittent lead exposure are scarse and standardized animal models are non-existent. This pattern of exposure has been increasing in the last years due to migrations, implementation of school exchange programs and/or residential changes. Therefore, the overall purpose of this work was to evaluate lead effects on mammal’s physiology along different profiles of lead exposure, including a new animal model of intermittent low-level lead exposure. Animal models of lead exposure were developed by replacing the tap water of seven-day pregnant Wistar females with 0.2% (p/v) solution of lead acetate. After being weaned at 21 days, rat pups, both sexes, were divided into 3 groups of lead exposure: long-term (exposure from foetal period until 28 weeks of age), short-term (exposure from foetal period until 12 weeks) and intermittent (exposure from foetal period until 12 weeks, lead-free period until 20 weeks and a second exposure between 20 and 28 weeks of age). At 12, 20 and 28 weeks of age, behavioural tests were performed for anxiety (Elevated Plus Maze Test), locomotor activity (Open Field Test), spatial working memory (Y-Maze) and episodic long-term memory (Novel Object Recognition test) assessment. Blood pressure (BP), electrocardiogram (ECG), heart rate (HR) and respiratory frequency (RF) were recorded at the same timepoints in the acute experiment. Baroreflex gain (BRG), chemoreflex sensitivity (ChS), cardiovascular variability were also evaluated. Immunohistochemistry studies for neuronal nuclear antigen (NeuN), Synaptophysin (Syn), ionized calcium binding adapter molecule-1 (Iba-1) and Glial fibrillary acidic protein (GFAP) stainings were performed in brain slices, and confocal imaging acquired and stainings quantified at dentate gyrus (DG) of the hippocampus. Blood lead levels were assessed by atomic absorption spectroscopy and metabolic evaluation of all groups was done using metabolic cages. A control group of Wistar rats without lead exposure, of both sexes, with the same number of individuals, underwent the same protocol and were evaluated in the same time points. Student T-test and one-way ANOVA with Tukey’s multiple comparison between means were used (significance p<0.05) for statistical analysis. Our data showed a clear association between lead exposure, hypertension and cardiorespiratory reflexes impairment, without heart rate changes, independently of the type of lead exposure profile. We also demonstrated for the first time that lead intermittent exposure causes adverse health effects, i.e, hypertension, sympathetic overactivity, increased chemoreflex sensitivity and baroreflex impairment, similar to a chronic exposure, however less pronounced. In fact, at 28 weeks, PbI group, the intermittent animal model of lead exposure developed, had a less severe hypertension when compared to the long-term exposure group (PbP), which might suggest that the duration of Pb exposure is more relevant than the time of exposure. Moreover, the effect on diastolic blood pressure produced by lead exposure was more evident than that of systolic blood pressure. Lead exposure from foetal period until 12 weeks of age causes long lasting hypertension and chemoreceptor reflex dysfunction even after a 16 weeks period without exposure. However, the clearance of lead promoted an improvement in baroreceptor reflex function, with repercussions on blood pressure values, since these values decreased, but did not reached the normotensive values. Regarding the autonomic data, in our study, the overactivity of the sympathetic nervous system, evaluated by the LF band, is concomitant with baroreceptor reflex impairment and/or hypertension. This means that the sympathetic nervous system may be involved in the modulation of the baroreceptor reflexes responses or in the hypertension development due to lead exposure. Concerning the effect of lead at behavioural level, all groups exposed to lead, evaluated in the three different time points, had behavioural changes, namely anxiety, hyperactivity and/or long-term memory impairment and molecular changes in the hippocampus region, more specifically, reactive astrogliosis and microgliosis were detected, indicating the presence of neuroinflammation. However, these alterations seem to reverse after lead abstinence for a certain period (single exposure) and are enhanced when a second exposure occurs (intermittent exposure), along with a synaptic loss. In summary, this study shows, that exposure to lead during the developmental phase can alter the normal course of development, with lifelong health consequences. Since