Cellular migration is an essential process for establishing neural connections during development. The MIG-10/RIAM/Lamellipodin signaling proteins are thought to send positional information from guidance cues to actin polymerization machinery, promoting the polarized outgrowth of axons. In C. elegans, mutations in the gene mig-10 result in the truncation of the migration of the mechanosensory neurons. Biochemical analysis demonstrates that MIG-10 interacts with abelson-interactor protein 1 (ABI-1), and therefore investigation into whether these proteins work together in the neuron to promote migration was completed. To demonstrate MIG-10 cell autonomy in the neuron, transgenic strains with specific expression of mig-10 were created. mig-10 mutants were rescued in the mechanosensory, anterior lateral microtubule neuron (ALM) by neuron specific expression of mig-10 but not by epithelial expression, suggesting that MIG-10 is acting cell autonomously. To determine ABI-1 cell autonomy, transgenic strains with specific neuronal expression of abi-1 were compared to the wild type strain. abi-1 mutants were rescued by neuron specific expression of abi-1 in the ALM, suggesting that ABI-1 also functions cell autonomously in the ALM during this migration. Further investigation into the MIG-10/ABI-1 relationship was done by feeding RNAi of abi-1 in a mig-10(ct41) mutant strain. The ALM migration was not more severely truncated in the double mutant, suggesting that MIG-10 and ABI-1 work in the same pathway. Taken together, this evidence supports a model where MIG-10 and ABI-1 work together autonomously within the ALM to promote migration.

A estrutura plástica do cérebro tem a capacidade de se adaptar a diversas condições e estímulos. No entanto, isso também pode facilitar a emergência de instabilidades, o que acarreta na necessidade de mecanismos de homeostase que previnam que a dinâmica da rede neural chegue a estados patológicos. A plasticidade associativa é considerada a principal base para o desenvolvimento de funções como memória e aprendizado, a realimentação positiva potencialmente leva à saturação de sinapses e instabilidades de atividade, especialmente em arquiteturas om conectividades recorrentes tais como em microcircuitos cerebrais. Neste trabalho investigamos a difícil interação entre a codificação de informação e o controle da atividade através da plasticidade Hebbiana e do escalonamento sináptico homeostático. O objetivo é a determinação de propriedades, como por exemplo a inibição e a conectividade, que proporcionam o desenvolvimento de codificação de informação de uma maneira confiável e fisiologicamente relevante através de plasticidade sináptica, prevenindo comportamento patológico. Após uma breve revisão bibliográfica de tópicos básicos da neurofisiologia e da modelagem de redes neurais, a primeira parte dos resultados apresenta uma rede que, sob uma forma específica de esc alonamento sináptico, desenvolve associatividade de padrões de disparo espaço-temporais e discute a afetação da capacidade de separação e confiabilidade de acordo om escalas de tempo de plasticidade, limitações sobre a eficácia sináptica e a dinâmica das interações inibitórias. A segunda parte define condições para manter o escalonamento sináptico homeostático sem instabilidades dinâmicas, om foco em fenômenos pouco explorados, como o escalonamento de sinapses inibitórias e o alcance efetivo da plasticidade. Em direção a outros mecanismos que podem influenciar esse balanço, a última parte descreve os efeitos do local de expressão da plasticidade de longa duração sobre a dinâmica de aprendizado, o que é demonstrado diferir de acordo om a codificação do estímulo.