all exposed Pb groups had the same route of exposure (i.e. exposure to lead by water) and the same dose and, despite the different time of exposure, all were exposed to lead since foetal period until adulthood, the most susceptible period to adverse health effects. Therefore, we can conclude that the different effects of lead toxicant between groups mainly depends on the total duration of lead exposure. This comparative study brings new insights on the environmental factors that influence nervous and cardiovascular systems during development, which can help creating public policy strategies to prevent and control the adverse effects of Pb toxicity.A identificação de agentes potencialmente tóxicos e a avaliação dos seus efeitos sobre o organismo humano constituem um tema importante de saúde pública. O chumbo encontra-se neste grupo de agentes, sendo bastante utilizado em todo o mundo, devido às suas propriedades únicas, como a alta maleabilidade, baixo ponto de fusão, suavidade, ductilidade e resistência à corrosão. O vasto uso deste metal pesado em indústrias, como a automóvel, cerâmica, de tintas e do plástico levou ao aumento da quantidade de chumbo livre no ambiente e a sua ocorrência nos sistemas biológicos, devido à sua natureza não biodegradável. A toxicidade do chumbo, como resultado da sua ingestão, inalação ou por contacto direto, mesmo em pequenas quantidades, pode evocar efeitos adversos irreversíveis em várias funções do corpo, afetando principalmente os sistemas cardiovascular (sendo uma das causas da hipertensão, promovendo aterosclerose, trombose, arteriosclerose e doenças cardiovasculares), hematopoiético, reprodutivo e renal. O chumbo é também uma neurotoxina já bem estudada, que induz alterações na neurogénese, nas células gliais e neurodegeneração. Estas alterações nos mecanismos celulares e moleculares, quando ocorrem durante as fases de desenvolvimento, provocam alterações cognitivas e comportamentais, que persistem durante toda a vida. Em termos de classificação, dois tipos de toxicidade de chumbo podem ser definidos: a toxicidade aguda, que geralmente ocorre pela exposição ocupacional a níveis elevados de chumbo, sendo esta bastante incomum, e a toxicidade crónica, uma exposição a níveis baixos de chumbo, mais comum no ambiente familiar. A maioria dos estudos realizados até à data em seres humanos e animais, focam-se na exposição crónica contínua e/ou permanente ao chumbo e nas consequências adversas na saúde deste tipo de exposição. Existem já, vários modelos animais descritos para a exposição contínua a níveis baixos de chumbo. No entanto, em determinadas situações, como nas migrações, nos programas de intercâmbio escolar e/ou nas mudanças residenciais, a exposição intermitente ao chumbo pode ocorrer, mas os estudos disponíveis em seres humanos são escassos e os modelos animais padronizados inexistentes para este tipo de exposição, que tem vindo a crescer exponencialmente nos últimos anos. Posto isto, o objetivo geral deste trabalho consistiu em avaliar os efeitos de diferentes perfis de exposição a níveis baixos de chumbo na fisiologia de ratos Wistar, incluindo o desenvolvimento de um novo modelo animal de exposição intermitente a chumbo. Os modelos animais de exposição ao chumbo foram desenvolvidos substituindo a água dos biberões das fêmeas Wistar grávidas de sete dias por uma solução de acetato de chumbo a 0,2% (p/v). Após os 21 dias de desmame, as crias, de ambos sexos, foram divididas em 3 grupos de exposição ao chumbo: de longo prazo (PbP - exposição do período fetal até às 28 semanas de idade), de curto prazo (Short-term PbS - exposição do período fetal até às 12 semanas, com abstinência ao chumbo até às 28 semanas) e intermitente (PbI - exposição do período fetal até 12 semanas, seguida por um período sem chumbo até 20 semanas e uma segunda exposição entre 20 e 28 semanas de idade). Em três diferentes pontos temporais (12, 20 e 28 semanas de idade), os diferentes grupos de animais foram sujeitos a testes comportamentais, para a avaliação dos níveis de ansiedade (EPM), da atividade locomotora (OFT), da memória espacial de trabalho (Y-Maze) e da memória episódica de longo prazo (NOR). Para avaliação dos parâmetros fisiológicos nos diferentes pontos temporais, os animais foram sujeitos a uma experiência aguda, onde foram registados os seguintes parâmetros: pressão arterial (PA), eletrocardiograma (ECG), frequência cardíaca (FC) e frequência respiratória (FR). Nesta experiência também se avaliaram os reflexos baro- e quimiorrecetores e obtiveram-se registos para a análise da variabilidade da FC e da PA sistólica. Após o término da experiência aguda, os animais foram sacrificados e os cérebros extraídos para estudos de imunohistoquímica em secções coronais, nas quais se analisou a morfologia das células e se quantificou a perda neuronal (neuronal nuclear antigen - NeuN), a astrogliose (Glial fibrillary acidic protein – GFAP) e a microgliose (ionized calcium binding adapter molecule-1 - Iba-1), bem como alterações na transmissão sináptica (Synaptophysin – Syn) no girus dentado do hipocampo. Os níveis de chumbo no sangue foram avaliados por espectroscopia de absorção atómica e a avaliação metabólica realizada através do uso de gaiolas metabólicas. Um grupo controlo de ratos Wistar sem exposição ao chumbo, de ambos os sexos e com o mesmo número de indivíduos, foi submetido ao mesmo protocolo e foi avaliado nos mesmos pontos temporais (12, 20 e 28 semanas de idade). Para a análise estatística foi utilizado o teste T de Student e a análise de Variância (ANOVA) unidirecional com o teste post-hoc de Tukey, considerando-se significativas diferenças com p < 0,05. Os resultados deste estudo mostram que, independentemente do tipo de perfil de exposição ao chumbo, existe uma associação clara entre exposição a chumbo, hipertensão e diminuição do ganho do barorreflexo, sem alterações de frequência cardíaca. Também demonstramos, pela primeira vez, que uma exposição intermitente a chumbo provoca efeitos adversos para a saúde, como hipertensão, hiperatividade simpática, aumento da sensibilidade quimiorreflexa e diminuição do ganho do barorreflexo, efeitos adversos semelhantes ao de uma exposição crónica permanente (PbP), porém menos pronunciada. De facto, às 28 semanas, o grupo PbI, o modelo animal intermitente de exposição ao chumbo desenvolvido, apresentou uma hipertensão menos grave em relação ao grupo de exposição de longo prazo (PbP), o que pode sugerir que a duração da exposição ao chumbo é mais relevante do que o tempo de exposição. Além disso, o efeito da exposição ao chumbo sobre a pressão arterial diastólica foi mais evidente do que sobre a pressão arterial sistólica. A exposição ao chumbo, desde o período fetal até as 12 semanas de idade, provoca hipertensão e disfunção quimiorreflexa duradoura, mesmo com um período de 16 semanas sem exposição. No entanto, a abstinência do chumbo promoveu uma melhoria na função barorreflexa, com repercussões nos valores da pressão arterial, uma vez que estes valores diminuíram, apesar de não atingirem os valores de normotensão. Em relação à avaliação autonómica, os dados indicam que quando existe um aumento do tónus simpático, avaliado pela banda LF, este é concomitante com disfunção barorreflexa e/ou hipertensão arterial. Isso significa que o sistema nervoso simpático deve estar envolvido na modulação da resposta barorreflexa ou no desenvolvimento da hipertensão decorrente da exposição ao chumbo. Relativamente ao efeito do chumbo a nível comportamental, todos os grupos expostos ao chumbo, avaliados nos diferentes pontos temporais, apresentaram alterações comportamentais, nomeadamente ansiedade, hiperatividade e / ou défices de memória a longo prazo, bem como alterações moleculares, mais especificamente, astrogliose e microgliose reativa, que indicam a presença de neuroinflamação. No entanto, estas alterações parecem reverter após a abstinência do chumbo durante um determinado período (PbS - exposição de curta duração), sendo mais evidentes quando ocorre uma segunda exposição a chumbo (PbI - exposição intermitente), levando mesmo a perda sináptica mais pronunciada. Em resumo, este estudo mostra, que exposições a chumbo durante as fases de desenvolvimento podem alterar o seu curso normal, com consequências adversas para a saúde que podem persistir para toda a vida. Uma vez que todos os grupos expostos a chumbo tiveram a mesma via de exposição (isto é, exposição ao chumbo através da água) e a mesma dose e, apesar do tempo de exposição diferente, todos foram expostos ao chumbo desde o período fetal até a idade adulta, período em que são mais suscetíveis a efeitos adversos na saúde. Portanto, podemos concluir que os diferentes efeitos tóxicos do chumbo entre os grupos dependem principalmente da duração total da exposição ao chumbo. As novas evidências obtidas por este estudo comparativo permitem-nos contribuir para o esclarecimento sobre os fatores ambientais que influenciam os sistemas nervoso e cardiovascular durante o desenvolvimento, o que pode ajudar a criar estratégias de políticas públicas para prevenir e controlar os efeitos adversos da toxicidade do chumbo