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Modifications of perineuronal nets to regulate plasticity Heleen Merel van 't Spijker Perineuronal nets (PNNs) are macromolecular structures formed by neurons after closure of critical periods of plasticity. During development, the central nervous system (CNS) goes through critical periods of plasticity; a period when substantial changes occur to adapt to the environment, during which many synapses are formed and also discarded. When a region of the CNS has finished its development and reached an efficient neuronal circuit, the capacity for plasticity needs to be reduced to preserve the formed circuit. PNNs are formed around neurons during this period of reduced plasticity. PNNs consist of a backbone of hyaluronan, bound by chondroitin sulfate proteoglycans (CSPGs). Here, I present my studies on the possible modifications of PNNs to regulate plasticity. Firstly, I have investigated the potential use of 4-methylumbelliferone (4-MU) to reduce PNN formation in vivo. 4-MU reduces the formation of hyaluronan. Since hyaluronan is the backbone of PNNs, I hypothesized 4-MU treatment would reduce PNN formation. For this study, I developed a method to orally administer 4-MU to rats. Subsequently, I investigated whether 4-MU treatment can improve recovery of rats after spinal cord injury, both with behavioural tests and with immunohistochemistry. Secondly, I have investigated a new binding partner of PNNs, neuronal pentraxin 2 (Nptx2). Nptx2 is secreted by neurons and regulates AMPA receptor diffusion. Nptx2 knockout mice show a prolonged critical period of plasticity in the visual cortex. Here, I have identified Nptx2 as a new binding partner of PNNs. Nptx2 is found in isolated PNN protein preparations and is removed from the surface of neurons by digestion of PNNs with chondroitinase ABC. I also determined Nptx2 facilitates PNN formation in vitro. Addition of Nptx2 to the medium of cortical neurons leads to an increase of neurons that start to form PNNs, as well as an increase in size and density of PNNs. These findings indicate Nptx2 may be used as a modulator of PNNs. Thirdly, I investigated the interaction between Nptx2 and PNNs. I developed a sandwich ELISA to determine which glycan chains from PNNs bind to Nptx2. Nptx2 binds to chondroitin sulfate E and hyaluronan. To investigate the binding properties of Nptx2, I performed quartz crystal microbalance with dissipation monitoring for Nptx2 films. Furthermore, I developed crystals of purified Nptx2 and hyaluronan for x-ray crystallography. The here presented results provide new insights in potential approaches to modulate PNN formation. Both lines of research provide a further understanding of the factors which regulate PNNs and may allow for the development of treatments for PNN related disorders.

Dans cette thèse, je propose plusieurs modèles et outils numériques afin de mieux comprendre et prédire le comportement et le développement de cultures et dispositifs neuronaux.Les cultures de neurones ont en effet été un outil précieux durant les 20 dernières années : elles ont permis de mieux comprendre la manière dont le cerveau traite les différentes informations qui lui parviennent en donnant aux scientifiques la possibilité de tester les effets de médicaments sur les neurones, ainsi que d'obtenir leurs réponses détaillées à diverses perturbations et stimuli.De plus, de récentes avancées en microfluidiques ont ouvert la voie à la conception de dispositifs neuronaux plus élaborés, rapprochant encore un peu plus la perspective du traitement de signaux complexes via des neurones in vitro.Dans une première partie, je propose un mécanisme pour expliquer les bouffées d'activité épileptiformes présentes dans les cultures, mécanisme que je formule via un modèle théorique concis. J'effectue ensuite une vérification expérimentale des prédictions du modèle sur des cultures et montre que celles-ci sont effectivement compatibles avec le comportement observé in vitro.Dans une seconde partie, je décris plus en détail la description de la dynamique spatio-temporelle du phénomène, notamment le fait que les bursts nucléent en des zones bien précises du réseau neuronal.Comme les prédictions et analyses effectuées dépendent fortement de la structure de ce réseau, je présente ensuite la réalisation d'une plateforme de simulation afin de permettre de modéliser efficacement le développement des réseaux neuronaux. Ce logiciel prend en compte les interactions entre les neurones et leur environnement et constitue la première plateforme à fournir des modèles polyvalents et complets pour décrire l'intégralité du processus de croissance neuronal. Je montre ensuite que ce simulateur est capable de générer des morphologies valides et l'utilise pour proposer des nouvelles topologies de réseaux afin de décrire les cultures de neurones. Je reproduis également des dispositifs neuronaux existants et montre que les activités entretenues par ces structures sont compatibles avec les observations expérimentales. Enfin, je discute plusieurs directions de recherche possibles, pour lesquelles l'utilisation de dispositifs neuronaux spécifiques permettrait de contourner les limitations des cultures neuronales et fournirait ainsi de nouvelles informations sur les processus sous-tendant le développement et la plasticité cérébrale / In this thesis, I provide models and numerical tools to better understand and predict the behavior and development of neuronal cultures and devices.Neuronal cultures have proven invaluable in improving our understanding of how the brain processes information, by enabling researchers to investigate neuronal and network response functions to various perturbations and stimuli.Furthermore, recent progress in microfluidics have opened the gate towards more elaborated neuronal devices, bringing us one step closer to complex signal processing with living in vitro neurons.In a first part, I propose a mechanism to explain the epileptiform bursts of activity present in cultures, mechanism which I formulate as a concise theoretical model. I subsequently test the predictions of this model on cultures and show that they are indeed compatible with the behavior observed in vitro.I further develop this description in the second part of the thesis, where I analyze its spatiotemporal dynamics and the fact that burst nucleate in specific areas in the network.Since predictions and analysis of these nucleation centers strongly depends on the network structure, I develop a simulation platform to enable efficient modeling of the network development. This software takes into account the interactions between the neurons and their environment and is the first platform to provide versatile and complete models to simulate the entire growth process of neurons. I demonstrate that this simulator is able to generate valid neuronal morphologies, then use it to propose new network topologies to describe neuronal cultures, as well as to reproduce existing neuronal devices. I then show that the activities sustained by these structures are compatible with the experimental recordings.Eventually, I discuss several future directions for which the use of neuronal devices would enable to circumvent current limitations of neuronal cultures, thus providing new information on the processes which underlie brain development and plasticity.