Functional evaluation of the diaphragm with a noninvasive test

Abstract Cardiac surgery with median sternotomy causes iatrogenic damage to the function of the diaphragm muscle that is both temporary and permanent. Myocardial infarction itself causes diaphragmatic genetic alterations, which lead the muscle to nonphysiological adaptation. The respiratory muscle area plays several roles in maintaining both physical and mental health, as well as in maximizing recovery after a cardiac event. The evaluation of the diaphragm is a fundamental step in the therapeutic process, including the use of instruments such as ultrasound, magnetic resonance imaging (MRI), and computed axial tomography (CT). This article reviews the neurophysiological relationships of the diaphragm muscle and the symptoms of diaphragmatic contractile dysfunction. The authors discuss a scientific basis for the use of a new noninstrumental diaphragmatic test in the hope of stimulating research

Aerospace medicine and biology: A continuing bibliography with indexes, supplement 128, May 1974

This special bibliography lists 282 reports, articles, and other documents introduced into the NASA scientific and technical information system in April 1974

Site-specific abnormalities in the visual system of a mouse model of CDKL5 deficiency disorder

CDKL5 deficiency disorder (CDD) is a neurodevelopmental disorder characterized by a severe global developmental delay and early-onset seizures. Notably, patients show distinctive visual abnormalities often clinically diagnosed as cortical visual impairment. However, the involvement of cerebral cortical dysfunctions in the origin of the symptoms is poorly understood. CDD mouse models also display visual deficits, and cortical visual responses can be used as a robust biomarker in CDKL5 mutant mice. A deeper understanding of the circuits underlying the described visual deficits is essential for directing preclinical research and translational approaches. Here, we addressed this question in two ways: first, we performed an in-depth morphological analysis of the visual pathway, from the retina to the primary visual cortex (V1), of CDKL5 null mice. We found that the lack of CDKL5 produced no alteration in the organization of retinal circuits. Conversely, CDKL5 mutants showed reduced density and altered morphology of spines and decreased excitatory synapse marker PSD95 in the dorsal lateral geniculate nucleus and in V1. An increase in the inhibitory marker VGAT was selectively present in V1. Second, using a conditional CDKL5 knockout model, we showed that selective cortical deletion of CDKL5 from excitatory cells is sufficient to produce abnormalities of visual cortical responses, demonstrating that the normal function of cortical circuits is dependent on CDKL5. Intriguingly, these deficits were associated with morphological alterations of V1 excitatory and inhibitory synaptic contacts. In summary, this work proposes cortical circuit structure and function as a critically important target for studying CDD

Stimulation Paradigms and Transduction Patterns for Optogenetic Intervention of Astrocytes

Gliosis observed in several neurological disorders is associated with neuroinflammation and enhanced astrocytic Ca2+ levels. The inherent multicellular nature of this neuroinflammation poses challenges in deciphering the exact role of astrocytic Ca2+ signaling and whether it leads to the generation and/or exacerbation of neuroinflammation. These challenges are aggravated by the dearth of systematic characterization of a regulated method for eliciting astrocytic Ca2+ increases. The primary goal of this dissertation is to address the lack of a characterized method by studying optogenetics for eliciting astrocytic Ca2+ increases. As part of this analysis, we aim to identify light stimulation paradigms resulting in consistent astrocytic Ca2+ increases and assess optogenetic construct serotypes yielding maximum target cell transduction. Firstly, a novel protocol was devised to perform simultaneous optogenetics and astrocytic Ca2+ imaging in adult murine brain slices. Neocortical astrocytes exhibited synchronous patterns of Ca2+ activity upon light stimulation, drastically different from resting spontaneous activity, and based on the effect of various light paradigms; we identified ix those conducive for robust astrocytic signaling. Secondly, a theoretical model was constructed to study the effect of short and long-term light stimulation of optogenetically-enabled (ChR2-expressing) astrocytes on their Ca2+ spiking activity and basal level. We further investigated how ChR2 gating dynamics, buffering, and coupling coefficient of Ca2+ influence astrocytic activity in a single cell and a network. The response of select variants of ChR2 to varying light stimulation paradigms and key parameters to design future constructs was explored. A preliminary evaluation revealed model similarities to our in situ experimental data. Finally, to facilitate future translational work and eventual comparison to current disease models, astrocytic transduction of various serotypes of an AAV optogenetic construct was assessed in vivo, and the serotype with maximal transduction efficiency was identified. Overall, we identified light stimulation paradigms that lead to repeated robust activation of astrocytes and AAV serotypes with high astrocytic transduction efficiency, thereby verifying that via an analysis of light stimulation paradigms and serotype transduction patterns, optogenetics can be implemented for inducing astrocytic Ca2+ increases in a controlled and tunable manner

Opinions and Outlooks on Morphological Computation

Morphological Computation is based on the observation that biological systems seem to carry out relevant computations with their morphology (physical body) in order to successfully interact with their environments. This can be observed in a whole range of systems and at many different scales. It has been studied in animals – e.g., while running, the functionality of coping with impact and slight unevenness in the ground is "delivered" by the shape of the legs and the damped elasticity of the muscle-tendon system – and plants, but it has also been observed at the cellular and even at the molecular level – as seen, for example, in spontaneous self-assembly. The concept of morphological computation has served as an inspirational resource to build bio-inspired robots, design novel approaches for support systems in health care, implement computation with natural systems, but also in art and architecture. As a consequence, the field is highly interdisciplinary, which is also nicely reflected in the wide range of authors that are featured in this e-book. We have contributions from robotics, mechanical engineering, health, architecture, biology, philosophy, and others