Neurons need to be maintained to persist throughout adulthood for proper brain function. However neuronal activity, injury and aging exert physical stress on the nervous system, which compromise nervous system function. Healthy neurons are able to maintain their integrity throughout the lifespan of the animal, suggesting the existence of a maintenance mechanism that allows neurons to sustain or even repair damage. A forward genetic screening in Drosophila identified mutations in a gene called nmnat that cause a rapid and severe neurodegeneration immediately post neuronal differentiation and development. NMNAT protein was required to maintain neuronal integrity in an activity-dependent manner. When probing for the exact role of NMNAT in neuronal maintenance, a novel stress responsive chaperone function was identified, in addition to its essential housekeeping NAD synthase role. In this work, the mechanism of NMNAT-mediated neuroprotection is investigated. First, the transcriptional regulation of Drosophila NMNAT during acute stress is analyzed. Here, both stress transcription factors heat shock factor (HSF) and hypoxia inducible factor alpha (HIF1-α) have been shown to upregulate NMNAT during stress through a heat shock element in the nmnat promoter. In addition, the role of NMNAT for stress tolerance in Drosophila is revealed. Second, to elucidate the neuroprotective capacity of NMNAT in neurodegenerative disease, mouse models of tauopathy have been used. In the P301L Tau-transgenic mouse model, the levels of endogenous NMNAT2 have been studied at various ages to link a reduction in NMNAT2 as a precursor for neurodegeneration. The underlying mechanism of NMNAT2 downregulation is further studied in this model. Third, using Drosophila model of Tauopathy, the protective capacity of both wild type and enzyme-inactive NMNAT in ameliorating the pathological and behavioral impairments from Tau-induced neurodegeneration were studied extensively. The possible protective mechanism of NMNAT is uncovered by identifying novel interactions of NMNAT with hyperphosphorylated and ubiquitinated Tau in regulating the levels of toxic Tau species. Finally, this study also identified endogenous proteins that NMNAT interacts with to provide insight into a neuroprotective chaperone role of NMNAT. Together, these studies improve our understanding of the mechanisms of neuronal maintenance, by providing a comprehensive investigation of the stress-responsive regulation of NMNAT in both Drosophila and mammalian models, and its role as a chaperone both in protein foldopathies and in healthy neurons.

Programmed cell death (PCD) is an essential feature during the development of the central nervous system in Drosophila as well as in mammals. During metamorphosis, a group of peptidergic neurons (vCrz) are eliminated from the larval central nervous system (CNS) via PCD within 6-7 h after puparium formation. To better understand this process, we first characterized the development of the vCrz neurons including their lineages and birth windows using the MARCM (Mosaic Analysis with a Repressible Cell Marker) assay. Further genetic and MARCM analyses showed that not only Myoglianin (Myo) and its type I receptor Baboon is required for neuron cell death, but also this death signal is extensively regulated by endocytic trafficking in Drosophila melanogaster. We found that clathrin-mediated membrane receptor internalization and subsequent endocytic events involved in Rab5-dependent early endosome and Rab11-dependent recycling endosome differentially participate in TGF-β [beta] signaling. Two early endosome-enriched proteins, SARA and Hrs, are found to act as a cytosolic retention factor of Smad2, indicating that endocytosis mediates TGF-β [beta] signaling through regulating the dissociation of Smad2 and its cytosolic retention factor.

Cannabinoids (CBs) are compounds that activate the CB1 and CB2 receptors. CB receptors mediate many different physiological functions, and cannabinoids have been reported to decrease tumor cell viability, proliferation, migration, as well as to modulate metastasis. In this thesis, the effects of cannabinoids on human colorectal carcinoma Caco-2 cells (Paper I) and mouse P19 embryonal carcinoma (EC) cells (Paper III) were studied.  In both cell lines, the compounds examined produced a concentration- and time-dependent decrease in cell viability. In Caco-2-cells, HU 210 and the pyrimidine antagonist 5-fluorouracil produced synergistic effects upon cell viability. The mechanisms behind the cytocidal effects of cannabinoids appear to be mediated by other than solely the CB receptor, and a common mechanism in Caco-2 and P19 EC cells was oxidative stress. However, in P19 EC cells the CB receptors contribute to the cytocidal effects possibly via ceramide production. In paper II, the association between CB1 receptor immunoreactivity (CB1IR) and different histopathological variables and disease-specific survival of colorectal cancer (CRC) was investigated. In microsatellite stable (MSS) cases there was a significant positive association of the tumor grade with the CB1IR intensity. A high CB1IR is indicative of a poorer prognosis in MSS with stage II CRC patients. Paper IV focused on the cytotoxic effects of cannabinoids during neuronal differentiation. HU 210 affected the cell viability, neurite formation and produced a decreased intracellular AChE activity. The effects of cannabinoids on embryonic development and survival were examined in Paper V, by repeated injection of cannabinoids in fertilized chicken eggs. After 10 days of incubation, HU 210 and cannabidiol (without CB receptor affinity), decreased the viability of chick embryos, in a manner that could be blocked by α-tocopherol (antioxidant) and attenuated by AM251 (CB1 receptor antagonist). In conclusion, based on these studies, the cannabinoid system may provide a new target for the development of drugs to treat cancer such as CRC. However, the CBs also produce seemingly unspecific cytotoxic effects, and may have negative effects on the neuronal differentiation process. This may be responsible for, at least some of, the embryotoxic effects found in ovo, but also for the cognitive and neurotoxic effects of cannabinoids in the developing and adult nervous system.

Efficient simulation of large-scale mammalian brain models provides a crucial computational means for understanding complex brain functions and neuronal dynamics. However, such tasks are hindered by significant computational complexities. In this work, we attempt to address the significant computational challenge in simulating large-scale neural networks based on the most biophysically accurate Hodgkin-Huxley (HH) neuron models. Unlike simpler phenomenological spiking models, the use of HH models allows one to directly associate the observed network dynamics with the underlying biological and physiological causes, but at a significantly higher computational cost. We exploit recent commodity massively parallel graphics processors (GPUs) to alleviate the significant computational cost in HH model based neural network simulation. We develop look-up table based HH model evaluation and efficient parallel implementation strategies geared towards higher arithmetic intensity and minimum thread divergence. Furthermore, we adopt and develop advanced multi-level numerical integration techniques well suited for intricate dynamical and stability characteristics of HH models. On a commodity CPU card with 240 streaming processors, for a neural network with one million neurons and 200 million synaptic connections, the presented GPU neural network simulator is about 600X faster than a basic serial CPU based simulator, 28X faster than the CPU implementation of the proposed techniques, and only two to three times slower than the GPU based simulation using simpler spiking models.

Frequenin (Frq) and its mammalian homologue, Neuronal Calcium Sensor 1 (NCS-1), are calcium-binding proteins, which regulate neurotransmitter release. However, reports are contradictory, and little is known about Frq's cellular mechanisms. The Drosophila nervous system can be used to gain a better understanding of the function of Frq. There are two Frq-encoding genes in Drosophila. The temporal and spatial expression patterns of the two genes are very similar, and the proteins they encode, Frq1 and Frq2, are 95% identical in amino acid sequence. Loss-of-function phenotypes were studied using three different procedures: creating a deletion designed to remove the entire frq1 gene and part of the frq2 gene; using an interfering C-terminal peptide to prevent Frq binding to its intracellular targets; and using RNAi to reduce frq1 and frq2 transcript levels. Deletion of the entire frq1 gene and part of the frq2 gene resulted in impaired neurotransmitter release and enhanced nerve terminal growth. To discriminate chronic from acute loss-of-function effects, the effects of transgenic expression and forward-filling an interfering C-terminal peptide into presynaptic terminals were compared. In both cases, a reduction in quantal content per bouton occurred, demonstrating that this trait does not result from homeostatic adaptations during development. The chronic treatment also enhanced nerve terminal growth. Conversely, gain-of-function conditions yielded an increase in quantal content and a reduction in nerve terminal growth. Frqs' effects on transmitter output were not due to changes in the number of active zones, nor were they due to changes in the size of the readily releasable pool of vesicles. Oregon Green 488 BAPTA-1 conjugated to 10 kDa Dextran was forward-filled into presynaptic boutons to detect changes in presynaptic Ca2+ signals. Ca2+ responses to presynaptic nerve impulses demonstrated that Frq modulates neurotransmitter release by regulating Ca2+ entry. Gain-of-function phenotypes remained present in a PI4KB null background, demonstrating that Frq's effects were not due to an interaction with PI4KB. All effects seen for all studies were identical for both Frqs, indicating that the two Frq proteins are likely functionally redundant. Overall, Frqs have two distinct functions: one on neurotransmission, primarily by regulating Ca2+ entry, and another on axonal growth and synaptic bouton formation.